EFFECT OF CINNAMALDEHYDE ON GROTH AND INTERNAL ORGANIZATION OF FRESHWATER BIOINDICATOR (P. tetraurelia)

http://doi.org/10.65281/642885

BOUCHIHA Hanene^1*1_;ACIDI Aanisa²_;RIZI Aicha ²_;MECHERI Hind¹.

1-University of Echahid Cheikh Larbi Tebessi -Tebessa, Algeria.

2-University of Badji Moktar -Annaba, Algeria.

Submission date: 01.03.2025. Accepted 02.09.2025 Publicaion.10.10.2025

ABSTRACT

Essential oils have received attention in recent years as potential ‘natural’ alternatives due to their positive impact. They are used in a wide variety of consumer such as pharmaceuticals, perfumes, cosmetics, confectionery food products, soft drinks……etc. Although accessible to all, Essential oils can represent some dangers and despite their image of natural products they are not devoid of toxicity. In fact, researchers report health problems every year due to the use of Essential oils. While many studies refer to the toxicity of many products on the market, the toxicity of essential oils is less investigated. The objective of this study was to examine in vitro the effects of essential oil (cinnamaldehyde) and to describe their specific effects on, the population growth, percentage of answer, proportioning of protein and proportioning of carbohydrate on a Biological model wich is Paramecium tétraurelia. Three doses were chosen, at time of 08 days .Among the effects of cinnamaldehyde on the (Paramecium tétraurelia), we find that different doses have direct and rapid effects on the living organism development and highlighted a inhibition of the growth of the protozoa as well as a disturbance of the contents of total proteins and a reduction in the proportioning of carbohydrate.

Keywords: Essential oils, Cinnamaldehyde, Paramecium tétraurelia, growth, total proteins, proportioning of carbohydrate.

INTRODUCTION

Essential oils (EOs) are important aromatic components of herbs and spices that can be extracted from plants by distillation methods, in particular steam distillation (Greathead, 2003). And have received much attention (Benchaar and al., 2007; Fraser and al., 2007). Their biological activities have been known and utilized since ancient times in perfumery, food preservation, flavouring and medicine. And have received much attention (Benchaar and al., 2007). Some of their biological activities include antibacterial, antifungal, anti-oxidant and anti-inflammatory effects amongst others. Belongs to the Lauraceae family these plants are of great economic importance. Indeed, cinnamon, a spice obtained from the inner bark is widely used in food. Also, these plants are an important source of essential oils (EOs), as they are synthesized in barks, leaves, and seeds (Revindran and al., 2003) to ensure the protection of the plants against undesirable insects and microorganisms. EOs is complex mixtures of secondary metabolites. Their composition depends on numerous factors, including the species, the geographical localization, and the part of the plant that is used (Burt, 2004). The most exploited Cinnamomum EOs are those from Cinnamomum verum, Cinnamomum cassia, and Cinnamomum camphora (Revindran and al., 2003). EOs extracted from the leaves or bark from trees of the genus Cinnamomum possesses a powerful antibacterial effect against a wide spectrum of pathogenic bacteria (Yang and al., 2012). In the United States, bark and leaf EOs of Cinnamomum cassia have Generally recognized as safe status as a food additive (Barceloux, 2009). Contrary to their name, EO is not true oils (lipids) and are most commonly associated with the fragrance, the Quinta essentia of plants. Chemically, EO are secondary metabolites composed primarily of isoprenes or terpenes (C10H16) and may contain mixtures of diterpenes (C20), triterpenes (C30), tetraterpenes (C40), hemiterpenes (C5), and sesquiterpenes (C15). When isoprenes are associated with additional elements, usually oxygen, they are termed terpenoids (8). The research for study models for different disciplines of the applied biology becomes an imperative scientific requirement. Paramecium tétraurelia is a very large (120 μm) eukaryotic cell covered with vibrating cilia. It belongs to the Ciliate phylum (Ciliophora). The use of Paramecium species as a model of survey has been reported by several authors in some disciplines; in ecology, toxicology. The growth constitutes the basic criteria that can make from an organism a model of survey. Growth of microorganism can be quantified by an increase of the size, the weight or the number. Nevertheless, there are several agents limiting this important criterion. The limiting factor is the one that conditions the speed or the amplitude of a phenomenon that depends on several other parameters (Liebig and al., 1844). Paramecium sp were used to study environmental qualities and toxic effects of industrial, agricultural and domestic chemicals (Edmiston and al., 1985); in genetic, because it’s sequencing genome is well known, researchers used Paramecium tétraurelia for genetic analysis, gene expression and mutation (Mayer and al., 1998; Haynes and al., 2000). Essential oils have been shown to have antimicrobial properties against different types of microorganisms including bacteria, protozoa, and fungi (Greathead, 2003). A number of in vitro studies have been recently published on the effects of EO and their components (EOC) on ruminal fermentation and metabolism (McIntosh and al., 2003). Results from those studies revealed variable effects of EO and their derivatives on rumen bacteria and ruminal fermentation (Busquet and al., 2006; Castillejos and al., 2006). Discrepancies between studies were attributed to different types and doses of EO, but also to the in vitro technique (batch versus continuous culture) used (Fraser and al., 2007). The objective of this study was to examine in vitro the effects of essential oil (cinnamaldehyde) and to describe their specific effects on, the population growth, percentage of answer, proportioning of protein and proportioning of carbohydrate.

MATERIALS AND METHODS

Paramecium culture

The habitual culture of Paramecium tétraurelia was done at 27°C, in test tubes using 10 ml of the culture medium: we prepared culture medium by mixing several dried vegetables (hay 7.5 g, wheat plant 7.5 g, lettuce 10 g, cucumber rind 5 g, potato rind 5 g, dash of yeast and source of sterol (2 g of peanut or almond). Mixture was boiled throughout one hour in 1.5 liters of distilled water. The broth was filtered, sterilized by boiling at 100°C during 30 min in thermo-resistant bottle and conserved to the shelter of light described by (Azzouz and al., 2012).

Chemical material

Cinnamaldehyde is the aldehyde that gives cinnamon its flavor and odor. It is occurs naturally in the bark of cinnamon trees and other species of the genus Cinnamomum like camphor and cassia. These trees are the natural source of cinnamon, and the essential oil of cinnamon bark is about 90% cinnamaldehyde. Cinnamaldehyde is also used as a fungicide and used primarily in the flavor and fragrance industries for imparting a cinnamon flavor and/or fragrance to various types of foods, beverages, medical products, and perfumes. This chemical is used in the liquor industry for flavoring liqueurs and cordials. Cinnamaldehyde has also been used as a rubber reinforcing agent, a filtering agent, an attractant for termites, a corrosion inhibitor for sulfuric acid baths to clean galvanized iron and zinc (Kirk-Othmer, 1979).

Treatment of the cultures with cinnamaldehyde

Chosen concentrations of cinnamaldehyde were selected based on preliminary test (Bouchiha and al., 2015). The lower concentration of each cinnamaldehyde that resulted in any noticeable change in growth was selected for further studies in the present experiment. The maintain concentrations were 2, 5 and 7 µl.ml-1. The treatment with cinnamaldehyde was done at the beginning (at t = 0). The experiment was repeated at three times for each repetition experiment with no additive (Control) studied. The growth monitoring lasts up to 08 days. After exposition to the cinnamaldehyde, paramecium population responds in a dose-response relationship (Bouchiha and al., 2015).

Parameters Measured

-Kinetics of cell multiplication of Paramecium tétraurelia:

The kinetic growth of the Paramecium tétraurelia is carried out according to the method of (Sbartai and al., 2009) by the measurement of the optical density as using a spectrophotometer (Jenway 6300).

– Number measurement

The growth kinetics study was realized by the cell counting, after fixation with lugol, under optic microscope using Malassez blade. The count was repeated at least three times for each repetition (Liebig and al., 1844).

-Percentage of answer

After exposition to cinnamaldehyde, Paramecium tétraurelia population responds in a dose-response relationship. The assessment of this response in percentage is calculated by the following formula (Sbartai and al., 2009) : Percentage of answer : (%) = [(Nc – Ne) / N C]*100. Where Nc is the number of cells in the control treatment and Ne is the number of cell in the treatment that received the cinnamaldehyde. The positive values of the percentage of answers indicate an inhibition of the growth while the negative values indicate a stimulation of growth. (Sbartai and al., 2009)

– Proportioning of proteins

The proteins are quantified according to the method of (Bouchiha and al., 2015; Bradford, 1979)

– Proportioning of Carbohydrates

Carbohydrates is performed according to the method of (Bouaricha and al., 2012; Duchateau and al., 1959)

– Statistical analyses

The results are presented as mean ± standard error, the results are compared by MINITAB software version 16.0, the level of significance chosen was p <0.05 (Bouchiha and al., 2015; Dagnelie, 1999).

RESULATS AND DISCUSSION

Impact of cinnamaldehyde on the pH of culturs

Figure 1: Effect of cinnamaldehyde on THE pH of culture of P. tétraurelia

The values of pH in control and after treatment with cinamaldehyde presented by (Figure 1) the values indicate that pH did not change statistically (p> 0.05) during test, remaining between 6 and 7. (Beaumont and Cassier, 1998) Consider these values very accurate and ideal for growth since they are between 6 and 7. We note the impact of pH on the biological activity of cinnamaldehyde and their ability of affecting the growth of some microorganism species specially the matrix in which the EO are present affects their hydrophobicity which will influence their interaction with the microbial cell membrane, and therefore, affect the action of cinnamaldehyde.

Impact of the cinnamaldehyde on the P. tétraurelia growth

Figure 2: Effect of cinnamaldehyde on Kinetics growth of P. tétraurelia.

The curves of growths offer quantitative information allowing a reliable analysis of the impact of the cinnamaldehyde on the Paramecium tétraurelia. Figure (2) represent the variations of the growth of the cells treated by the cinnamaldehyde. We note that the presence of the cinnamaldehyde slow down the cell multiplication of the cells treated compared to the control cells. This inhibiting effect (p <0.05) is not significant for short times (hours).

Figure 3: Effect of cinnamaldehyde on Kinetics growth of P. tétraurelia.

The effects of cinnamaldehyde on the growth kinetics for 8 days are representing in Figure 3. We note that the presence of the cinnamaldehyde slow down the cell multiplication of the cells treated compared to the control cells. This inhibiting effect is significant for long times (days) (p <0.05). Curves recorded in that Figure show that the concentration of the curves of growths offer quantitative information allowing a reliable analysis of the impact of the cinnamaldehyde on the Paramecium tétraurelia has a negative effect on the paramecia growth. The concentration that produces the maximal growth of Paramecium tétraurelia was with the control. Whereas a less concentration in the curves of growths we noted an impact no considered of the cinnamaldehyde on the Paramecium tétraurelia. However, in high concentration the growth was almost null. Comparisons between densities were significantly different It is therefore not likely that the activity of EOs can be attributed to one specific mechanism, but rather to variety of parameters in the targets cell. This makes it difficult to predict the effects susceptible of EOs in species studied. For example Monoterpene phenols, such as thymol and carvacrol, interact with the cell membrane by hydrogen bonding, rendering the membranes and mitochondria more permeable and disintegrating the outer cell membrane. They can inhibit the growth of Escherichia coli O157:H7, Staphylococcus aureus, Salmonella enteric, serovar Typhimurium, Pseudomonas fluorescens, and Brochothrix thermosphacta (Di Pasqua and al., 2007).

Impact of cinnamaldehyde on the P. tétraurelia Percentage of answer (%)

Figure 4: Effect of cinnamaldehyde on Percentage of answer (%) of P. tétraurelia.

Figure 5: Effect of cinnamaldehyde on percentage of answer (%) of P. tétraurelia.

The results obtained concerning the response percentage confirm those of kinetics growth. We note that the inhibitory effect was dose dependent and proportional to the increasing concentrations (Figure 5).Thus, the response percentage was positive and shows a strong inhibition of microorganisms growth. In fact, it varies from (16%) to (78%) for 2µL/ml and 7µL/ml respectively.

Impact of cinnamaldehyde on the proportioning of protein of P. tétraurelia

According to Figure 6, we note dependent increase of proportioning of protein (µg/ml) rate in the presence of different doses of cinnamaldehyde. About The statistical analysis we find that in the Treaties, the rate of total protein tends to increase in a dose-dependent and highly compared to witnesses for the concentration. In fact, it varies from 0,62 µg/ml to 0,09 µg/ml for 2µL/ml and 7µL/ml respectively.

Figure 6: Effect of cinnamaldehyde on proportioning of protein (µg/ml) of P. tétraurelia.

Impact of cinnamaldehyde on the Proportioning of carbohydrate of P. tétraurelia

Figure 7: Effect of cinnamaldehyde on Proportioning of carbohydrate (µg/ml) of P. tétraurelia.

However, according to Figure 7 the rate of carbohydrate tends to decrease in a dose-dependent and not significant for the treated concentration compared to controls. In fact, it varies from 2,96 µg/ml to 2,89 µg/ml for 2µL/ml and 7µL/ml respectively.

DISCUSSION GENERAL

Extensive application is usually companied with serious problems and health hazard. It is established that many chemicals, in common use, can produce some toxic effects on biological systems when tested on various type of experimental models through their mode of action or by production of free radicals that damage all cell compounds (Azzouz and al., 2012). In fact, these chemicals act as pro-oxidants (Zeriri and al., 2012). Cinnamaldehyde is biochimical product It is occurs naturally in the bark of cinnamon trees and other species of the genus Cinnamomum like camphor and cassia. And used as a fungicide and used primarily in the flavor and fragrance industries for imparting a cinnamon flavor and/or fragrance to various types of foods, beverages, medical products, and perfumes. This chemical is used in the liquor industry for flavoring liqueurs and cordials. Paramecium is one of the most commonly used ciliated for laboratory research to investigate the direct toxicity of compounds (Benbouzid and al., 2012). And more that they represent a basic component of aquatic environment, where they play critical roles both quantitatively and qualitatively (Azouz and al., 2012). In this context, the ciliate assay has become a valuable tool for detection of environmental disturbance and for assessment of the trophic state (Zeriri and al., 2012). For all the reason and plus we have chose Paramecium tétraurelia as a biological model for elucidating cinnamaldehyde toxicity. In this study, we were interested in the effect of cinnamaldehyde on population growth, percentage of answer, Proportioning of protein and Proportioning of carbohydrate. Our result showed an inhibition in the growth of microorganisms especially for the highest concentrations. Similar results were reported in studies (Benbouzid and al., 2012, Bouaricha and al., 2013) that investigate the effect of different chemicals on the physiology and morphology of Paramecium sp. (Azzouz and al., 2012) Reports that toxics may affect the survival of protozoa in a variety of ways, as the concentration of toxicants in the cell membrane increase and destroy their integrity causing cell death. Toxic affects freshwater ciliates; these effects are perceptible at the population level by reducing the number of cells and on the cellular level by a structural behavioral and physiological damage. On the other hand, the positive value of response percentage demonstrated the inhibitory effect of cinnamaldehyde. Indeed, cinnamaldehyde as a lipophilic compound can penetrate into cell, disturbing phospholipid orientation and causing changes in fluidity of membrane (Benbouzid and al., 2012). So oxidative stress is believed to occur when there is an imbalance in the biological oxidant-antioxidant ratio; this can result in oxidative damage to lipid, proteins, carbohydrates and nucleic acids. In most cases, the abnormal generation of ROS, which can result in significant damage to cell structure, is considered an important signal of oxidative damage (El-Demerdash and al., 2007). (Benbouzid and al., 2012) Indicate that cells are equipped with both the enzymatic and non enzymatic antioxidants for combating oxidative stress, which may be either due to increased production of free radical or impaired antioxidant defense or both. Proteins are one of the major energy reserves present in all organisms, these reserves will be affected by toxicant exposure (Azzouz and al., 2012). In this work, we noted an increase of total protein rate in a dose dependent manner and very highly significant. This finding is in agreement with those of (Bouaricha and al., 2012) who showed an increase in the rate of total proteins of paramecia treated with increasing concentrations of Bifenazole and Proclaim, for (Rouabhi and al., 2007) the antioxidant system activity may undergo an increase or depletion under the effect of a chemical stress. For (Winston and al., 1991) this increase could be related to the induction of the detoxification process elaborated by this control system which is composed of enzymes, proteins and antioxidant molecules. The antioxidant defense systems are present in all aerobic cells and neutralize the intermediate chemical reactions produced endogenously and/or metabolism of xenobiotics.

CONCLUSIONS

In summary, under the current experimental conditions, cinnamaldehyde is toxic to the freshwater ciliate Paramecium tetraurelia. Exposure to fort concentrations of cinnamaldehyde showed decreasing considerable on growth supported by the positives values of percentages of answer and the intensification of the antioxidant enzymes supported by the increasing in total protein. It showed be mentioned that other xenabiotics have an oxidative stress proved by lipid peroxidation supported by values measured of carbohydrate. Biomarqueurs and genotoxic study may provide more answers concerning the effects of cinnamaldehyde on Paramecium tetraurelia.

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