The effect of derum (bark of Juglans regia tree) extract on oral mucosa: An in vivo study based on epithelial atypia in rabbit model

Abstract

Background and objectives

Derum, the bark of walnut tree (Juglans regia) has been used as a traditional tooth cleanser and chewed for its ability to bestow purple color to oral mucosa, tongue and lips Studies have shown that derum extract could affect oral epithelium after long term exposure, causing dysplasia. The aim of this in-vivo study was to evaluate the degree of epithelial dysplasia caused by varying durations and frequencies of topical derum application on oral mucosa of rabbits.

Materials and Methods

Following ethical approval, derum extract was applied to the buccal vestibule of New Zealand white rabbits over three different periods (60 days, 120 days, 180 days) and two different protocols were used (daily application and once every 3 days). Accordingly, the animals were divided into four groups (A – daily derum application/B – derum applied once in 3 days/C – Positive control and acetone applied every alternate day/D – negative control), and three batches (I – 60 days/II – 120 days/III – 180 days). The animals were sacrificed, and oral biopsies prepared and examined under light microscope. The magnitude of epithelial changes was evaluated using epithelial atypia index (EAI) based on Smith and Pindborg histological grading system (1969) for epithelial dysplasia.

Results

Mild dysplastic changes were detected in animals treated with derum extract regardless of the period of application. Similar results were noted among positive control group, and highest score was recorded in group A followed by group B with high tendency towards long-term derum application. Moderate changes were encountered only in group A that received derum for 180 days. Statistically, long-term derum application (180 days / Batch III in groups A and B), irrespective of the frequency of application, resulted in significantly higher mean EAI scores than all other groups or batches.

Conclusion

Based on this study, prolonged and frequent use of derum can induce dysplastic changes in rabbit oral mucosa, ranging from mild to moderate dysplasia. Further studies with extended times of exposure of derum to oral mucosa are recommended to document these adverse effects as an evidence base.

1. Introduction

Chewing sticks derived from natural plants have been used for many years as an oral hygiene aid. The beneficial effects of these sticks in reducing plaque accumulation by mechanical action and antibacterial activities are reported widely over the past many years (Almas and T. R. al-Lafi, , 1995, Elvin-Lewis, 1974, Olsson, 1978). Miswak (Salvadora persica) and Derum (Juglans regia) are two common plant derivatives used as a chewing stick or substitute, as a part of traditional medicinal practices (Eid and Selim, 1990, Darmani et al., 2006). While the miswak tree branches are used for chewing, the barks of derum tree are used for similar purposes. Derum is the local name for the barks of the walnut tree (Juglans regia), popularly used as a herbal chewing stick in the middle-eastern and north-African regions (Faden 2016). Originally grown in the European and Balkan regions since 2500–3000 years ago, walnut trees have spread far and wide, in recent centuries, extending up to the Himalayan regions, China, Africa, South America, East Asia and North America (Osman et al., 1987, Darmani et al., 2006, Faden, 2016). Apart from walnuts, which are a popularly consumed nut variety, the tough and fibrous barks of the tree are chewed as an efficient mechanical oral cleanser and tooth whitening agent (Khattak et al., 2022).

Chemically, the derum bark contains “Juglone”, a plant-based alkaloid, as its main and most important constituent, and is known to possess anti-viral, anti-parasitic, anti-fungal, anti-bacterial, anti-inflammatory, and anti-cancerous properties (Darmani et al., 2006, Salimi et al., 2012, Faden, 2016, Ibrahim et al., 2023). In addition, it also contains certain volatile phenols, phenolic acids like gallic acid, tannins and saponins which are responsible for the soapy texture, unique color and bitter taste (Al-Snafi 2018). Furthermore, the inherent ash content combined with a semi-rigid fibrous texture, enable the walnut tree bark or derum to be chewed on for a considerable amount of time, and then be used as a natural abrasive on teeth and oral surfaces (Al-Snafi, 2018, Khattak et al., 2022). Over the last century, the usage of derum as a chewing material has been reported more frequently from parts of North Africa, Saudi Arabia, Kuwait, and the Indian subcontinent (Chakraborty et al., 2016).

Interestingly, in the aforementioned geographical regions, derum is more frequently chewed by women for an additional cosmetic purpose, because of its ability to impart a purplish to brown-orange pigmentation on oral mucosal surfaces after use (Gazi, 1986, Osman et al., 1987, Ashri and Gazi, 1990). Apparently, the tannins present in the walnut tree bark not only give the material a characteristic reddish-brown color, but also help precipitate mucosal proteins and provide a favorable pigmentation on the lips, tongue and cheek (Gazi, 1986, Ashri and Gazi, 1990, Bonamonte et al., 2001, Al-Snafi, 2018). In a survey conducted in Saudi Arabia, the prevalence of derum chewing among Saudi females was reported to be as high as 31.9 %, with most of them in the age group of 25–39 years and the central provincial region of Riyadh representing the highest usage (20.6 %) among all the provinces in the country (Mandorah et al., 2021). Fig. 1 shows a representative specimen of the walnut tree bark (derum) as it is available in the local markets of Saudi Arabia and consumed directly as a chewing material.

Fig. 1.

Derum, dried bark of the walnut tree, in its commercially available natural form.

Several studies have reported the beneficial oral cleansing properties of chewing derum and its ability to inhibit growth of harmful microorganisms in culture (Darmani et al., 2006, Faden, 2016, Al-Snafi, 2018, Khattak et al., 2022, Ibrahim et al., 2023). Nevertheless, instances of contact dermatitis, hypersensitivity and cytotoxicity have been reported with the use of derum barks or its extracts therein (Osman et al., 1987, Bonamonte et al., 2001, Neri et al., 2006, Salimi et al., 2012). Most of the aforementioned adverse effects are attributed the chemical composition of derum comprising juglone, a phenolic compound, and juglonic acid, the phenolic acid derivative of juglone (Salimi et al., 2012). In fact, extracts of the walnut tree bark containing juglone and juglonic acid have been used in agriculture to kill harmful weeds and undesired plants (Choudhary et al., 2022). It is pertinent to note that development of oral mucosal white lesions are associated with the analogues of tumor-promoting agents (Van Duuren et al., 1978). Incidentally, studies have shown that both juglone and juglonic acid present in the walnut tree barks or derum, are capable of being classified as tumor-promoting substances (Silverman et al., 1984, Schepman et al., 1998, Shiu and Chen, 2004).

Researchers evaluating gingival lesions among patients who habitually chewed on derum, diagnosed them as leukoplakia. Histological evaluation of the lesions showed varying degrees of epithelial dysplasia, which manifested as stratified squamous epithelial layers with spongiosis, parakeratosis, and slight basal cell hyperplasia (Gazi, 1986, Osman et al., 1987, Ashri and Gazi, 1990). It is highly likely that these histopathological changes in the oral mucosa of derum chewers are due to the effects of juglone and juglonic acid (Eid and Selim, 1990). Therefore it would be alluring to surmise that the extracts of walnut tree barks or derum, which are released as a result of chewing and sucking, may contain chemical substances that act synergistically to promote mucosal irritation, inflammation, epithelial dysplasia and tumorigenesis. Moreover, it is imperative that there be no delay in diagnosis of precancerous dysplastic oral mucosal lesions, as this precludes prevention of oral squamous cell carcinoma and contributes towards successful treatment and favorable outcomes (Schepman et al., 1998). One way of early monitoring and mitigation for disease progression associated with oral potentially precancerous lesions is through assessment of epithelial dysplastic changes on biopsied tissue specimens (Ranganathan and Kavitha 2019). The degree of epithelial dysplasia could further be quantified using a scoring/grading system proposed by Smith and Pindborg in 1969, called the Epithelial Atypia Index (EAI), which not only helps classify epithelial atypia, but also enables statistical comparison (Geetha et al., 2015).

In recent years, there has been increasing interest towards in-depth understanding about the chemical compounds present in derum bark, their possible applications in dentistry and potential for causing harm (Osman et al., 1987, Salimi et al., 2012, Al-Snafi, 2018, Khattak et al., 2022). However, the lack of evidence regarding the effects of chewing derum on oral tissues and the need for examining if the bioactive components in derum are responsible for epithelial dysplastic changes, mandates in vivo researches on animal models. Therefore the aim of the present in vivo study was to quantify EAI and evaluate the degree of epithelial dysplasia caused by varying durations and frequencies of topical derum application on the oral mucosa of rabbits.

2. Materials and methods

2.1. Ethical approval and study sample

Institutional approval for ethical conduct of the study was obtained from King Saud University, College of Dentistry Research Center, Riyadh, Kingdom of Saudi Arabia (Approval # F1167). All experimental procedures on animals were conducted in a compliant manner based on NIH guidelines laid down for use and care of laboratory animal models (NIH Publication #85–23 Rev.1985) (Binsalah et al., 2019).

The study sample included 84 white New Zealand rabbits, aged about 8–10 months. The sample size was arrived at based on a statistical sample size calculation assuming 80 % power and 95 % level of significance. Accordingly, the sample size was estimated to be five animals per group, for a total number of 12 groups (4 intervention and 3 time periods). The final sample size of seven animals per group was ascertained after overestimation for animal mortality during the experiment. All through the experimental period, the study animals were housed in individual cages and cared for under veterinary supervision in the Center for Laboratory Animals and Experimental Surgery at College of Medicine, King Saud University, Riyadh, Saudi Arabia. Prior to the experiment, the animals were accustomed to the laboratory environment by keeping them under standard room temperature and humidity conditions, with 12 hourly dark-light cycles. All the animals were cleared off any oral or systemic illnesses by the veterinarian and had ad libitum access to food and water throughout the period of experiment.

2.2. Derum extract preparation and experimental application

Derum material for use in the study was obtained from a local herbal store in Al-Ahsa region of Saudi Arabia (Fig. 1). Nearly three kilograms of raw derum (bark) was sourced to extract active ingredients. The procedure for synthesis of derum extract was carried out using an ethanol extraction procedure, and was done as per standard laboratory protocols for extraction of natural plant based derivatives, at the Botanical Department in College of Sciences, King Saud University (Faden 2016). In short, large derum barks were washed in distilled water and shredded into smaller pieces, measuring approximately 3–5 mm in size. The shredded derum particles were washed again and dried to remove impurities. Following which derum particles were immersed in 90 % ethanol and allowed to soak for 24 h. This mixture was then homogenized in a blender, filtered through a sterile micro-filter paper and centrifuged to obtain the supernatant derum extract. Ethanol was removed from the extract by evaporation under a stream of air at room temperature and pressure. The resultant extract was then dissolved in 100 % acetone at a 3:2 ratio (3 cc of extract in 2 cc of acetone), to make it soluble and dispensable.

The final prepared derum extract was applied in the oral mucosa of the rabbits, accordingly the study animals were grouped to either receive daily derum application (Group A), derum application once in three days (Group B), acetone application every alternate day (Group C – Positive control) or receive no topical applications (Group D – Negative control). The distribution of study animals and sample sizes in the groups are described in Table 1. During each application, both derum and acetone were applied at the same site, an area of oral mucosa, measuring approximately 5 mm by 5 mm, in the lower right buccal vestibule, posterior to the incisors. The application was done using an etchant brush wherein the brush saturated with either derum extract or acetone was passively stroked 10 time in the same direction over the mucosal area of interest, and a new brush was used at each application (Fig. 2). For each of the study groups, the respective topical mucosal applications were carried out for a period of either 60 days (Batch I), 120 days (Batch II) or 180 days (batch III) (Table 1).

Table 1.

Study animal distribution and sample size per group.

Study Batches (based on duration of application)	Study Groups (based on topical application)
	Group A (daily application of derum extract)	Group B (derum extract applied once in 3 days)	Group C (Positive control − acetone applied every alternate day)	Group D (Negative control – no topical application)
Batch I (60 days)	7	7	7	7
Batch II (120 days)	7	7	7	7
Batch III (180 days)	7	7	7	7
Study animals per group	21	21	21	21

Fig. 2.

Representative image showing application of derum extract to the buccal vestibule, in rabbit animal model.

2.3. Study animal sacrifice and histological assessment for epithelial dysplasia

Upon completion of the period of topical application according to the study group, the study animals were sacrificed either after 60 days (Batch I), 120 days (Batch II) or 180 days (Batch III). Euthanasia of the animals was done by administering a lethal dose of ketamine hydrochloride (75–90 mg/Kg body weight; Ketamine CIII®, Dechra Veterinary Products, Overland Park, Kansas, USA) and xylazine hydrochloride (12–15 mg/Kg body weight; Xzin, Nicosia Biolabs Int. Pvt. Ltd., Ludhiana, India), through intramuscular injection. Following study animal sacrifice, an incision biopsy was obtained from the oral mucosa including the site of application of either derum extract or acetone (Groups A, B and C). A similar mucosal biopsy specimen was also obtained from the oral cavity of animals in the negative control group (Group D). The collected soft tissue specimens were fixed in 10 % formalin for seven days, embedded in paraffin blocks and sectioned into 4 µm histological sections. The histological sections mounted on a glass slide were stained using hematoxylin and eosin (H & E) stain and photomicrographs of the stained slides were obtained for histopathological assessment and scoring.

The histological interpretation was conducted by two independent examiners using Smith and Pindborg grading system (1969) for scoring of epithelial dysplasia and determination of epithelial atypia index (EAI) scores (Geetha et al., 2015, Ranganathan and Kavitha, 2019). Both investigators underwent training for identification and evaluation of epithelial dysplasia by a qualified histopathologist, and based on a pilot assessment, they had an inter-examiner agreement (Cohen’s kappa) score of 0.87. The histological assessment and grading were done based on 13 characteristic features of epithelial dysplasia, outlined by Smith and Pindborg (1969). For each characteristic feature, either a score of “0″ was given when not present (none), or a weighted score was assigned when present in “slight” or “marked” fashion. The scoring criteria and features of epithelial dysplasia are enumerated in Table 2. The severity of the dysplastic changes (EAI score) was a sum of scores for all the 13 features of epithelial dysplasia and could range between 0–75. Based on EAI score, epithelial dysplasia in each sample was further categorized according either as none (<11), mild (11–25), moderate (26–45) and severe (>45).

Table 2.

Smith and Pindborg grading system (1969) for scoring of epithelial dysplasia and determination of epithelial atypia index scores (Geetha et al., 2015, Ranganathan and Kavitha, 2019).

Characteristic features of dysplastic change in the epithelial cells	Severity scores for each characteristic feature of epithelial dysplasia
	None	Slight	Marked
Drop-shaped rete pegs	0	2	4
Irregularly stratified epithelium	0	2	5
Cellular keratinization below keratinized layer	0	1	3
Hyperplasia of basal cells	0	1	4
Intercellular adhesion loss	0	1	5
Loss of cellular polarity	0	2	6
Nuclear hyperchromatism	0	2	5
Increased nuclear-cytoplasmic ratio in the cells of basal and prickle layers	0	2	6
Anisonucleosis and Anisocytosis	0	2	6
Cellular and nuclear pleomorphism	0	2	6
Increased mitotic activity in cells	0	1	5
Differing levels of mitoses among cells	0	3	10
Presence of abnormal mitoses among cells	0	3	10

Descriptive statistical analysis was done for the measured quantitative variable (EAI Score) to estimate mean values and frequencies for the different grades of epithelial dysplasia. The differences in dysplastic changes between the groups (Groups A, B, C and D) and within each group between different periods of applications (Batches I, II and III) were examined using the multivariate analysis of variance (ANOVA) at 0.05 level of significance (p < 0.05), and utilizing statistical software package (IBM SPSS Statistics Version 22, IBM Corp., Armonk, NY, USA).

3. Results

Among the experimental animals, 12 rabbits deceased during the experimental study, due to unexplained reasons, and were therefore excluded from final statistical analysis. These included three animals in group B, four animals in group C and five animals in group D. The final experimental sample size per group and batch, and the frequency of epithelial dysplasia, and its severity based on EAI scores, are given in Table 3. While severe epithelia dysplasia was not observed in any of the study animals, mild dysplastic changes were observed in 30.55 % (n = 22) of the animals, moderate dysplastic changes in 2.81 % (n = 2) of the animals and no dysplastic change in the remaining 66.66 % (n = 47) of the animals. Substantial numbers of animals in group A and B, and few animals in group C demonstrated mild epithelial dysplastic changes. Especially in groups A and B, this finding was pronounced among animals which received topical derum application for a longer period of time (180 days). Interestingly this was irrespective of the frequency of application of derum extract (daily or once in three days). Both the animals which showed moderate epithelial dysplasia, belonged to group A and incidentally received daily topical application of derum extract for 180 days. Fig. 3 shows representative images of the different grades of epithelial dysplasia (none, mild and moderate) based on EAI scores.

Table 3.

Final distribution of the study animals in each group and batch, along with level of epithelial atypia index (EAI) scores and frequency for each level. (Based on Smith and Pindborg grading system for epithelia dysplasia).

Study Groups (based on topical application)	Study Batches (based on duration of application)	Final no. of Animals	Incidence of epithelial dysplasia according to EAI score
			None (EAI<11)n (%)	Mild (EAI=11–25)n (%)	Moderate (EAI=26–45)n (%)	Severe (EAI>45)n (%)
A	Batch I (60 days)	7	5 (71.42 %)	2 (28.57 %)	0	0
	Batch II (120 days)	7	4 (57.14 %)	3 (42.85 %)	0	0
	Batch III (180 days)	7	0	5 (71.42 %)	2 (28.57 %)	0
B	Batch I (60 days)	6	4 (66.66 %)	2 (33.33 %)	0	0
	Batch II (120 days)	6	5 (83.33 %)	1 (16.66 %)	0	0
	Batch III (180 days)	6	0	6 (100 %)	0	0
C	Batch I (60 days)	6	5 (83.33 %)	1 (16.66 %)	0	0
	Batch II (120 days)	5	4 (80 %)	1 (20 %)	0	0
	Batch III (180 days)	6	5 (83.33 %)	1 (16.66 %)	0	0
D	Batch I (60 days)	5	5 (100 %)	0	0	0
	Batch II (120 days)	5	5 (100 %)	0	0	0
	Batch III (180 days)	6	6 (100 %)	0	0	0
Total		72	48 (66.66 %)	22 (30.55 %)	2 (2.81 %)	0
EAI – Epithelial atypia index

Fig. 3.

Representative histological images (Hematoxylin and Eosin; x30 magnification) of specimens obtained from the oral mucosa of animals in the different study groups and graded according to Smith and Pindborg epithelial atypia index, showing no dysplastic changes in (a) negative control group, (b) positive control group; (c) and (d) showing examples of mild dysplastic changes observed in the experimental group animals and three animals in the positive control group; some of these findings include drop-shaped rete pegs, irregularly stratified epithelial layers, increased mitoses, nuclear-cytoplasmic ratio and nuclear hyperchromatism, slight hyperplasia of basal cell layers and loss of cellular polarity; (e) and (f) showing examples of moderate dysplastic changes observed in two animals of the experimental group with daily derum application for 180 days; in addition to the aforementioned findings seen in mild dysplasia, these histological images further reveal the presence of keratinization of cells in the prickle layer, loss of intercellular adhesion, anisonucleosis, anisocytosis, different levels of mitotic activity in multiple cells and occasional abnormal mitoses.

Using Smith and Pindborg grading system (1969) for scoring epithelial atypia, higher EAI scores were generally consistent with animals that received topical application of derum extra on a daily basis (Group A). This was followed by animals in group B, which received topical derum application once in three days. The mean EAI scores were lower for animals in the control groups (Group C – positive control and Group D – negative control). While in all groups, the greatest degree of epithelial atypia evidenced by EAI score was seen in batch III (180 days application), only in group C, batch II (120 days application) had a slightly higher mean EAI score than that of batch III. Table 4 shows the mean values (with standard deviation) of EAI scores in all the study groups and in different batches, along with the statistical differences and p-values based on multivariate ANOVA. Comparing the mean EAI scores between batches in each group, significant differences were observed only in derum topical applications groups, A and B. Within both of these two groups, the degree of epithelial atypia was mostly similar after 60 days and 120 days of topical application of the derum extract. However, further prolonged application up to 180 days resulted in a highly significant degree of epithelial dysplasia. Moreover, long term exposure of the oral mucosa to derum (180 days), irrespective of the frequency of application (daily or once in three days), resulted in significantly higher EAI scores than any other group or batch. There was no significant difference in mean EAI scores between the different batches in groups C (positive control – acetone) and D (negative control). Similarly, after topical application for 60 days and 120 days, the difference in mean EAI scores between the groups was not statistically significant. Graphical representation of the difference in mean EAI scores are further shown in Fig. 4.

Table 4.

Means and standard deviations of epithelial atypia index (EAI) scores, in each group and batch, and their statistical differences based on multivariate analysis of variance (ANOVA).

Study Groups (based on topical application)	Study Batches(based on duration of application)			p-value(difference between batches in each group)
	Batch I (60 days)	Batch II (120 days)	Batch III (180 days)
A	8.01 ± 6.66a	8.71 ± 3.21b	20.71 ± 8.94ab *	< 0.01
B	6.17 ± 4.17c	6.51 ± 5.01d	19.83 ± 4.02 cd #	< 0.001
C	4.17 ± 3.76	8.41 ± 3.29	7.17 ± 3.13*#	0.1371
D	4.41 ± 2.32	5.61 ± 2.61	6.33 ± 1.63*#	0.3741
p-value (difference between groups for each batch)	0.4553	0.4452	< 0.001	−
a, b, c, d – indicates significant difference between mean values in a row *, # − indicates significant difference between mean values in a column

Fig. 4.

Bar graphs with error bars, respectively representing mean and standard deviation of EAI scores in each group and batch.

4. Discussion

Long term exposure to exogenous carcinogens, present in smoked and chewed tobacco, betel nut, betel quid, alcohol…etc., is believed to be a predisposing factor for initiation of oral leukoplakia, a dysplastic condition involving the oral epithelium (Schepman et al., 1998, Erugula et al., 2020). Leukoplakia clinically presents as a non-scrapable white lesion afflicting the oral mucosal surfaces and is considered as a precancerous or premalignant lesion, which could undergo malignant transformation to de novo oral squamous cell carcinoma (Silverman et al., 1984, Gazi, 1986, Ashri and Gazi, 1990, Eid and Selim, 1990). Traditional plant based chewing derivatives such as miswak sticks (Salvadora persica) and derum barks (Juglans regia) have been used as oral cleansers and are believed to possess medicinal benefits (Darmani et al., 2006). Although naturally derived from plants and trees, the chemical composition of these substances are not fully understood (Al-Snafi 2018). In several instances, these substances are additionally used for indications other than oral cleansing, like the case of miswak being used for habitual chewing and derum being chewed for its ability to cause pigmentation of the oral mucosa (Gazi, 1986, Ashri and Gazi, 1990, Eid and Selim, 1990). Such parafunctional uses of plant based products can cause potential adverse effects, which are more commonly reported in the case of derum use and these include contact dermatitis, irritation, inflammation and epithelial changes, to name a few (Osman et al., 1987, Bonamonte et al., 2001, Neri et al., 2006, Salimi et al., 2012). On the other hand, in vitro studies have also showcased the antimicrobial and anticancer properties of derum extract, when cultured with bacterial and cancer cell lines, respectively (Zhang et al., 2012, Faden, 2016). In light of these contradicting evidences regarding the beneficial and deleterious biological effects of derum and its extracts, it was imperative to evaluate the outcomes of their long-term usage through in vivo animal models, which formed the premise for the present study.

In the present in vivo study, animals were exposed to topical application of derum extract on the oral mucosa, at different frequencies (daily and once in three days) and for varying durations (60, 120 or 180 days). The study parameters mimicked oral chewing of derum for both dental cleansing and pigmentation purposes, as reported in the literature (Mandorah et al., 2021). The use of “epithelial atypia index” derived from Smith and Pindborg grading system (1969) for quantifying the effect of derum exposure on oral mucosa, was based on the robustness of the tool (EAI) for assessing and reporting epithelial dysplasia (Geetha et al., 2015, Ranganathan and Kavitha, 2019). Accordingly, dysplastic epithelial changes in the oral mucosa of animal models were capable of being classified as none, mild, moderate or severe (Table 3) and in addition were also amenable to statistical comparison (Table 4 and Fig. 4). Apart from the experimental groups (A and B), the positive control (Group C) animals were exposed to topically applied acetone, as it was used as a solvent for derum extract. Studies from the literature have reported the efficiency of alcohols, like ethanol and methanol, and acetone as effective solvents for extraction of plant based phytochemical compounds (Tanih and Ndip, 2012, Faden, 2016, Tourabi et al., 2023). These findings conceptually reflect the extraction procedure followed in the present study from the Juglans regia tree barks, wherein ethanol was used for initial extraction and acetone was used for subsequent carriage and application. Furthermore, the method of alcoholic extraction used in the present study is superior to aqueous extraction for releasing the predominant phenols and phenolic acids in the derum barks (Tanih and Ndip, 2012, Faden, 2016, Tourabi et al., 2023). This would have probably ensured an optimum availability of the chemical substance from derum, namely juglone and juglonic acid, which are predominantly implicated to cause adverse effects (Al-Snafi 2018). Nevertheless, a complete analysis of the chemical composition of the prepared derum extract could have added value, and should be a supplemental objective for future researches.

Derum like other chewing sticks, is regarded as a timeless natural toothbrush and people over the ages have found it useful for oral cleansing and tooth brushing. Additionally, as studies show, women have preferred chewing it, as the bark when chewed for long enough bestows favorable pigmentation to the oral mucosa (Gazi, 1986, Ashri and Gazi, 1990). In spite of its several therapeutic benefits, the hazardous effects of chewing derum are not only sparsely reported in the literature, but is also an uncommon knowledge among people who use it routinely (Osman et al., 1987, Neri et al., 2006). These changes may be related to the active chemicals in derum, juglone and juglonic acid, which are known to have tumor promoting activity (Neri et al., 2006), and this could be in addition to the possible synergistic effects of other phytochemical compounds present in derum. Although the mechanisms behind juglone toxicity are not fully understood, cell death and cell cycle disruption, along with molecular changes such as DNA modification and decreasing levels of tumor suppresser gene P53 have been reported as potential effects of derum extract on living tissues and cells in culture (Salimi et al., 2012). Interestingly, Juglans regia has also been suggested to have promising roles as a natural anticancer therapeutic agent and as a promoter for soft tissue healing (Zhang et al., 2012, Catanzaro et al., 2018). It was suggested that the anticancer effects were achieved by blocking molecular pathways that are involved in cancer development and increasing the rate of apoptosis (Zhang et al., 2012, Catanzaro et al., 2018). Similarly, Darmani et al., (2006) reported based on an in vitro study, that derum extract had a significant effect in increasing the proliferation of fibroblasts in a dose dependent manner (Darmani et al., 2006). Nevertheless, the anticancer activity of derum extract does not necessarily translate into juglone activity because of what is called as a matrix effect, which needs to be taken into consideration (Sestili et al., 2018). According to the matrix effect, plants are basically formed of complex structures that store many phytochemical compounds, which are capable of generating a certain effect due to synergistic activities and the final effects of the mixture need not necessarily mirror the sum of the effects of the single compounds (Sestili et al., 2018).

In this study, the impact of derum application to the oral mucosa of rabbits resulted in significant epithelial dysplastic changes, especially when it was applied at shorter frequencies (daily) and when the duration of exposure was longer (180 days). It must be noted here that irrespective of daily or once in three days application, long-term derum exposure (180 days) was always detrimental for the mucosa, leading to moderate epithelial dysplasia. The above findings are in agreement with that of Osman et al., (1987), who reported that continuous use of derum tends to produce neoplastic changes in the oral mucosa (Osman et al., 1987). It is indeed well known that a single application of a carcinogenic substance does not by itself give rise to a tumor, even though it causes latent genetic damage. This genetic mutation could however be promoted further into a malignancy through repeated exposure to carcinogens, particularly when it exceeds a certain threshold (Schepman et al., 1998). Although, chronic alcohol exposure has been implicated as a cause for epithelial dysplasia (Shiu and Chen 2004), no alcoholic substance was applied on the oral mucosa of rabbits in this study. Nevertheless, acetone application in the positive control group did lead to a certain degree of epithelial dysplasia, which could be classified as mild in nature. Unlike with derum, these changes were noticed early on, after 120 days of application, but did not vary significantly after 180 days of application. Moreover, at all instances of examination, the mean EAI scores in the positive control/acetone application group were lower than that of either of the derum application groups (Groups A and B) (Fig. 4).

The clinical implications of the present study outcomes would be a valuable case in point for educating patients who are habitual users of derum as a chewing aid. Although, the epithelial dysplastic changes in the animal model were observed only after long-term application (180 days), the same may not be applicable for patients, wherein the use may be equivalent to prolonged exposure even after using for a short time frame, like days or weeks. This is a major limitation of in vivo studies in general, attributed to the differences in human and experimental animal biology (Ramalingam et al., 2016), and the present study is no exception as well. Therefore the clinical extrapolation of the present study results need to be carefully considered based on the risks of developing epithelial dysplasia, when derum is chewed for longer durations and the extracts of which are in contact with the oral mucosa for a prolonged amount of time. Nevertheless, the present study results shall definitely be regarded as a baseline for dental public health recommendations relating to the use of derum as a chewing stick and also for the basis for future studies.

5. Conclusion

Based on the outcomes of the present in vivo study, it may be concluded that prolonged and frequent use of derum extract can induce dysplastic changes of rabbit oral mucosa. Such changes could range from mild to moderate dysplasia and are consistent with epithelial atypia described by Smith and Pindborg. Severe changes might be expected if the period of application extended up to or beyond 180 days. Further studies with extended times and varying frequencies of derum exposure/application to oral mucosa are recommended to identify and formulate an evidence base for the reported adverse effects.

Funding

This research was ethically approved and financially supported by the College of Dentistry Research Center, King Saud University (Approval # F1167).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.