A Potential Natural Alternative to Chlorhexidine Mouthwashes: An in Vivo Study in Rats 

Vol 11 No 1 (2023)    DOI 10.5195/d3000.2023.503 

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A Potential Natural Alternative to Chlorhexidine 
Mouthwashes: An in Vivo Study in Rats 

Mariana Bezamat1,2, Alice Spitz1*, Daniela Sales Alviano3, Dayanne Lopes da Silva1, Vicente Telles1,2, Marcia Grillo Cabral4, 

Ana Maria Bolognese1 

1 Department of Pediatric Dentistry and Orthodontics, Federal University of Rio de Janeiro. 

2 Department of Oral and Craniofacial Sciences, University of Pittsburgh School of Dental Medicine. 

3 Department of Microbiology, Federal University of Rio de Janeiro. 

4 Department of Oral Pathology, Federal University of Rio de Janeiro.

Abstract 

Background: In the Brazilian northeast region, the leaves of a plant scientifically known as Aristolochia 
cymbifera (A. cymbifera) are therapeutically used to treat different conditions, including fevers, ulcers 
and stomach disorders.  

Objective: To explore its effects in the oral cavity, this study aimed to evaluate the presence of 
morphological, cellular, and tissue modifications in the oral mucosa of rats treated with different A. 
cymbifera solutions. 

Materials and Methods: The rats were divided into 6 groups: Group 1 was treated with 0.3 ml of vehicle 
(DMSO and water); Group 2 treated with 0.3 ml of A. cymbifera-ethanolic extract 4mg/ml; Group 3 
treated with 0.3 ml of A. cymbifera-hexane extract 4mg/ml; Group 4 treated with 0.3 ml of A. cymbifera-
ethanolic extract 10mg/ml; Group 5 treated with 0.3 ml of A. cymbifera-hexane extract 10mg/ml; and 
Group 6 treated with 0.3 ml of chlorhexidine gluconate 0.12%. All groups were treated twice daily by 
applying the solutions into the oral cavity, over a total period of 24 days. To analyze cytotoxicity on cells, 
the micronucleus test was performed, and an additional histopathologic analysis was conducted in order 
to evaluate tissue changes such as inflammatory infiltration, desquamation, and degeneration of cells. 

Results: Results showed that the cytotoxicity of the A cymbifera -based solutions was similar to those of 

 chlorhexidine, and all groups showed morphological characteristics of normal mucosa after treatments. 

Conclusion: It is concluded that no 
cytotoxicity was present after treatment with 
extracts of Aristolochia cymbifera, showing its 
potential for the development of future 
clinical trials. 

Keywords: Alternative medicine, Cytotoxicity, 
Plant extract, Aristolochia cymbifera, Oral 
medicine. 

Citation:  Bezamat, M et al. (2023) A Potential Natural 
Alternative to Chlorhexidine Mouthwashes: An in Vivo 
Study in Rats. Dentistry 3000. 1:a001 
doi:10.5195/d3000.2023.503 
Received:  May 1, 2023 
Accepted: May 13, 2023  
Published: October 20, 2023 
Copyright: ©2023 Bezamat M, et al. This is an open 
access article licensed under a Creative Commons 
Attribution Work 4.0 United States License. 
Email:  mbl29@pitt.edu

Introduction 

The daily use of toothbrushes, 

toothpaste and dental floss is 

essential to control plaque, 

avoiding the occurrence of 

dental caries and periodontal 

disease [1]. Nevertheless, in 

periods of higher cariogenic 

risk, when quantitative and 

qualitative changes of oral 

microbiota occur [2], the use of 

mouthwashes is highly 

recommended. The anti-plaque 

action of these mouthwashes 

consists of the reduction of 

bacterial adhesion to the teeth 

surfaces, inhibition of growth 

and proliferation of 

microorganisms and plaque’s 

intercellular matrix formation 

[3]. Furthermore, mouthwashes 

have also shown to modify 

bacterial biochemistry, leading 

to a less pathogenic biofilm 

overall [4].  

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For everyday plaque control in 

chronic situations of cariogenic 

challenge, such as in patients 

subjected to orthodontic 

appliances, regular mouthwash 

with fluoride and triclosan are 

commonly indicated [5]. 

However, the incidence of 

white spot lesions around 

brackets is still extremely high, 

highlighting the need for new 

therapies to prevent this 

outcome [6]. For short periods 

of increased cariogenic 

challenge, such as during 

treatment of periodontal 

disease and pericoronitis, the 

gold standard is the use of 

chlorhexidine rinses for 14 

days. Chlorhexidine 

mouthwashes, although 

extremely effective, have 

significant side effects such as 

alteration of taste perception 

and teeth staining that limit 

their long term use [7]. Because 

of the side effects of these 

mouthwashes commonly used 

in dentistry, there is a need for 

safe, effective and affordable 

alternatives [8]. 

In the pharmaceutical industry, 

rinses derived from essential 

oils and plant extracts have 

been explored due to their 

ability to inhibit growth of fungi 

and bacteria and the 

effectiveness in reducing 

plaque and changing the 

composition of biofilm [9]. For 

everyday use and for long 

periods, these natural rinses 

might be preferable in 

comparison to chlorhexidine in 

controlling plaque and mild to 

moderate gingivitis because 

they may present less side-

effects. Among the essential 

oils, an in vitro study performed 

by our group with the alcoholic 

extract of Aristolochia 

cymbifera (A. cymbifera) 

showed high effectiveness, 

reducing the microbial 

population by about 94.2% 

after one-hour in vitro tests 

with the reproduction of 

biofilms of human saliva [10]. 

Aiming to translate this 

research into future clinical 

application of the A. cymbifera 

extract, the objective of this 

study was to assess the 

cytotoxicity of this substance in 

the oral mucosa of rats, as the 

experimental animal model. 

Materials and Methods 

Experimental design 

All procedures have been 

conducted according to the 

guidelines described by the 

ethics committee of the Federal 

University of Rio de Janeiro 

(CEUA-UFRJ).  

The sample consisted of 30 

Wistar rats, approximately 8 

weeks old and weighing 

approximately 250g. The 

animals were kept under 

controlled temperature (22º ± 

2ºC), humidity (50 ± 10%) and 

cycle of 12 hours light/ dark. 

Food and water were available 

ad libitum. The rats were 

divided into 6 groups: Group 1 

(negative control) was treated 

with 0.3 ml of vehicle (DMSO 

and water); Group 2 treated 

with 0.3ml of A. cymbifera -

ethanolic extract 4mg/ml; 

Group 3 treated with 0.3 ml of 

A. cymbifera -hexane extract 

4mg/ml; Group 4 treated with 

0.3 ml of A. cymbifera -

ethanolic extract 10mg/ml; 

Group 5 treated with 0.3 ml of 

A. cymbifera -hexane extract 

10mg/ml; and Group 6 treated 

with 0.3 ml of chlorhexidine 

gluconate 0.12% (positive 

control). All groups were 

treated twice daily with the 

solutions applied into the oral 

cavity by syringes. The 

experiment was carried out for 

24 days. Collection of cells 

occurred three times, with 8 

days interval between them: T1 

– after 8 days, T2 – after 16 

days, and T3 after 24 days. 

Cells were collected from the 

right and left buccal mucosa 

and hard palate by scraping 

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these areas using sterile 

spatulas. The collection was 

done in two different ways: a) a 

smear directly on the slide, 

wetted with PBS; b) cells 

collected from each animal 

were placed in an Eppendorf 

with 0.5 ml of PBS. For fixation 

of the cells to the Eppendorf, 

0.5 ml of methanol: acetic acid 

(3:1 v / v) was added. 

Thereafter, the resulting 

solution of each collection was 

distributed into two slides (0.5 

mL in each slide). The slides 

were dried for 24 hours at 

room temperature before being 

stained. At the end of the 

experimental period all animals 

were sacrificed, and tissue 

samples were collected from 

the right and left buccal 

mucosa, palate, and tongue for 

histopathology.  

Cytology and Imaging 

 We performed an 

analysis of micronucleus 

formation since its appearance 

has been shown to be a reliable 

marker for cytogenetic damage 

[11, 12].  

The Feulgen-Fast Green 

method was used for staining. 

Initially the slides were dipped 

in 1N HCl solution for 15 

minutes for acid hydrolysis of 

nitrogenous bases purine and 

pyrimidine, allowing 

subsequent staining of the 

nuclear material. The slides 

were then washed with distilled 

water, where they remained for 

15 minutes. Subsequently they 

were immersed in Schiff 

reagent (Merck, Darmstadt, 

Hessen, Germany) for 90 

minutes, which is a specific dye 

for the nuclear chromosomal 

material exposed by acid 

hydrolysis. After removal of the 

Schiff reagent, the slides were 

dipped in 0.5% Fast Green for 

cytoplasmic staining (Merck, 

Darmstadt, Hessen, Germany) 

for 60 seconds and washed 

with distilled water again.  

The slides were then subjected 

to four batteries of absolute 

alcohol in a row for 2 minutes 

each, and after dehydration in 

alcohol, the slides were 

subjected to three batteries of 

xylol for 5 minutes each. Final 

preparation of the slides with 

Entellan was then performed 

(Merck, Darmstadt, Hessen, 

Germany), covering them with 

coverslips to avoid bubble 

formation. The slides dried for 

24 hours at room temperature.  

Finally, the cells were evaluated 

with the Nikon Eclipse E600 

microscope (Nikon Instruments, 

Melville, New York, USA) at a 

400x magnification, to 

determine the frequency of 

micronucleated cells indicating 

mutagenicity. Figure 1 

illustrates possible nuclear 

changes that indicate 

genotoxicity and cytotoxicity. 

Data were recorded in tables 

and analyses of variance with 

post hoc Tukey (intergroup) and 

the Student t test (intragroup) 

were performed. 

Histopathology  

The tissue samples collected 

from right and left buccal 

mucosa, palate, and tongue 

were processed, embedded 

with paraffin, cut into 5μm 

sections and adhered to glass 

slides. Finally, the slides were 

stained by hematoxylin and 

eosin and analyzed by light 

microscopy. Histopathology 

analysis evaluated the presence 

of tissue changes such as 

inflammatory infiltration, 

desquamation, degeneration of 

cells and other injuries. 

Photomicrographs were taken 

using a microscope coupled to 

a Leica DM500 digital camera of 

5 mega pixels ICC50. 

  

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Figure 1. Schematic of potential nuclear changes. 

Results

In the cytologic analysis we were able to observe the presence of different nuclear changes, such as cells 

without nucleus and with or without granules, illustrated in Figure 2. 

 

 

 

 

 

 

 

Figure 2. Nuclear changes (100x): A) Control cell, B) pyknosis, C) cell without nucleus and without granules, D) 

karyolysis, E) cell without nucleus and with granules. 

 

 

 

 

Exposure

Genotoxicity

Chromosomal breakage
Classic Micronucleous

Spindle’s 
Interference

Apoptosis
Pyknosis, chromatin 

condensation, cariorrexis
cytotoxicity

Cytotoxicity
Necrosis 

Keratinization

Pyknosis, chromatin 
condensation, cariorrexis, 

karyolysis

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The A. cymbifera -hexane 

extract 10mg/ml (group 5) 

showed a significant increase in 

the count of cells without 

nucleus with time. However, no 

other treatment groups 

showed the same trend (Table 

1).  On the other hand, when 

the count of cells without 

nucleus and without granules at 

different intervals was 

analyzed, group 2 treated with 

4mg/ml of the A. cymbifera -

ethanolic extract showed a 

significant increase count from 

day 8 to day 16 and a significant 

decrease from day 16 to day 24 

(Table 2). The cytotoxicity 

capacity of the A. cymbifera -

based solutions was similar to 

those of chlorhexidine and no 

significant differences 

intergroups were observed 

(Tables 1 and 2). 

 

Table 1. Count of cells without nucleus at intervals of 8, 16, and 24 days. 

GROUP 

8 days (T8) 16 days (T16) 24 days (T24) 

Average 

(SD) 

intergroup 

comparison* 

Average 

(SD) 

intergroup 

comparison* 

Average 

(SD) 

intergroup 

comparison* 

Group 1 
59.50 

(62.93)a 
A 

31.50 

(30.40)a 
A 

54.50 

(19.09)a 
A 

Group 2 
60.50 

(33.23)a 
A 

59.50 

(21.92)a 
A 

40.50 

(9.19)a 
A 

Group 3 
69.50 

(24.74)a 
A 

42.50 

(0.70)a 
A 

51.00 

(19.79)a 
A 

Group 4 
38.00 

(18.38)a 
A 

32.00 

(5.65)a 
A 

41.00 

(16.97)a 
A 

Group 5 
36.00 

(11.31)a 
A 

45.50 

(10.60)b 
A 

62.00 

(14.14)b 
A 

Group 6 
66.50 

(27.57)a 
A 

80.50 

(57.27)a 
A 

44.50 

(7.77)a 
A 

a, b, c, - Different lowercase letters mean that there is statistically significant difference (p <0.05) between 

different time in the same group (same row). *Different uppercase letters mean that there is a statistically 

significant difference (p <0.05) between groups at each time (same column). SD – Standard deviation. 

 

 

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Table 2. Count of cells without nucleus and without granules at intervals of 8, 16, and 24 days 

GROUP 

8 days (T8) 16 days (T16) 24 days (T24) 

Average 

(SD) 

intergroup 

comparison* 

Average 

(SD) 

intergroup 

comparison* 

Average 

(SD) 

intergroup 

comparison* 

Group 1 
16.50 

(3.53)a 
A 

15.50 

(0.70)a 
A 

23.50 

(6.36)a 
A 

Group 2 
9.50 

(6.36)a 
A 

29.00 

(7.07)b 
A 

21.00 

(16.97)ab 
A 

Group 3 
12.50 

(3.53)a 
A 

18.50 

(16.26)a 
A 

24.50 

(20.50)a 
A 

Group 4 
11.00 

(1.41)a 
A 

13.00 

(1.41)a 
A 

10.00 

(7.07)a 
A 

Group 5 
7.00 

(1.41)a 
A 

13.50 

(4.94)a 
A 

23.00 

(2.82)a 
A 

Group 6 
12.50 

(12.02)a 
A 

24.00 

(7.07)a 
A 

10.50 

(9.19)a 
A 

a, b, c, - Different lowercase letters mean that there is statistically significant difference (p <0.05) 

between different time in the same group (same row). *Different uppercase letters mean that there 

is a statistically significant difference (p <0.05) between groups at each time (same column). SD – 

Standard deviation. 

 

 

  

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Upon examination of the tissues previously submitted to treatments, all groups showed morphological 

characteristics of normal mucosa in the histopathologic analysis. Figure 3 shows the most representative 

photomicrographs of each group.  

 

 

 

 

 

 

 

 

 

 

 

Discussion 

In the northeast of Brazil, 

leaves of A. cymbifera are used 

to treat liver and stomach 

disorders, snake bites, fever 

and ulcer [13]. The root of A. 

cymbifera is believed to have 

therapeutic properties [13]. 

This study aimed to evaluate 

the presence of morphological, 

cellular, and tissue changes in 

the oral mucosa of 30 Wistar 

rats after topical application of 

different A. cymbifera extract 

solutions.  

Our results showed that 

cytologically, most of the 

extract concentrations tested 

did not show significant nuclear 

changes that would indicate 

cytotoxicity. Histologically, all 

tissue samples were also 

considered normal. Both the 

efficacy and the absence of 

cytotoxicity of this plant extract 

suggests the possibility of a 

natural alternative for 

chlorhexidine mouthwashes. A 

disadvantage of chlorhexidine 

is the alteration of taste 

perception that it causes after a 

few days of use. Chlorhexidine 

is amphiphilic, which means it’s 

both lipophilic and hydrophilic, 

a characteristic suggested to be 

the main cause of the taste 

alteration as side effect [7]. On 

the other hand, A. cymbifera 

has a strong bitter taste that we 

have tried to overcome with 

different dilutions. Even though 

natural and herbal substances 

A B C D E 

F G H I J 

Figure 3. Selection of tissue structures in different treatment groups: A) Tongue (group 4 – 4x), B) Tongue 
(group 3 – 40x); C) Tongue (group 1 – 4x), D) Palate (group 4 – 40x), E) Palate (group 5 – 40x), F) Palate 
(group 2 – 40x); G) Palate (group 3 -10x), H) Buccal mucosa (group 4 – 10x), I) Buccal mucosa (group 3 – 
10x), J) Buccal mucosa (group 5 -10x). 

 

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popularly used for nutritional 

purposes, medications and 

even mouthwashes are taken 

considerably well by users, 

additional experiments are 

warranted in order to provide 

the extract with a more 

pleasant taste before 

commercialization. Additional 

tests to determine whether A. 

cymbifera would cause any 

teeth staining would also be 

desirable as the next step. 

Using the kill-kinetics assay, the 

A. cymbifera alcoholic extract 

has previously demonstrated 

favorable results in 

microorganism’s growth 

inhibition after 1 hour of in 

vitro exposure [10]. In 

conclusion, although the 

extract of A. cymbifera showed 

no cytotoxicity, no abnormal 

tissue characteristics, there is a 

need for clinical trials to 

confirm the usability and 

efficacy of this substance in 

humans before it is applied 

clinically or commercialized.  

Disclosure of interest 

The authors declare that they 

have no competing interests. 

Authors' contributions 

These authors contributed 

equally to this work. 

 

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