
==== Front
J Taibah Univ Med Sci
J Taibah Univ Med Sci
Journal of Taibah University Medical Sciences
1658-3612
Taibah University

S1658-3612(24)00074-X
10.1016/j.jtumed.2024.08.002
Original Article
Cytotoxic properties of Pelargonium graveolens leaf extract and its green-synthesized gold nanoparticles (in vitro study)
Asker Ahmed Y.M. BDS ahmed.Mahdi2202@codental.uobaghdad.edu.iq
⁎
Al Haidar Aseel H.M.J. MSc
Department of Pediatric and Preventive Dentistry, College of Dentistry, University of Baghdad, Iraq
⁎ Corresponding address: Department of Pediatric and Preventative Dentistry, College of Dentistry, University of Baghdad, P.O-Box 1417, Baghdad, Iraq. ahmed.Mahdi2202@codental.uobaghdad.edu.iq
21 8 2024
8 2024
21 8 2024
19 4 901909
10 5 2024
18 7 2024
8 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Objective

This study aimed to assess the cytotoxic effects of an extract from Pelargonium graveolens leaves and green-synthesized gold nanoparticles (AuNPs) in a mouthwash when used as a substitute for commercial mouthwashes. Human dermal fibroblasts from neonates (HDFn) were used in this study that done in vitro, because their characteristics were nearly identical to those of human gingival fibroblasts.

Method

In this study, the green synthesis of AuNPs using extracts from P. graveolens leaves was investigated as a sustainable and economical method. Then, using a range of analytical techniques, the physicochemical properties were evaluated, such as transmission electron microscopy (TEM), X-ray diffraction (XRD), and ultraviolet visible absorption spectroscopy (UV–Vis), so the preparations and analytical techniques of P. graveolens gold nanoparticles (AuNPs) take little time is about 10 days. The current study used the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl-tetrazolium bromide Mosmann's Tetrazolium Toxicity (MTT) to study the cytotoxic effects of P. graveolens leaf extract with P. graveolens gold nanoparticles (AuNPs) utilizing a human fibroblast-derived standard cell line.

Results

Various doses (1000, 500, 250, 100, 50, 25, 12.5, 6.25, and 3.125 μg/mL) of P. graveolens AuNPs were used to assess cytotoxicity, demonstrating little cytotoxic effects (approximately below 20% toxicity). A high level of biocompatibility was observed between the P. graveolens AuNPs and normal human fibroblasts.

Conclusion

The mouthwash made using green synthetic AuNPs obtained from P. graveolens leaf extract show high level of biocompatibility and has low cytotoxicity. Therefore, herbal mouthwash formulations can serve as a viable substitute for chemical mouthwashes.

الملخص

اهداف البحث

هدفت هذه الدراسة إلى تقييم التأثيرات السامة على الخلايا الحية لمستخلص من أوراق نبات العطرة وجسيمات الذهب النانوية الحيوية المصنعة بطريقة التوليف الاخضر لتكوين غسول الفم لاستخدامه كبديل لغسولات الفم التجارية. تم استخدام (الخلايا الليفية الجلدية البشرية من الاجنة) في هذه الدراسة التي أجريت في المختبر، لأن خصائصها كانت متطابقة تقريبًا مع خصائص الخلايا الليفية اللثوية البشرية.

طرق البحث

في هذه الدراسة، تم دراسة التوليف الأخضر لـجسيمات الذهب النانوية الحيوية باستخدام مقتطفات من أوراق نبات العطرة كوسيلة مستدامة واقتصادية. بعد ذلك، وباستخدام مجموعة من التقنيات التحليلية، تم تقييم الخواص الفيزيائية والكيميائية، مثل المجهر الإلكتروني النافذ و حيود الأشعة السينية و ومطياف الامتصاص المرئي فوق البنفسجي، وبالتالي استغرقت الاستعدادات والتقنيات التحليلية لتكوين جسيمات الذهب النانوية وقتًا قصيرًا يبلغ حوالي عشرة أيام. وقد استخدمت الدراسة الحالية ٣-[٤,٣-ثنائي ميثيل ثيازول-٢-ييل]-٢,٥ بروميد ثنائي فينيل-تترازوليوم (سمية التترازوليوم لموسمان) لدراسة التأثيرات السامة للخلايا لمستخلص أوراق نبات العطرة مع جسيمات الذهب النانوية الحيوية لنفس النبات باستخدام خط الخلايا القياسية المشتقة من الخلايا الليفية البشرية.

النتائج

تم استخدام جرعات مختلفة (١٠٠٠، ٥٠٠، ٢٥٠، ١٠٠، ٥٠، ٢٥، ١٢,٥، ٦,٢٥، و ٣,١٢٥ ميكروجرام/مل) من جسيمات الذهب النانوية لنبات العطرة لتقييم السمية الخلوية، وقد دلت النتائج على تأثيرات سامة خلوية قليلة (أقل من ٢٠٪ سمية تقريبًا). وقد لوحظ مستوى عالٍ من التوافق الحيوي بين جسيمات الذهب النانوية لنبات العطرة والخلايا الليفية البشرية الطبيعية.

الاستنتاجات

أظهر غسول الفم المصنوع باستخدام جزيئات الذهب النانوية الحيوية من مستخلص أوراق نبات العطرة المصنع بطريقة التوليف الاخضر, مستوى عالٍ من التوافق البيولوجي وله سمية خلوية منخفضة. لذلك، يمكن أن تكون تركيبات غسول الفم العشبية بمثابة بديل قابل للتطبيق لغسولات الفم الكيميائية.

الكلمات المفتاحية

ننبات العطرة
جزيئات الذهب النانوية
غسول الفم
السمية الخلوية
لخلايا الليفية البشرية الطبيعية
سمية التترازوليوم لموسمان
Keywords

Cytotoxicity
Gold nanoparticle
Mosmann's tetrazolium toxicity
Mouthwash
Normal human fibroblast
Pelargonium graveolens
==== Body
pmcIntroduction

Dental plaque (dental biofilm) is the primary etiological factor responsible for the development of periodontal diseases and tooth caries.1 Periodontal disease is a widespread oral inflammatory disease often observed in middle-aged and elderly individuals. Prevention of periodontal disease may be achieved by early identification facilitated by advancements in clinical understanding and procedures.2 Thus, it is essential to utilize efficient strategies for plaque management, for example, items like toothbrushes and dental floss to stop plaque formation and build-up on tooth surfaces. Mouthwashes can also be used as adjuncts to these mechanical methods, particularly in situations where people lack the necessary physical skills and drive, such as children. Chlorhexidine is generally recognized as the preferred mouthwash and is highly recommended as an effective antibacterial and antiseptic agent. However, the use of chlorhexidine has been associated with many undesirable consequences, including tooth discoloration, burning sensation, oral ulcers, and alteration in taste perception. These documented side effects require exploration of natural alternatives that are safe and minimize adverse reactions.3

Nanoparticles are widely used in biomedicine, medication administration, materials chemistry, and pollution prevention. Biosynthesized nanoparticles have been shown to have notable antibacterial and photocatalytic properties. Antibacterial activity, biosensing, optics, catalysis, and the treatment of diseases are just a few of the many uses for metal nanoparticles, such as iron oxide, zinc oxide, and gold nanoparticles.4 In recent years, considerable interest has been demonstrated in the research of gold nanoparticles (AuNPs). AuNPs have unique properties, such as the ability to change size, large surface area relative to volume, remarkable optical properties, excellent compatibility with living organisms, low toxicity, and easy surface alteration.5 Furthermore, nanoparticles have distinguishing properties, such as small size, large specific surface area, and the capacity to induce quantum phenomena.6

Due to their tiny size, nanomaterials possess a much higher surface area per unit mass in comparison to larger particles. As a consequence, there is a higher level of attraction between molecules and an exceptional capacity to evenly distribute in liquid mixtures. In addition, they have a significant affinity for proteins, plaque, and bacteria. Moreover, the enlarged surface area of the substance amplifies its antibacterial characteristics.7

Various methods have been used to fabricate nanomaterials, including chemical reduction, electrochemical synthesis, laser ablation, lithography, and pyrolysis. Generally, these methods are distinguished by their high expense and inherent hazards, mostly because of the use of hazardous reducing agents. The use of bio-fabrication by plants and other organisms to create nanoparticles offers a feasible, eco-friendly, economical, and sustainable method. Plant extracts provide prospective benefits in terms of the rapidity, ease, and appropriateness for nanoparticle production. Furthermore, plant phytoconstituents often serve as both lowering and stabilizing agents, resulting in decreased costs and improved effectiveness.8 AuNPs have been synthesized from extracts of various wild plant species, including Potamogeton pectinatus,9 Ferula persica,10 Phragmites australis,11 and Pelargonium graveolens.12

A variety of optical and physical methods are available for characterizing AuNPs. The distinctive optical and physical characteristics of AuNPs are mainly due to their peculiar shape, sizes, forms, and colloidal stability. Therefore, it is essential to characterize the produced AuNPs thoroughly utilizing a range of techniques in order to preserve their quality. In order to avoid unclear interpretations, a number of different approaches are usually advised for acceptable and complimentary characterizations.13

Nanoparticles material can be characterized by the following tests, Ultraviolet–Visible Spectroscopy (UV–VIS), Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS) and X-ray Photoelectron Spectroscopy XPS.14

The plant species P. graveolens (Geraniaceae) is well-recognized for its production of a commercially popular essential oil, known as rose geranium. Its aerial components have been extensively used in traditional medicine. The oils and extracts of P. graveolens have been studied pharmacologically both in vitro and in vivo. The extracts' marker constituents include citronellol, guaia-6,9-diene, geranyl formate, linalool, isomethone, citronellyl formate, and geraniol.15

The 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay is used for the in vitro evaluation of prospective pharmaceuticals and to investigate drug resistance in cellular models. Similarly, it aids in evaluating the effects of drugs in controlled laboratory environments and forecasting their actual use in clinical settings. This test is a method that is known for its high sensitivity, precision, and widespread usage in detecting cytotoxicity and assessing cell viability following exposure to harmful chemicals. Therefore, it is an invaluable tool for assessing the toxicity of substances and differentiating between various cell lines. It is efficient in terms of cost, provides a large amount of data rapidly, and has exceptional sensitivity and reproducibility.3

Tests conducted in vitro can be used to estimate the toxicity of NPs in an initial manner. Realistically speaking, though, the intricate biological interaction seen in in vivo models does not correspond with the results obtained in laboratories. Consequently, a comprehensive biocompatibility testing program that includes an evaluation of the effects on organs and the immune system is necessary for the assessment of NMs. Any nanomaterial that might be used in the human body needs to be studied to find out if it can enter cells or tissues and how it gets dispersed, breaks down naturally, and gets eliminated. Therefore, it is indisputable that the use of animal models is significant and ought to be included in preclinical toxicity studies of nanoformulations.16

The current research seeks to examine the effects of AuNPs synthesized from P. graveolens leaf extract on a normal human cell line by assessing growth viability using the MTT assay. Therefore, this study has the potential to serve as a valuable resource for the development of sophisticated natural antibacterial treatments.

Materials and Methods

Materials and chemicals

The compound chloroauric acid (HAuCl4·3H2O) was acquired from Sigma-USA. Ethanol was obtained from Duksan (Seoul, Korea). Trypsin/EDTA (Ethylene diamine tetraacetic acid) reagent was procured from Capricorn, Germany, and DMSO (Dimethyl sulfoxide) was acquired from Santa Cruz Biotechnology (USA). The experiment utilized the minimum essential medium (MEM) obtained from Capricorn, Germany. The MTT stain used in the investigation was acquired from Bio-World-USA. Fetal bovine serum used in the experiment was sourced from Capricorn, Germany. The microtiter reader was sourced from Gennex Lab-USA, and cell culture plates were obtained from Santa Cruz Biotechnology, USA. All supplementary chemicals and reagents used in this study were of analytical grade about the characterization of the synthesized nanoparticles, using a Japanese X-ray diffract meter, Transmission electron microscopy TEM from PHILIPS in the USA is used to measure the size and shape of nanoparticles. A double-beam UV–Vis spectrophotometer was utilized to analyze beams ranging from 200 to 1100 nm for the experiment. The Zetasizer-Nano from ZS-Malvern Instruments was used to analyze the zeta potential. All materials and devices were summarized in Table 1.Table 1 Materials and Equipments that used for cytotoxicity assay of Pelargonium graveolens leaf extract and its green-synthesized gold nanoparticles.

Table 1Equipment and materials	Company	Origin	
Autoclave	Gallenkamp	United Kingdom	
CO2 Incubator	Gallenkamp	England	
Crystal violate	BDH	England	
Deep Freezer	The Electron Corporation	U.S. A	
DMSO	Santacruz	USA	
DPPH	Sigma	U.S. A	
ELISA	Quik Fit	Germany	
Fetal Bovine Serum	Sigma	U.S. A	
Filter unit 0.22 μm	Millipore	Spain	
Giemsa stain	Thomas Baker	India	
Hot air oven	Memmert	Germany	
Incubator	Memmert	Germany	
Inverted Microscope	Olympus	Japan	
Laminar Flow Hood	K & K	Korea	
MEM	Capricorn	Germany	
Micro Centrifuge	Hermle	Germany	
Micropipette	Volac	England	
Microtiter reader	Gennex Lab	USA	
MTT Dye Sigma aldrich	Sluka	Germany	
MTT stain	Bia-World	USA	
HAuCl4	Sigma–Aldrich	U.S. A	
pH-Meter	Radiometer	Denmark	
Plastic Flasic For Tissue Culture	Falcon	U.S. A	
RPMI-1640	Sigma	U.S. A	
Sensitive Balance	Sartorius	Germany	
Shaker Incubator	Selecta	Spain	
Trypsin∖Versine	Sigma	U.S. A	
Trypsin∖EDTA	Capricorn	Germany	
Vortex	Griffin	England	
Water Bath	Gallenkamp	England	
An X-ray diffract meter (XRD-6000	SHEMADZU	Japan	
Transmission electron microscopy	PHILIPS CM-120	U.S. A	
UV–Vis spectrophotometer	SHEMADZU	Japan	
Zetasizer Nano ZS	Malvern Instruments	Germany	

Cell culture

For maintenance, a human dermal fibroblast from neonates (HDFn) cell line displaying typical features was grown in the minimum essential medium (MEM) enriched with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin. The cells were propagated using Trypsin–EDTA and then replanted twice a week until they reached a confluence level of 50%. Afterwards, the cells were placed in an incubator set at a temperature of 37 °C.17 The cell lines used in this study were obtained from the Cell Bank Unit of the Experimental Therapy Department at the Iraqi Center for Cancer and Medical Genetic Research (ICCMGR). The cells are regularly assessed to determine their developmental features and undergo constant authentication.

Preparation of leaf extract from P. graveolens and the green synthesis of its AuNPs

The leaves of P. graveolens were collected from Diyala Governorate in Iraq. The leaves were carefully washed with sterilized water to remove any remaining chemicals and lime. Following a meticulous rinse, P. graveolens leaves left to dry overnight at a temperature of 37 °C. Following incubation, the thoroughly dried leaves were finely ground into a powder for extraction purposes. Then using 100 g of powder of plant material was extracted using aqueous alcohols as the solvent. The solvents used were methanol, water, and ethanol (1:1:3) 80% v/v, with a volume of 500 mL. The samples underwent Soxhlet extraction for 8 h in a water bath. Afterwards, the extracts underwent solvent removal by decreased pressure using a rotary evaporator at a temperature of 45 °C. Afterwards, the amounts of the dried, unrefined concentrated extracts were measured to calculate the yield. Subsequently, the powder was diluted to attain a concentration of 1 mg/mL, equivalent to the bactericidal concentration (MBC) of the plant extract. Subsequently, the filtered extracts were combined with 50 mL of gold chloride to serve as a reducing agent for the synthesis of gold nanoparticles. The gold ion solution was made following the approach described by Elia et al. (2014), with some alterations. A 10 mg/mL solution was prepared by dissolving (1 g of HAuCl4) in 100 mL of distilled water (DW). Gold nanoparticles (Au NPs) were synthesized by mixing a 1 mg/mL HAuCl4 gold ion solution with 50 mL of plant extract. The solution was vigorously stirred using a magnetic stirrer for 30 min while being heated slowly to a temperature ranging from 35 °C to 45 °C. Consequently, the mixture had a rapid alteration in color, shifting to a rich purple/red shade in a little duration. The color shift that was seen might be attributed to the production of 15.7 parts per million (ppm) of gold nanoparticles (AuNPs). The presence of a purple color, which arises from the amalgamation of the plant extract with the HAuCl4 solution, signifies the creation of P. graveolens AuNPs,18 This substance is known for its high absorption in the ultraviolet (UV) region with a peak at 527 nm. The average size of nano crystallites in AuNPs is determined to be 69.587 nm using X-ray diffraction testing. TEM has shown the average size of AuNPs to be 19 nm, as seen in Figure 1. The zeta potential of AuNPs was determined to be 27 mV. This measurement indicates that the nanoparticles were stable and had a decreased tendency to clump together and form bigger particles. The preparing of P. graveolens AuNPs mouthwash is inexpensive, for example, the cost of preparing one liter was $30, so the economic effectiveness of this mouthwash within everyone's reach.Figure 1 A-Distribution of particle size of Pelargonium graveolens AuNPs. B-TEM of Pelargonium graveolens AuNPs.

Figure 1

MTT assay

The cytotoxic assay is a screening test to evaluate the biocompatibility of the examined substances (P. graveolens leaves extract and it,s AuNPs) in normal cell lines. In vitro evaluation of cytotoxicity can be performed by Mosmann's tetrazolium toxicity assay, which is considered the most common, rapid, and simple assay. This assay uses a yellow water-soluble dye, which converts to insoluble purple formazan.19,20 The reaction occurs due to the fracturing of the tetrazolium ring by a mitochondrial enzyme called succinate dehydrogenase, which is exclusively present in healthy, viable cells.21 This assay was conducted at the Biology Department of the College of Science, University of Diyala. For the cell suspension, 50 mL of tissue culture were mixed with a trypsin-versene solution. Afterwards, 20 mL of the culture medium, which was supplemented with the prepared serum, was added to the mixture. The cell suspension was mixed well and 0.2 mL was put into each well of the 96-well micro titer plates using a precise micropipette. Therefore, each well contained 1 × 105 (cells/well). The surface of the micro titer plate was covered with opaque transparent adhesive paper and the plate was moved gently for homogeneous distribution of cells in the wells. The plates had been Subjected to incubation at a temperature of 37 °C for a period of 12–18 h until the cells attached to the well. Consequently, the prior cultural medium was taken out from the wells. Then, 0.2 mL of the simultaneously formulated solutions of P. graveolens leaves crude alcoholic extract and P. graveolens AuNPs was added using serum free media (SFM) (6.25, 12.5, 25, 50, 100, 200, 250, 500,1000, 2000,4000,8000) μg/mL and deionized water as control negative, with three replicates for each concentration of each solution. After 72-h exposure time, after taking out the plate from the incubator, a precise volume of crystal violet dye, 50 μL, was carefully added to each well. The plate was left to incubate for 20 min. Afterwards, the contents were taken out, and the plate was rinsed with a phosphate buffered saline (PBS) solution as long as the excess dye was removed and the cells were allowed to dry. The crystal violet assay relies on the staining of cells that are adhered to cell culture plates. This process involves the separation of adherent cells from cell culture plates when cell death occurs. As part of the assay, non-viable cells are removed through a washing process, leaving behind only viable cells that are then stained using crystal violet. The crystal violet dye is solubilized, and its measurement is obtained by absorbance at 492 nm after the washing process. Cell biomass may be used to estimate cell viability along with cytotoxicity. The crystal violet test operates on the assumption that all cells adhered to the plate are viable, whereas any cells that detach are considered nonviable.

We used the following equation to calculate the cell growth inhibition rate (percentage of cytotoxicity):22Cellviability%=AbsorbanceoftreatedcellsAbsorbanceofuntreatedcellsx100

Cytotoxicity % = 100 − cell viability

Statistical analysis

The significance level was determined using one-way analysis of variance (ANOVA) with a p-value of 0.05 to describe and analyze the data. GraphPad Prism version 8 (GraphPad Software Inc., La Jolla, CA, USA) was used to display the results as mean, standard deviation, and statistical significance.

Results

The formation of formazan crystals, which are produced when living cells reduce MTT tetrazolium, was used to measure cell viability. This research examined the impact of various doses of P. graveolens extract on the survival of normal human fibroblast cells by dose–response analysis. The objective was to calculate the IC50 value, which represents the concentration at which 50% of cell growth is inhibited. The results of this investigation are summarized in Table 1, where the data are shown as means accompanied by their corresponding standard deviations. The MTT test was employed to evaluate the cytotoxicity of various concentrations (8000, 4000, 2000, 1000, 500, 250, 200, 100, 50, 25, 12.5, 6.25, and 3.125 μg/mL) of P. graveolens leaves extract on normal human fibroblast cells during a 72-h period. The concentrations of P. graveolens leaves extract at 250, 200, 100, 50, 25, 12.5, and 6.25 μg/mL exhibited reduced cytotoxicity towards the cells. The cytotoxicity of P. graveolens against typical human fibroblasts increased as its concentration increased. Furthermore, the test doses (50, 25, 12.5, and 6.25) μg/mL exhibited greater cell viability compared with that at 100, 200, 250, and 500 μg/mL concentrations, indicating a dose-dependent cytotoxic impact of the drug. As shown in Table 2, a noticeable increase in the mean fibroblast inhibition percentage was observed as the concentration of P. graveolens leaf extract increased. This increase was statistically significant, indicating that the cytotoxic impact of the extract depended on its concentration, as seen in Figure 2. Tukey's multiple pairwise comparison showed that the difference between each group with the other was significant except for between 100 vs. 200, 200 vs. 250, and 250 vs. 500 μg/mL (Table 3). In relation to the cytocompatibility of P. graveolens leaf extract, the determination of the half lethal concentration (LC50) required to cause the death of 50% fibroblast cells was achieved through the utilization of a simple linear regression equation (Y = 0.0081x + 6.355). This calculation was performed under the assumption that the value of Y corresponded to 50 (Figure 2). LC50 was determined to be 5388 μg/mL (Figure 3).Table 2 Descriptive and statistical test of % of inhibition among agent and concentration (one way ANOVA).

Table 2Conc
μg/mL	Pelargonium graveolens Extract Inhibition% ± SD	Pelargonium graveolens
AuNPs Inhibition% ± SD	T test	p value	
6.25	0 ± 0.0	0 ± 0.0			
12.5	0 ± 0.0	0 ± 0.0			
25	0 ± 0.0	0 ± 0.0			
50	0 ± 0.0	0 ± 0.0			
100	9.03 ± 1.1	0 ± 0.0			
200	10.30 ± 1.3	0 ± 0.0			
250	11.50 ± 1.5	0 ± 0.0			
500	16.35 ± 1.6	7.03 ± 1.1	8.313	0.001HS	
1000	22.82 ± 1.8	9.03 ± 1.8	9.383	0.0007HS	
2000	30.71 ± 1.9	15.71 ± 1.9	9.669	0.0006HS	
4000	39.15 ± 2.0	20.15 ± 2.0	11.635	0.0003HS	
8000	67.86 ± 2.3	40.86 ± 2.3	14.377	0.0001HS	
F	399.678	158.149			
p value	0.000	0.000			
Sig. = significant at p < 0.05.

Figure 2 Mean of fibroblast inhibition percentage at different Pelargonium graveolens Leaves Extract and Pelargonium graveolens AuNPs concentration.

Figure 2

Table 3 Multiple comparisons of % inhibition between groups in Pelargonium graveolens Extract using Tukey HSD method.

Table 3Groups
μg/mL	100	200	250	500	1000	2000	4000	8000	
MD	p	MD	p	MD	p	MD	p	MD	p	MD	p	MD	p	MD	p	
100			−1.27	0.98	−2.47	0.65	−7.32	0.002HS	−13.79	0.000HS	−21.68	0.000HS	−30.12	0.000HS	−58.65	0.000HS	
200					−1.2	0.98	−6.05	0.01HS	−12.5	0.000HS	−20.41	0.000HS	−28.85	0.000HS	−57.38	0.000HS	
250							−4.85	0.052	−11.32	0.000HS	−19.21	0.000HS	−27.65	0.007HS	−56.18	0.000HS	
500									6.47	0.006HS	14.36	0.000HS	22.80	0.000HS	−51.33	0.000HS	
1000											−7.89	0.000HS	−16.33	0.000HS	−44.86	0.000HS	
2000													−8.44	0.000HS	−36.97	0.000HS	
4000															−28.53	0.000HS	

Figure 3 LC50 of Pelargonium graveolens Leaves Extract determination by simple linear regression graph.

Figure 3

The MTT test was employed to explore the cytotoxicity of various concentrations (8000, 4000, 2000, 1000, 500, 250, 200, 100, 50, 25, 12.5, 6.25, and 3.125(μg/mL of P. graveolens AuNPs on normal human fibroblast cells in a 72-h period. The test doses 1000, 500, 250, 200, 100, 50, 25, 12.5, and 6.25 μg/mL exhibited greater cell viability compared with that at 8000, 4000, and 2000 μg/mL, indicating a dose-dependent cytotoxic impact of the drug. As shown in Table 2, there was a noticeable increase in the mean fibroblast inhibition percentage as the concentration of the P. graveolens leaf extract increased. Tukey's multiple pairwise comparison results showed significant differences between groups except for between 500 vs. 1000 and 2000 vs. 4000 μg/mL (Table 4). Regarding the cytocompatibility of P. graveolens AuNPs mouthwash, the LC50 was a very high concentration and all tested mouthwash concentrations killed less than 50% of fibroblasts, for example, a higher concentration of 8000 μg/mL killed 40% of fibroblasts as shown in Figure 4 the determination of the half lethal concentration (LC50) required to cause the death of 50% fibroblast cells was achieved through the utilization of a simple linear regression equation (Y = 0.0051x + 0.8278). This calculation was performed under the assumption that the value of Y corresponded to 50 (Figure 2). LC50 was determined to be 9641 μg/mL of P. graveolens AuNPs as shown in (Figure 4). And the cells visualized under phase contrast inverted microscope for normal morphology of human dermal fibroblasts cells as shown in Figure 5.Table 4 Multiple comparisons of % inhibition between groups in Pelargonium graveolens AuNPs using Tukey HSD method.

Table 4Groups
μg/mL	500	1000	2000	4000	8000	
MD	p	MD	p	MD	p	MD	p	MD	p	
500			−2	0.689	−8.68	0.001HS	−13.12	0.000HS	−33.83	0.000HS	
1000					−6.68	0.009HS	−11.12	0.000HS	−31.83	0.000HS	
2000							−4.44	0.089HS	−25.15	0.000HS	
4000									−20.71	0.000HS	

Figure 4 LC50 of Pelargonium graveolens AuNPs determination by simple linear regression graph.

Figure 4

Figure 5 Normal morphology of human dermal fibroblasts cells (A) Untreated cells, (B) treated cells.

Figure 5

Also, the determination of the half lethal concentration (LC50) can be done using the most appropriate method) The probit analysis (is conducted by calculating the percentage of dead cells and then converting this percentage to a probit value using a recommended table. Then, will employ log values for the concentrations that were used to create a scatter diagram for the probability percent relationship. Subsequently, the equation (the Y values) is displayed, and the Y value is replaced with 5 (corresponding to a 50-lethal dosage). The X value (the log concentration) is then calculated using the equation (y = ax + b), and the log value is then converted to the absolute concentration, wherefore, Table 5 show that LC50 of P. graveolens leaves extract was about 2818.38 μg/mL, while, Table 6 show that LC50 of P. graveolens AuNPs was about 14791.08 μg/mL.Table 5 The probit analysis to calculating the LC50 for different concentrations Pelargonium graveolens leaves extract.

Table 5Concentrations
Pelargonium graveolens extract
μg/mL	log 10 (concentration)	% dead	Probit	
6.25	0.795880017	0	0	
12.5	1.096910013	0	0	
25	1.397940009	0	0	
50	1.698970004	0	0	
100	2	9.03	3.66	
200	2.301029996	10.3	3.78	
250	2.397940009	11.5	3.77	
500	2.698970004	16.35	4.01	
1000	3	22.82	4.23	
2000	3.301029996	30.71	4.48	
4000	3.602059991	39.15	4.72	
8000	3.903089987	67.86	5.44	


	
Intercept	−1.805683154			
X Variable 1	1.977674462			


	
y = ax + b				
y = 1.97x + (−1.80)				
5 = 1.97x − 1.80				
5 + 1.80 = 1.97x				
x=(5 + 1.80)/1.97				
x = 3.45				
LC50 = antilog x				
LC50 = antilog 3.45				
LC50 = 2818.38 μg/mL				

Table 6 The probit analysis to calculating the LC50 for different concentrations Pelargonium graveolens AuNPs.

Table 6Concentrations
Pelargonium graveolens AuNPs μg/mL	Log 10 (concentration)	% dead	Probit	
6.25	0.795880017	0	0	
12.5	1.096910013	0	0	
25	1.397940009	0	0	
50	1.698970004	0	0	
100	2	0	0	
200	2.301029996	0	0	
250	2.397940009	0	0	
500	2.698970004	7.03	3.52	
1000	3	9.03	3.66	
2000	3.301029996	15.71	3.96	
4000	3.602059991	20.15	4.16	
8000	3.903089987	40.86	4.75	


	
Intercept	−2.644881412			
X Variable 1	1.836876908			
Y = ax + b				
y = 1.83x + (−2.64)				
5 = 1.83x − 2.64				
5 + 2.64 = 1.83x				
x=(5 + 2.64)/1.83				
x = 4.17				
LC50 = antilog x				
LC50 = antilog 4.17				
LC50 = 14791.08 μg/mL				

Discussion

The MTT assay is widely used as a means of determining the cytotoxic impact of different chemicals over a range of circumstances or concentrations. This study used Human dermal fibroblasts from neonates (HDFn) because their characteristics are comparable to those of human gingival fibroblasts.23 The IC50 value (half inhibitory concentration) of the applicable drugs can be calculated by examining the survival rates of the drug-treated and control groups.19

Therefore, we conducted laboratory tests to evaluate the cytotoxic effects of P. graveolens leaf extract AuNPs at different concentrations. The results of the experiments conducted on normal human dermal fibroblast cells over a 72-h period showed that the outcome was influenced by the dose of leaf extract administered. Small amounts of both tested solutions had minimal effects on cell viability. In contrast, when human cells were exposed to the IC50 dose, a significant decrease in cell viability was noted.

These findings suggest a direct relationship between the concentration of P. graveolens leaf extract and its level of toxicity. Concentrations of the herbal substance over 5330 μg/mL were specifically identified as more toxic. The results align with a previous study that investigated the cytotoxicity of P. graveolens leaf extracts using in vitro tests.24 Furthermore, the use of P. graveolens extract has gained extensive recognition in the food industry owing to its established safety and approval by the American Food and Drug Administration (FDA) for use in food products.25 The use of botanical medicines for the management and prevention of oral ailments might provide sporadic benefits to rural communities or individuals with limited financial resources, owing to the cost-effectiveness and widespread accessibility of herbal resources throughout the nation. Consequently, medicinal plants have the potential to serve as feasible alternatives to conventional antibacterial agents.26 In addition, some adverse effects are linked to the chemical substances included in some drugs currently used, and should be avoided.

Several studies have investigated the use of P. graveolens extract for the green syntheses of AuNPs and have demonstrated its antibacterial properties. Therefore, this research seeks to investigate the possible harmful effects of these medications. This investigation is relevant for determining its appropriateness for patient use and for establishing its efficacy as a mouthwash at a specified concentration, which is considered the standard.

The main aim of the research was to produce AuNPs to examine their potential use in biological systems. P. graveolens AuNPs were thoroughly tested for biocompatibility and cytotoxicity. Different dosages (100, 50, 25, 12.5, 6.25, and 3.125 μg/mL) of P. graveolens AuNPs were employed to measure cytotoxicity and demonstrated slight cytotoxicity (approximately 20% toxicity). The findings presented in this study indicate a high level of biocompatibility between the AuNPs and normal human fibroblasts. The results align with the findings presented by Elia et al., in 2014.18

Furthermore, over the last few years, several pharmaceutical firms have obtained permission from FDA for the development of nanotechnology-based pharmaceuticals. This is primarily driven by the pressing need for substantial investment in the creation of novel nanotechnology-based medical devices for therapeutic purposes.27

Study limitation

1. Due to lack of facilities and abilities make it difficult to expose any hazardous medications that could potentially induce a condition resembling periodontitis, which will make the experiment's applicability more realistic by testing the cytoprotective effect of prepared nano-based mouthwash after infecting cells with a harmful medication, as this will simulate the patient's condition. Thus, it could be adapted for clinical applications. This will need more studies in future.

2. Unavailable kits and materials at Iraqi laboratories; Therefore, it needs to be imported from outside Iraq, and this takes a lot of time, outside the specified study time.

3. Conducting research with many repetitions the cytotoxicity test can be expensive and resource-intensive and this research has limited funds.

4. Unavailable the following tests in Iraq (Flow cytometry–fluorescent assays–Luminescent assays–Dye exclusion assay–Dye uptake and release assay) employing any of these techniques in future studies will improve the validity and reliability of cytotoxicity measurements. Other studies will be conducted if these capabilities are available in our country. Note that it is a novel study that is being conducted for the first time.

Conclusion and recommendations

In the current study, examination of the alcoholic crude extract of P. graveolens and it's green synthetic AuNPs revealed varying levels of cell viability based on the doses used. Additionally, we assessed the effect of these substances on cell viability, and found that a normal number of human fibroblasts remained stable with a very small decrease in response to low doses., indicating a dose-dependent hazardous impact. P. graveolens AuNPs demonstrated minimal cytotoxicity in human fibroblast cells, allowing their use in dentistry based on recommendations due to their potential as a viable substitute for chlorhexidine gluconate 0.12% (a common oral antibacterial mouthwash) for both the prevention and treatment of bacteria-induced diseases.

Source of funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Conflict of interest

The authors have no conflict of interest to declare.

Ethical approval

The ethical committee of the University of Baghdad's-College of Dentistry, gave consent for the study (No. 771233, 26-1-2023 with Ref. number: 771).

Authors contributions

The research report was written by AYM and AHMJ, who also helped with the study's design, implementation, and interpretation of data. All authors have critically reviewed and approved the final draft and are responsible for the content and similarity index of the manuscript.

Acknowledgment

The authors would like to thank everyone who took part in the research for their helpful feedback.

Peer review under responsibility of Taibah University.
==== Refs
References

1 Al-Awadi R.N. Al-Casey M. Oral health status, salivary physical properties and salivary Mutans Streptococci among a group of mouth breathing patients in comparison to nose breathing J Baghdad Coll Dent 325 2210 2013 1 8
2 Rawdhah R. Mahmood M.S. Salivary lactate dehydrogenase and salivary total protein as potential biomarkers for screening periodontal disease Saudi Dent J 35 7 2023 827 833 38025598
3 Ali S.H. Jafar Z.J. In vitro cytotoxic effect of annona squamosa pulp ex-tract as a mouthwash for children on human normal cell line J Baghdad Coll Dent 34 1 2022 60 66
4 Patil T.P. Vibhute A.A. Patil S.L. Dongale T.D. Tiwari A.P. Green synthesis of gold nanoparticles via Capsicum annum fruit extract: characterization, antiangiogenic, antioxidant and anti-inflammatory activities Appl Surf Sci Adv 13 2023 100372
5 Patil T. Gambhir R. Vibhute A. Tiwari A.P. Gold nanoparticles: synthesis methods, functionalization and biological applications J Clust Sci 34 2 2023 705 725
6 Shakir D.A. Abdul-Ameer F.M. Effect of nano-titanium oxide addition on some mechanical properties of silicone elastomers for maxillofacial prostheses J Taibah Univ Med Sci 13 3 2018 281 290 31435335
7 Al-Bazaz F.A. Radhi N.J. Hubeatir K.A. Sensitivity of Streptococcus mutans to selected nanoparticles (in vitro study) J Baghdad Coll Dent 30 1 2018 69 75
8 Hosny M. Fawzy M. Abdelfatah A.M. Fawzy E.E. Eltaweil A.S. Comparative study on the potentialities of two halophytic species in the green synthesis of gold nanoparticles and their anticancer, antioxidant and catalytic efficiencies Adv Powder Technol 32 9 2021 3220 3233
9 AbdelHamid A.A. Al-Ghobashy M.A. Fawzy M. Mohamed M.B. Abdel-Mottaleb M.M. Phytosynthesis of Au, Ag, and Au–Ag bimetallic nanoparticles using aqueous extract of sago pondweed (Potamogeton pectinatus L.) ACS Sustain Chem Eng 1 12 2013 1520 1529
10 Hosseinzadeh N. Shomali T. Hosseinzadeh S. Raouf Fard F. Pourmontaseri M. Fazeli M. Green synthesis of gold nanoparticles by using Ferula persica Willd. gum essential oil: production, characterization and in vitro anti-cancer effects J Pharm Pharmacol 72 8 2020 1013 1025 32319112
11 El-Borady O.M. Fawzy M. Hosny M.J. Antioxidant, anticancer and enhanced photocatalytic potentials of gold nanoparticles biosynthesized by common reed leaf extract Appl Nanosci 13 5 2023 3149 3160
12 Shankar S.S. Ahmad A. Pasricha R. Sastry M. Bioreduction of chloroaurate ions by geranium leaves and its endophytic fungus yields gold nanoparticles of different shapes J Mater Chem 13 7 2003 1822 1826
13 Urban D.A. Milosevic A.M. Bossert D. Crippa F. Moore T.L. Geers C. Taylor dispersion of inorganic nanoparticles and comparison to dynamic light scattering and transmission electron microscopy Colloid Interface Sci Commun 22 2018 29 33
14 Ielo I. Rando G. Giacobello F. Sfameni S. Castellano A. Galletta M. Synthesis, chemical–physical characterization, and biomedical applications of functional gold nanoparticles: a review Molecules 26 19 2021 5823 34641367
15 Sandasi M. Kamatou G. Mulaudzi N. Pelargonium graveolens The South African herbal pharmacopoeia 2023 Elsevier 387 406
16 Kus-Liśkiewicz M. Fickers P. Ben Tahar I. Biocompatibility and cytotoxicity of gold nanoparticles: recent advances in methodologies and regulations Int J Mol Sci 22 20 2021 10952
17 Al-Shammari A.M. Alshami M.A. Umran M.A. Almukhtar A.A. Yaseen N.Y. Raad K. Establishment and characterization of a receptor-negative, hormone-nonresponsive breast cancer cell line from an Iraqi patient Breast Cancer 2015 223 230
18 Elia P. Zach R. Hazan S. Kolusheva S. Porat Ze Zeiri Y. Green synthesis of gold nanoparticles using plant extracts as reducing agents Int J Nanomed 2014 4007 4021
19 Berridge M.V. Herst P.M. Tan A.S. Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction Biotechnol Annu Rev 11 2005 127 152 16216776
20 Stockert J.C. Horobin R.W. Colombo L.L. Blázquez-Castro A. Tetrazolium salts and formazan products in Cell Biology: viability assessment, fluorescence imaging, and labeling perspectives Acta Histochem 120 3 2018 159 167 29496266
21 Babu B. Palanisamy S. Vinosha M. Anjali R. Kumar P. Pandi B. Bioengineered gold nanoparticles from marine seaweed Acanthophora spicifera for pharmaceutical uses: antioxidant, antibacterial, and anticancer activities Bioprocess Biosyst Eng 43 2020 2231 2242 32683506
22 Al-Shammari A.M. Jalill R.D.A. Hussein M.F. Combined therapy of oncolytic Newcastle disease virus and rhizomes extract of Rheum ribes enhances cancer virotherapy in vitro and in vivo Mol Biol Rep 47 3 2020 1691 1702 31970625
23 Ebisawa K. Kato R. Okada M. Sugimura T. Latif M.A. Hori Y. Gingival and dermal fibroblasts: their similarities and differences revealed from gene expression J Biosci Bioeng 111 3 2011 255 258 21288768
24 El Aanachi S. Gali L. Nacer S.N. Bensouici C. Dari K. Aassila H. Phenolic contents and in vitro investigation of the antioxidant, enzyme inhibitory, photoprotective, and antimicrobial effects of the organic extracts of Pelargonium graveolens growing in Morocco Biocatal Agric Biotechnol 29 2020 101819
25 Boukhatem M.N. Kameli A. Saidi F. Essential oil of Algerian rose-scented geranium (Pelargonium graveolens): chemical composition and antimicrobial activity against food spoilage pathogens Food Control 34 1 2013 208 213
26 Karbach J. Ebenezer S. Warnke P. Behrens E. Al-Nawas B. Antimicrobial effect of Australian antibacterial essential oils as alternative to common antiseptic solutions against clinically relevant oral pathogens Clin Lab 61 1–2 2015 61 68 25807639
27 Hobson D.W. The commercialization of medical nanotechnology for medical applications 2016 405 449
