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Use of Strawberry Tree (Arbutus unedo) as a Source of Functional Fractions with Biological Activities

Arbutus unedo, commonly named ‘strawberry tree’ (ST), is a Mediterranean native plant that represents a relevant source of biologically active fractions and compounds. ST fruits, traditionally used with culinary and medicinal purposes, along with other components (leaves, roots, honeys, etc.), have been subjected to varied extraction procedures to obtain enriched and bioactive products. This work reviewed the scientific literature, searching for studies that evaluated the potential health implications of ST fractions and attending to the tested biological activities (antioxidant, antiproliferative, hypoglycemic, immune-modulatory, antihypertensive, antimicrobial, etc.), the part of the tree, the experimental model, the specific bioactive compounds and the selected extraction protocol. Furthermore, the strengths and weaknesses of the current state of the published evidence were critically analysed. Although in vitro results demonstrated the potential of ST fractions, further research is encouraged in order to obtain in vivo evidence (animal and clinical studies), assess additional activities (hypocholesterolemic, microbiome-modulatory), maximize the use of advanced extraction technologies, purify and isolate specific bioactive compounds and broaden the analysis investigating phenolic and non-phenolic molecules and their bioavailability.

Introduction
Arbutus genus belongs to the Ericaceae family and comprises 12 accepted species with different geographical distributions and interest (Table 1). Among them, Arbutus unedo, commonly named 'strawberry tree' (ST), is the most popular and investigated species due to its gastronomical and medicinal uses. ST (also called arbutus, strawberry madrone, Irish/Killarney ST) is an evergreen and flowering small tree or shrub whose fruits have been traditionally utilized in fermented and distilled beverages, jellies, jams, marmalades, sauces and vinegars and are even consumed fresh. This Mediterranean native plant can be found in Southern, Western and Central Europe, Canary Islands, Ireland, North-Eastern Africa and Western Asia, growing in regions with unusual intense dryness or frost [1,2]. Table 2. Traditional uses, biological activities and potential health implications (related disorders whose risk may be reduced) of the main parts of Arbutus unedo.
Among the investigated compounds and bioactivities, phenolics have attracted the greatest attention, particularly associated with antioxidant properties (with health and technological implications) but also with antiproliferative, hypoglycemic, anti-inflammatory, antihypertensive and antimicrobial actions [17,22,26,30,39,71]. While >90% of the works highlighted polyphenols as target compounds, a significantly lower number of publications were focused on the presence of other biologically active molecules such as ascorbic acid, tocopherols, carotenoids, fatty acids, lectins and other proteins, carbohydrates, etc. These non-phenolic compounds were particularly effective as antioxidant and antiproliferative or cytoprotective agents, and the reported activities encourage further investigation to broaden the spectrum of possibilities and efficiently exploit the versatility of ST composition [28,38,64,65].
Nevertheless, despite the promising in vitro results, just a few in vivo experiments have been carried out so far to validate the real impact of ST fractions on health. Among them, the hypoglycemic effect of roots extracts was confirmed in different rodent models [25,72,73], as well as the safety of leaf extracts administration in rats [84] and the anti-inflammatory and immune-modulatory effects in CD mice [75], but most of the activities have not been assayed in animals, and no clinical trials have been registered to date testing ST parts, extracts or isolated molecules. This lack of animal and human studies must be considered as a key point for the future to be able to formulate novel healthy products based on ST components.
In this article, the scientific literature was reviewed with the aim of identifying the recent insights into the obtention of functional fractions from ST, investigating the relevance of the different ST parts as raw materials, the described biological activities, the utilized experimental models and extraction methods (conventional and advanced) and the identification of the active molecules as well as the potential effects on human health in spite of the absence of clinical interventions.

Antioxidant Activity
When the production and accumulation of oxygen reactive species cannot be alleviated by the action of biological mechanisms in cells and tissues, the resultant imbalance is called 'oxidative stress', and the related damage may lead to cancer, cardiovascular, neurological, respiratory and renal diseases, among others [85]. Since the use of dietary antioxidants has been demonstrated to be effective in preventing the disturbances or restoring the balance, vegetal sources have been exhaustively investigated during recent decades to obtain potent antioxidant fractions [86,87].
ST components, particularly fruits and leaves, have been subjected to numerous and varied extraction procedures to obtain phenolics-rich fractions with high antioxidant power (Table 3). In general, solid-liquid extractions were carried out using solvents such as water, ethanol or methanol, although other solvents such as ethyl acetate, acetone or dichloromethane and more advanced technologies such as pressurized liquid extraction (PLE) were utilized for harder matrices such as roots [26,63].
Most of the works utilized methodologies that determined the ability of the phenolicsrich extracts to scavenge free radicals (DPPH•, ABTS•+, etc.), as well as other in vitro assays such as the β-carotene or crocin-bleaching assays, the measurement of lipid peroxidation inhibition, etc. Furthermore, more specific experiments were carried out by Mendes et al. (2011) [49] using human erythrocytes that were incubated with aqueous extracts obtained from ST fruits and leaves. These extracts exerted high antioxidant capacities by inhibiting oxidative hemolysis and lipid peroxidation [49]. In addition, a similar leaf fraction obtained by ultrasound-assisted water extraction also inhibited lipid peroxidation in human peripheral blood lymphocytes [59].
Apart from the already mentioned sources (fruits, leaves, roots), honeys have also been studied because of their antioxidant potential. In this sense, honeys with high levels of phenolic compounds such as arbutin and homogentistic acid demonstrated the ability to scavenge free radicals and impede cholesterol and liposomes oxidative degradation [21,61,62].
Although the main purpose of this research field is clinical and targets human health, the antioxidant potential of ST has been concurrently studied from a technological perspective. Thus, anthocyanins-rich fractions extracted from fruits using the acidified ethanol or water were tested, supplementing yoghurts [14] and wafers [41], respectively. Both experiments concluded that the addition of the extract can replace synthetic antioxidants and colorants and might lead to the design of healthy food products [14,41]. Similar results were obtained in other matrices such as cheese, sausages, pork burgers or limpet pâtés, showing that ST ingredients are promising candidates to be used in functional and novel foods [15,40,42,43].
However, despite the auspicious outcomes that have been reported, all the works have employed in vitro models to date. The validation in animal models and clinical trials is mandatory to reach clinical significance and to demonstrate the real impact of the described extracts on human health.

Antiproliferative/Antitumoral Activity
Cancer is still the second-leading cause of death worldwide, and it is accompanied by high morbidity, constituting a major concern that has motivated a vast amount of research to find and validate novel alternatives based on natural and edible products that can avoid the disadvantages of anticancer drugs such as the side effects and low specificity [87,88].
Accordingly, STs have also been included in the plethora of plant species that have been studied for this purpose. In this case, research identifying ST fruits as a source of antitumoral compounds is scarce, but a methanolic extract prepared by Guimarães et al. (2014) [32] was shown to be effective in inhibiting the growth of different tumoral cell lines, highlighting its activity against NCI-H460 (non-small lung cancer) cells and correlating the stronger potential of the ST fruit extract (other species such as Prunus spinosa, Rosa micrantha and Rosa canina were also tested) with its particular phenolic profile with a high abundance of galloyl derivatives [32]. Furthermore, Alexandre at al. (2020) obtained a set of extracts, subjecting the fruit residues that were discarded after distillation processes (to prepare alcoholic ST beverages) to supercritical-CO 2 technology using ethanol as a co-solvent and a range of temperatures and pressures [64]. Those obtained at 55 • C and 100 or 300 bar were the most effective as antiproliferative agents against HT-29 (human colorectal adenocarcinoma) cells, and correlations between the activity and the content in specific phenolic compounds such as pyrogallol were observed. Moreover, the growth of HT-29 cells was also inhibited by a protein-rich product with a high content of lectins that was prepared from ST leaves [65]. As a matter of fact, leaves were utilized to generate other extracts using different solvents (water, ethanol, methanol, hexane) that were able to inhibit specific animal and human cell lines such as L20B (rat embryonal rhabdomyosarcoma), Vero (monkey kidney carcinoma), KB (human epithelial carcinoma), HeLa (human cervical epithelial carcinoma), A431 (human epidermioid carcinoma), A375 (human malignant melanoma) and RD (human embryonal rhabdomyosarcoma) [17,66,67]. Besides fruits and leaves, honeys demonstrated cytotoxic effects against human colon adenocarcinoma (HCT-116) and metastatic (LoVo) cells [19] (Table 4). Regarding the involved mechanisms of action that ST compounds exerted against cancerous cells, further investigation is needed to explain the exact actions, although the published works reported that these effects are strongly correlated with the antioxidant activity of ST phenolics [32,64]. Moreover, their ability to scavenge free radicals and other pro-oxidant molecules encourage the conduction of future studies to confirm their ability to prevent carcinogenesis in vivo. Thus, animal and human trials are still required to really understand the power of these substances to be used in chemoprevention or anticancer therapies. Anti-inflammatory/ immune-modulatory Human red blood cells 50-500 µg/mL Leaves aqueous extract [56] A549/8, THP-1 0.9-59.2 µg gallic acid-equivalents/mL Leaves methanolic extract [75] MDA-MB-231 0.02-29.6 µg gallic acid-equivalents/mL [76] Human fibroblasts 3.7-14.8 µg gallic acid-equivalents/mL

Hypoglycemic Activity
Diabetes is a chronic metabolic disease primarily characterized by hyperglycemia as a consequence of a lack of insulin or resistance. Although diabetic patients are normally treated with therapies that involved drugs (metformin, insulin), novel treatments based on functional dietary ingredients can be certainly advantageous, facilitating administration and avoiding side effects [89]. Fruits, leaves and mainly roots of ST have been used within this approach to obtain hypoglycemic fractions: a methanolic extract from ST fruit with a high content in phenolic compounds inhibited α-glucosidase activity in vitro [31], as well as different ethanolic and hydroalcoholic extracts from fruits and leaves that were additionally able to inhibit α-amylase and contained significant levels of flavonoids and iridoids [55]. Regarding root extracts, pressurized water extraction produced a catechin-rich fraction that was able to inhibit α-glucosidase activity [74], and, besides the in vitro insights, other procedures using hot water resulted in antidiabetic extracts that validated their properties in vivo by inhibiting glucose absorption and enhancing glucose tolerance in healthy Sprague-Dawley rats [25] and also in healthy and diabetic Wistar rats [72,73] (Table 5). Wistar rats (males) 0.5 g/kg 30 min before glucose administration, oral administration [72] Wistar rats (males and females) 0.15 g/kg [73] Antiinflammatory/ immune modulatory CD mice (males) 0.02 g (gallic-acid equivalents)/kg 1 h before carragenan and saline administration, oral administration Leaves methanolic extract [75]

Anti-Inflammatory/Immune-Modulatory Activity
Inflammatory responses against infection, injury, irritation, etc. are non-specific immune mechanisms that can become chronic and are linked to specific conditions such as cardiovascular disease, cancer, arthritis or allergy. Within this context, a plethora of vegetal matrices have been investigated, searching for compounds that exert immune-modulatory or anti-inflammatory properties [90].
Just a few studies have been carried out using ST components to study immunemodulatory function, and most of them utilized leaves as the main source. For instance, Moualek et al. (2016) [56] reported that an aqueous extract obtained from ST leaves showed in vitro anti-inflammatory capacity by inhibiting heat albumin denaturation and showing a protective effect of human erythrocytes membranes against induced hemolysis. The authors attributed this to the ability of the ST extract to modify the calcium influx in red blood cells [56]. The extract was rich in phenolic compounds, as well as those recovered from leaves using methanol [75,76]. In this case, the methanolic fraction inhibited the activation of STAT1 elicited by IFN-γ in MDA-MB-231 (human breast cancer) cells, human fibroblasts and human alveolar epithelial (A549/8) cells. STAT1 is a complex protein that has to be finely controlled to regulate the inflammatory process, i.e., its excessive or erroneous activation might generate exacerbated inflammatory responses [75,76]. Furthermore, this bioactive fraction inhibited STAT3 activation mediated by IL-6 in THP-1 cells (human leukemia monocytes). This activity reduction is considered as a strategy to ameliorate inflammation and was correlated in this work with the down-regulated expression of iNOS and ICAM-1 (both inflammatory genes) in A549/8 and THP-1 cells [75]. The promising in vitro results were validated in vivo in male CD mice that were subjected to carrageenan-induced pleural and lung inflammation. The pleural exudates that were collected from the group that was orally treated with the ST extract showed reduced levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), and the lung injury in animals was also decreased [75].
Apart from leaves extracts, ST honeys and the phenolic fractions obtained from them (using Amberlite XAD-2 resin columns for purification) exerted potent in vitro inhibitory activities against hyaluronidase. Surprisingly, honeys showed a higher inhibition percentage than their extracts, suggesting that other non-phenolic compounds are involved in the anti-inflammatory mechanism of action [21].

Antihypertensive/Vasodilatory Activity
Hypertension is a long-term condition associated with persistent blood pressure levels, and it is considered a major risk factor for cardiovascular disease, which is still the leading cause of death worldwide [91]. Most antihypertensive approaches involve the study of the renin-angiotensin system and the inhibition of its enzymes, particularly angiotensinconverting enzyme (ACE). Therefore, many works have looked for ACE-inhibitory dietary ingredients [92,93], but, surprisingly, this is not the case with Arbutus unedo. However, a few studies were conducted that evaluated the effect of ST extracts as vasodilatory agents or as inhibitors of platelet aggregation. In this context, Legssyer et al. (2004) [77] observed an endothelium-dependent vasorelaxant effect of ST leaves extracts obtained with water or methanol and applied to Wistar rat aorta (ex vivo) [77]. These authors also fractionated the methanolic extract using semipreparative high-performance liquid chromatography (HPLC) or caffeine addition for tannins precipitation, and it was observed that polyphenolic compounds such as oligomeric condensed tannins and catechin gallate were the most active molecules. A similar effect was observed for an aqueous roots extract [80], but the vasodilation was not achieved with ST honey [22].
In addition, leaves extracts induced the inhibition of platelet aggregation in vitro. This antiaggregant activity was noticed for an aqueous fraction, but particularly for those obtained using methanol or ethyl acetate. When the methanolic fraction was subjected to tannin precipitation, it was elucidated that tannins were mainly responsible for the inhibitory action [78]. Regarding the mechanism of action, a later study demonstrated that the inhibitory effect exerted by hot water, diethyl ether and ethyl acetate extracts (from ST leaves) was likely mediated by their high antioxidant activity (attenuating ROS production) and their inhibitory capacity against protein tyrosine phosphorylation and calcium influx in the platelets [79].

Antimicrobial/Antiviral Activity
Many plant species have been tested as a source of natural antimicrobial compounds to prevent or eliminate the presence of pathogens or non-desirable microorganisms in food products or the human body, i.e., from a technological or clinical perspective [94].
ST components (fruits, leaves, roots and honeys) have been exhaustively investigated for this purpose. Regarding fruits, an ethanolic fraction with a high content of phenolic compounds showed a strong antimicrobial activity against Staphylococcus aureus, Bacillus subtilis and Pseudomonas aeruginosa [30]. Later, an aqueous extract of ST fruit was added to limpet pâtés, and it showed the capacity of controlling the microbial stability of the products along 90 days of preservation, conducting a reduced microbial development compared with the non-supplemented pâtés and with no presence of pathogenic species [15]. However, ST leaves were the material that led to the highest number of studies. A great diversity of extracts (using ethanol, methanol, hot water) constitute an interesting battery of antimicrobials, inhibiting the growth of S. aureus, P. aeruginosa, Escherichia coli, Enterococcus faecalis, Bacillus cereus and Mycobacterium spp. [52][53][54]80,81]. Moreover, silver nanoparticles prepared with an aqueous ST leave extract were also able to inhibit S. aureus, P. aeruginosa, E. coli and Staphylococcus epidermis [82]. Antiviral activity was also reported in the case of an ethanolic leave extract that was effective against herpes simplex virus type-1 [17].
Roots were also subjected to extraction procedures to obtain phenolic-rich products with antimicrobial activities. Dib et al. (2013) reported a poor capacity against S. aureus and P. aeruginosa for methanolic and aqueous extracts but a moderate capacity against E. coli in the case of the water extract [24]. However, when advanced extraction technologies such as PLE were applied to the ST roots, the antibacterial potential was increased, as was shown for the methanolic PLE product that was highly active against S. aureus, E. coli and Salmonella spp. [26].
Finally, a study that evaluated ST honey reported promising antibacterial activities against S. aureus and S. epidermis and antifungal activity against Candida spp. [20]. However, these results were not in concordance with those obtained by Osés et al. (2020) [21], which concluded that ST honeys were not effective against bacteria or fungi. This discordance might be due to the differences in the honeys composition, and when the phenolic fractions were purified from them, the antimicrobial activity was observed. The authors attributed this to the presence of non-phenolic compounds inhibiting the antimicrobial activity in honeys or just to the lower concentration of the active compounds in the original matrix [21].
Despite the significant number of publications addressing antimicrobial properties, further research is needed to elucidate the details of the molecular mechanisms. Nevertheless, it is widely described that phenolic compounds are synthesized in response to microbial infections by plants species such as ST, being effective against a broad range of microorganisms [24]. Moreover, when ST phenolics are orally consumed, gut microbiota species (e.g., Bifidobacterium spp.) can utilize glycoside forms exerting β-glucosidase activities and releasing aglycones that might be more active. In this sense, the antibacterial activity of arbutin and its aglycone (hydroquinone) was investigated, and after the Bifidobacterium β-glucosidase action, the bioavailability and bioactivity of aglicones were increased [95].

Other Biological Activities
Apart from all the bioactivities that were previously described, other particular properties must be highlighted for ST fractions.
For instance, ST leaves were subjected to hydroalcoholic extraction and water infusion, and the resulting products were effective against lithiasis and in the prevention of calcium oxalate's crystals formation [50]. Apparently, the mechanism of action seemed to not be directly related to the antioxidant activity of the extracts and was shown to be dependent on pH variations that estimulated the formation of soluble complexes between functional groups of the active ingredients (phenolics and, mainly, flavonoid derivatives) and calcium oxalate. The in vitro insights were promising enough to encourage further investigation to elucidate the litholytic mechanism of action and the potential applications of these fractions in the prevention or treatment of urolithiasis, a condition caused by urinary biochemical disturbances and characterized by the formation of stones in the kidneys or the urinary tract [50].
Furthermore, when leaves were macerated with ethanol, methanol or hexane, the produced infusions-particularly, the hexane fraction-were strongly active against Leishmania major, Leishmania tropia and Leishmania infantum, these being intracellular parasites responsible for leishmaniasis, a tropical and subtropical disease transmitted to humans by the bite of Phlebotomus and Lutzomyia insects that causes high mortality and morbidity, especially in Africa [16]. The mentioned work did not report correlations between the biological action and the concentration of the studied compounds (total phenolics and flavonoids) and suggested a possible synergistic effect of the minoritary components. Further studies are required to identify the responsible molecules [16].
An additional interest of A. unedo is the presence of arbutin. This phenolic compound, the glycosylated form of hydroquinone, was detected in ST, and, besides antioxidant, antiinflammatory, antiproliferative and antimicrobial properties, it has attracted the attention of the cosmetic industry due to its potential as a tyrosinase inhibitor in the melanogenesis pathway, i.e., reducing melanin pigments formation and, therefore, being a promising candidate to be used as a skin-whitening agent correcting dark spots or blemishes. However, laboratory procedures of arbutin synthesis have already been developed and are more frequently used than arbutin extraction and purification from natural sources such as ST [96].

Conclusions, Current Limitations and Future Perspectives
This work reviewed the scientific literature directly related to A. unedo as a source of bioactive fractions. Considering the vast knowledge about ST and the great number of works investigating its properties, it can be concluded that the obtained results are promising enough to encourage further studies that may elucidate the responsible molecules and build stronger evidence for the development of novel functional products.
Although the previously described activities were demonstrated in vitro, validation using animal models has not been carried out yet for most of the activities. In fact, only the hypoglycemic action of root extracts was confirmed in rats and mice [25,72,73], but antioxidant, antiproliferative, anti-inflammatory, antimicrobial, antiviral, antileishmanial and anti-urolithiasis activities were evaluated in vitro or ex vivo in all cases. No clinical trials have been published to date. In addition to the above, it seems that there is an imbalance in the study of the mentioned capacities, which is mostly focused on antioxidant activity to the detriment of the others (Table 3).
Moreover, some relevant properties have not been tested yet. For instance, no studies addressing the hypocholesterolemic potential of the fractions were found. The presence of phenolics, bioactive lipids and polysaccharides, among others, and the clinical impact of hypercholesterolemia in cardiovascular health suggest that further investigations must be carried out to target ST compounds with the capability of reducing cholesterol absorption and/or biosynthesis. Similarly, the ability to modulate human gut microbiota is still a pending issue that must be studied for ST fractions, since this factor may cause relevant effects on human health status [97,98].
In order to obtain ST bioactive products, several extraction procedures were described in the literature; however, advanced technologies have been used in just a few works. These methodologies are frequently recommended because of their advantages over traditional protocols: a higher specificity, lower solvent volumes, shorter extraction times, etc. These benefits allow for one to work in concordance with the principles of Green Chemistry and lead to environmentally friendly methods linked to sustainability. Therefore, although some of these techniques were applied to ST (supercritical fluids, pressurized liquids, ultrasound and microwave extractions), their utilization, as well as that of other novel ones (pulsed-electric fields, ionic liquids, deep eutectic solvents, solvent-free-microwave extraction, etc.), must be strongly advised [99].
Other weakness of the current status of the art is the lack of works carrying out the purification and isolation of single compounds (except for some phenolic compounds such as arbutin) that might determine compound-activity correlations. A large majority of the studies are only targeting phenolic compounds, without taking into consideration the great diversity of molecules that might exert relevant biological activities (proteins and peptides, carbohydrates, glycoproteins, lipids, secondary metabolites, etc.).
Moreover, since the obtained products may be dietary ingredients, bioaccesibility and bioavailability assays must be conducted, also defining the effect of the food matrix, digestion, interaction with microbiota, etc.
In summary, some points must be further investigated: animal and clinical studies to evaluate biological activities; the assessment of other properties such as hypocholesterolemic or microbiome-modulatory properties; the use of advanced extraction technologies; the purification and isolation of single compounds; the deeper investigation of non-phenolic components; and bioaccesibility and bioavailability studies. The resulting insights may be crucial in validating the auspicious results that were reviewed and described in this work.

Conflicts of Interest:
The authors declare no conflict of interest.


s2fieldsofstudy:
Agricultural And Food Sciences
Biology

provenance: AgriculturalAndFoodSciences,2022-2022-Agricultural And Food Sciences-2022.jsonl.gz.gz

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Determination of Chemical Compounds and Investigation of Biological Properties of Matricaria chamomilla Essential Oils, Honey, and Their Mixture

This exploratory investigation aimed to determine the chemical composition and evaluate some biological properties, such as antioxidant, anti-inflammatory, antidiabetic, and antimicrobial activities, of Matricaria chamomilla L. essential oils (EOs). EOs of M. chamomilla were obtained by hydrodistillation and phytochemical screening was performed by gas chromatography–mass spectrophotometry (GC-MS). The antimicrobial activities were tested against different pathogenic strains of microorganisms by using disc diffusion assay, the minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) methods. The antidiabetic activity was performed in vitro using the enzyme inhibition test. The antioxidant activity of EOs was tested using the free radical scavenging ability (DPPH method), ferrous ion chelating (FIC) ability, and β-carotene bleaching assay. The anti-inflammatory effects were tested in vivo using the carrageenan-induced paw edema method and in vitro using the inhibition of the lipoxygenase test. The analysis of the phytochemical composition by GC-MS revealed that camphor (16.42%) was the major compound of EOs, followed by 3-carene (9.95%), β-myrcene (8.01%), and chamazulene (6.54%). MCEO, honey, and their mixture exhibited antioxidant activity against the DPPH assay (IC50 ranging from 533.89 ± 15.05 µg/mL to 1945.38 ± 12.71 µg/mL). The mixture exhibited the best radical scavenging activity, with an IC50 of 533.89 ± 15.05 µg/mL. As antidiabetic effect, EO presented the best values against α-glucosidase (265.57 ± 0.03 μg/mL) and α-amylase (121.44 ± 0.05 μg/mL). The EOs and honey mixture at a dose of 100 mg/kg exhibited a high anti-inflammatory effect, with 63.75% edema inhibition after 3 h. The impact of EOs on the studied species showed an excellent antimicrobial (Staphylococcus aureus ATCC 29213 (22.97 ± 0.16 mm)), antifungal (Aspergillus niger (18.13 ± 0.18 mm)) and anti-yeast (Candida albicans (21.07 ± 0.24 mm) effect against all the tested strains. The results obtained indicate that the EOs of M. chamomilla could be a potential drug target against diabetes, inflammation and microbial infections; however, further investigations to assess their bioactive molecules individually and in combination are greatly required.

Introduction
The therapeutic benefits of plants have been proven for centuries. Many disease states have been successfully remedied using plant-derived medicines. These remedies are employed in the form of concoctions or concentrated extracts of plants, without separating the active components. Nevertheless, there are many health problems worldwide with disorders such as diabetes, hypertension, chronic inflammation, microbial infections, degenerative diseases, and cancer for which contemporary medicine is still trying to find appropriate treatments [1]. Indeed, the use of traditional medicine has been eclipsed by modern medicine as a way to cure and manage human illnesses [2]. However, in recent decades, the use of herbal medicines in promoting health and treating conditions has increased in several countries, including developing societies [3]. Natural products can be considered as sources of therapeutic ingredients for discovering innovative drugs [4][5][6]. They could, therefore, constitute a tool for identifying molecules with several pharmacological and biological characteristics, which will be available and effective for the management of different human and animal pathologies [7][8][9][10]. Regardless, the problem of drug discovery is multifaceted and needs the assessment of multiple endpoints of natural and synthetic agents, such as pharmacokinetics, safety, and efficacy, when screening drug compounds [1,11].
Currently, it is well known that natural molecules, in particular those derived from medicinal plants, have already demonstrated interesting outcomes, due to their antimicrobial actions against different pathogenic strains, their anti-diabetic effects in vitro and in vivo, and their anti-inflammatory properties through different molecular and cellular mechanisms [12][13][14][15].
Matricaria chamomilla L. (chamomile) is a popular herbal medicine from the Asteraceae family that is widely distributed throughout the world [16]. The plant can be found in North Africa, North and South America, Australia, New Zealand, and Asia. It is also cultivated in France, Germany, Brazil, Yugoslavia, Hungary, and Russia [16]. It is nowadays a very appreciated and highly used medicinal plant in popular and traditional medicine for the treatment of a certain number of pathologies, especially diabetes and chronic inflammation, but also can be used for treating colic, flatulence, hysteria and intermittent fever [17].
M. chamomilla contains a broad spectrum of secondary metabolites and active compound classes. Flavonoids, sesquiterpenes, polyacetylenes, and coumarins are identified as the major components of chamomile. Among the bioactive phenolic compounds present in chamomile extracts, we found chlorogenic acid and caffeic acid, luteolin and luteolin-7-Oglucoside, quercetin and rutin, apigenin and apigenin-7-O-glucoside, and naringenin [16]. As chamomile is an excellent natural source of medicinal products, this study was designed to highlight the phytochemical composition and valorize the EOs (terpene compounds) of this plant against different biological systems, namely antioxidant, antidiabetic, antiinflammatory, and antimicrobial effects.

Essential Oil Origin
The leaves of M. chamomilla were collected from the Taza region (north-west of Morocco) in April 2021. The plant material was identified and authenticated (voucher spec-imen: RAB 104230) from the Department of Botany and Plant Ecology of the Scientific Institute of Rabat, University of Mohammed V Rabat, Morocco. After collection, the leaves were then dried and M. chamomilla essential oil (MCEO) was extracted by hydrodistillation.

Honey Samples
Samples of honey were collected from natural hives in the Taza region (north-west of Morocco) during the month of April. Indeed, during this period, the hives become rich with honey. The samples were kept at room temperature and the different analyses were performed within a time period that did not exceed three months from the date of collection.

Physicochemical Properties of M. chamomilla Honey
Physicochemical properties of M. chamomilla honey have been estimated by many methods. All tests were performed as described by Mekkaoui et al. [18].

Chemical Analysis of Volatile Compounds
The chemical components of MCEO were determined using GC/MS analysis, as described previously [18]. Indeed, a Hewlett-Packard (HP6890) GC instrument coupled with an HP5973 MS and equipped with a 5% phenylmethyl silicone HP-5MS capillary column (30 m × 0.25 mm × film thickness 0.25 µm) was used in the GC analysis. The used column temperature increased from 50 • C for 5 min to 200 • C, with a 4 • C/min rate. Helium with a 1.5 mL/min flow rate and split mode (flow: 112 mL/min, ratio: 1/74.7) was the carrier gas used. The hold time was 48 min, while the temperature of the injector and detector was 250 • C.
The machine was led by a computer system type "HP ChemStation", which managed the functioning of the machine and allowed us to follow the evolution of the chromatographic analyses. Diluted samples (1/20 in methanol) of 1 µL were injected manually. In addition, 70 eV ionization voltage, 230 • C ion source temperature, and 35-450 (m/z) scanning range were the MS operating conditions. Finally, the qualitative quantification of the different compounds was based on the percent area of each peak of the sample compounds and confirmed by reference to their MS identities (Library of NIST/EPA/NIH MASS SPECTRAL LIBRARY version 2.0, built 1 July 2002).

Antioxidant Activity
The estimation of the antioxidant activity was carried out using three complementary methods.

Free Radical Scavenging Ability (DPPH Method)
The ability of EOs to scavenge the DPPH radical was measured using the method of Brand-Williams [19]. Aliquots (40 µL) of samples were added to 3 mL of DPPH solution (6 × 10 −5 mol/L) and the absorbance was determined at 515 nm after 60 min.

Determination of Ferrous Ion Chelating Ability (FIC Assay)
Firstly, solutions of MCEO at different concentrations were mixed with the FeSO 4 ·7H 2 O solution (2 mM), and then ferrozine solution (5 mM) was added to the vial to initiate the reaction. After 10 min, the optical density was read at 562 nm. The FIC was calculated according to the following equation: Ac is the absorbance of the reference (contains ferrous sulfate and ferrozine) and As is the absorbance of the EO (ferrous sulfate and ferrozine with EOs of M. chamomilla solution).

β-. Carotene Bleaching Assay (BCB)
The BCB assay was carried out as previously reported by Belmehdi et al. [20]. An emulsion of β-carotene, linoleic acid, and tween 40 was prepared. A specific volume of the emulsion was added to EOs of M. chamomilla solution and the absorbance was immediately read at 470 nm, with intervals up to 180 min. Using the following formula, the antioxidant activity (AA) was evaluated in terms of the bleaching of β-carotene percentage: where As 0 and Ac 0 are the respective absorbances of MCCO and control at zero time, As t and Ac t are the respective absorbances of EOs of M. chamomilla and control after 180 min.

5-Lipoxygenase (5-LOX) Inhibition Assay
The lipoxygenase inhibitory activity of M. chamomilla was evaluated by following the linoleic acid oxidation at 234 nm, according to a previous published method [21]. Briefly, 20 µL of oil and 20 µL of 5-LOX from Glycine max (100 U/mL) were pre-incubated with 200 µL of phosphate buffer (0.1 M, pH 9), at room temperature for 5 min. The reaction was started by the addition of 20 µL of linolenic acid (4.18 mM in ethanol) and followed for 3 min at 234 nm. The results correspond to the mean ± SD of three independent assays, each performed in triplicate. Quercetin was used as a positive control.

In Vivo Anti-Immflamatory
The anti-inflammatory effects were studied using the carrageenan-induced paw edema method previously described by Musa G.Rege et al. [22]. Briefly, Wistar rats (150 to 180 g) were randomly divided into eight groups (n = 6). The animals fasted for 18 h prior to testing. The groups of rats were orally administered with different concentrations of the studied drugs (MCEO, M. chamomilla honey, and a mixture of the EO and honey (1:1) (50 and 100 mg/kg). The control group receives distilled water, while the last group received indomethacin (10 mg/kg) as the reference drug. After 60 min, all rats were injected subcutaneously with carrageenan solution (0.05 mL of 1% carrageenan suspended in 0.9% NaCl) into the subplantar region of the left hind paw. The paw volumes of the rats were recorded using a LE 7500 digital plethysmometer, controlled by SeDaCOM software, just before the carrageenan injection (V 0 ), then at 1 h, 3 h, and 6 h after the carrageenan injection (V t ). The anti-inflammatory effect is calculated using the following equation: 2.7. Antimicrobial Activity 2.7.1. Tested Micro-Organisms The antibacterial activity was evaluated against the following six bacterial strains, representing Gram-positive and Gram-negative bacteria: Escherchia coli (E. coli) ATCC 25922, Proteus mirabilis (P. mirabilis) ATCC 25933, Salmonella Typhimurium (S. Typhimurium) ATCC 700408, (B. subtilis) ATCC 6633, Staphylococcus aureus (S. aureus) ATCC 29213, and Listeria monocytogenes (L. monocytogenes) ATCC 13932.

Disc Diffusion Assay
The antimicrobial activity of MCEO, honey and their mixture against the tested microorganisms was investigated using the disc diffusion method, according to the previously published protocols [23,24]. Briefly, the culture suspension was inoculated by swabbing on optimal culture media (Mueller-Hinton agar for bacteria and Sabouraud agar for yeast and fungi) and then the MCEO oil (mixed with 5% of DMSO), honey, or the mixture of MCEO and honey were deposited on each plate. Chloramphenicol (30 µg) was used as a positive control for bacteria and nystatin (100 I.U.) was used as a positive control for yeast and fungi, while DMSO (10 µL; 5%) was used as a negative control. The bacterial plates were incubated at 37 • C for 24 h, the yeast plates were incubated at 25 • C for 48 h, and the fungi plates were incubated at 25 • C for 72 h. After incubation, the inhibitory diameters were measured in millimeters and the results were expressed as means ± standard deviation of the three replicates.

Determination of MIC
The MIC corresponds to the minimum concentration of samples that can inhibit the growth of microorganisms. In fact, the determination of MIC against bacteria and yeast was performed according to the protocol described previously [25], with some modifications, in which Mueller-Hinton broth (Biokar, Beauvais, France) was used for bacteria and Sabouraud broth (Biokar, Beauvais, France) was used for yeast. The incubation was conducted at 37 • C for 24 h for bacteria and 25 • C for 48 h for yeast. However, the determination of MIC against the fungi was conducted by the gradient plate method according to the protocol described previously [26]. The chloramphenicol was used as a positive control for bacteria, while nystatin was used for yeast and fungi.

Determination of MBC
Minimum bactericidal concentration (MBC) corresponds to the minimum concentration of samples that can kill the bacteria. The same microdilution experiment derived from the determination of MIC was used. After the incubation, 5 µL of each tube that did not present visible growth was subcultured on tryptone soy agar (Biokar, Beauvais, France) and incubated at 37 • C for 24 h, and the lowest concentration that did not present any growth on media was considered as the MBC [27].

Physicochemical Properties of M. chamomilla Honey Sample
The physicochemical properties of the studied honey sample, including color, moisture, pH, free acidity, hydroxymethylfurfural (HMF), lactone acidity, electrical conductivity, density and ash content, are shown in Table 1. The moisture content is an important parameter to determine the quality of honey, as it is closely linked to climatic conditions, harvest seasons, honey storage, and nectar sources used by bees. Since the moisture content in M. chamomilla honey was 11.04 ± 0.02%, which was within the internationally recommended parameters (≤20%) for commercial honey quality [28,29]. These results suggest the appropriate handling and storage of the honey. The analyzed honey sample showed acidic pH values (4.02 ± 0.01). Similar values were reported for other Moroccan honey samples [30][31][32][33][34]. This acidity is due to the presence of many organic acids, such as gluconic acid and their lactones and esters. This parameter has an important effect on conservation, due to its capacity to limit and inhibit the growth and proliferation of several microorganisms, making it an important factor for honey texture and stability [35]. Free acidity, established by the Codex Alimentarius and European Community regulations, requires, in general, no more than 50 milliequiv acid/kg [28,29]. The free acidity of the honey sample in this study was 22.78 ± 0.01 meq/kg, while the lactone acidity value (considered as the acidity reserve when honey becomes alkaline) was 3.01 ± 0.05 meq/kg. This acidity is similar to other kinds of honey from other botanical and geographical origins and is due to the presence of organic acids and inorganic ions, such as phosphate [36][37][38]. The analyzed Chamomilla honey showed an electrical conductivity value greater than 0.48 ± 0.01 mS/cm, suggesting that the honey sample is from nectar honey, which is supported by the total ash content of less than 0.6%. The color of honey is related to the phenol and flavonoid content, mineral and pollen content and may be influenced by the storage and processing conditions. In this study, the honey sample presented an extra light amber color, which confirmed that Chamomilla honey has a higher content of these compounds.
The HMF content, an indicator of honey freshness that increases during processing and/or aging, is influenced by several factors, such as temperature, pH, storage conditions, and the concentrations of metallic ions, such as manganese, zinc, magnesium, and iron (II)-presenting honey. The analyzed sample has a level lower than the maximum established by international standards (<40 mg/kg) [29].

Chemical Composition MCEO
Regarding the analysis of the chemical composition of MCEO, it was carried out by the GC-MS method ( Table 2). In total, 49 molecules were identified in our study, with percentages ranging from 0.11 to 16.42%. Indeed, camphor (16.42%) was the major compound of our EO, followed by 3-carene (9.95%), β-myrcene (8.01%), and chamazulene (6.54%). Similarly, other main molecules were determined with percentages of 5.93, 5.34, 4.23% and 3.04% for 2,4,5-tetramethyl-1,3-cyclopentadiene, 3,6-dihydrochamazulene, αphellandrene, and β-sesquiphellandrene, respectively. On the other hand, many research works have investigated the chemical composition of this plant, as well as that of another species of the same family called Matricaria recutita. Indeed, concerning chamazulene, our results perfectly corroborate those obtained from M. chamomilla var. Chamomilla flower EO, in a study performed by Mahdavi et al. (2020) with a percentage of 6.46% [39]. Previous studies have revealed the great diversity in the chemical composition of the different extracts and EOs of this species [40][41][42][43]. This diversity has been associated with several factors, in particular the harvest region. In 2002, two Iranian researchers evaluated the phytochemical profile of this plant collected from three different Iranian cities (Tehran, Kazeroon, and Hammadan) during different periods [44]. Therefore, they detected several phytochemicals, including chamazulene with a concentration of 2.6%. Additionally, Ashnagar and collaborators separated and identified three main molecules, namely chamazulene (detected in our study), bisabololoxid A, and bisabolonoxid from M. chamomilla harvested in the northwest of Khuzestan, Iran [45]. This province, along with others in the same country, was the subject of a more recent study to compare the volatile compounds of twelve different oil samples from M. chamomilla growing in Iran [46]. The authors found a striking correlation between MCEO phytochemical profiles and geographic factors.
This correlation has already been recorded by Satyal and colleagues, who observed a strong diversity in the chemical composition of Nepalese chamomile EO [47]. It was discovered that in addition to these geographical factors, others are also involved in the variation in MCEO, such as the differences in irrigation regimes [48], the application of certain trace elements [49], the choice of extraction temperature [50] and method [51], the drying conditions [52], and agronomic interventions [53].

Antioxidant Activity
The antioxidant activity of MCEO, honey, and their mixture was analyzed using three different methods (DPPH, FIC ability, and BCB assay). The results are shown in Table 3 and the IC 50 values were calculated to compare these results to those of BHT, which was used as the reference standard. All samples had the capacity to reduce the stable violet DPPH radical to yellow DPPH-H, with 50% of the reduction values (IC 50 ) ranging from 533.89 ± 15.05 µg/mL to 1945.38 ± 12.71 µg/mL. The mixture exhibited the best radical scavenging activity, with an IC 50 of 533.89 ± 15.05 µg/mL and the best ferrous ion chelating ability, with an IC 50 value of 713.69 ± 03.02 µg/mL. The IC 50 value of synthetic antioxidant BHT was 14.24 ± 1.32 µg/mL in DPPH and 42.12 ± 0.07 µg/mL in FIC ability, indicating better antioxidant activity of BHT compared to the tested samples. The BCB assay honey sample presented the best activity with a percentage of 745.54 ± 8.03%, compared with the other samples and the BHT used as a reference standard. Several studies on honey indicate that the antioxidant activity of honey is due to the presence of the mentioned bioactive compounds, such as polyphenols and flavonoids and varies widely, depending on floral source and geographical origin [31,54]. Furthermore, no scientific exploration has measured the antioxidant ability of M. chamomilla honey but the previous antioxidant activity of MCEO showed the best antioxidant properties, with an IC 50 value of 2.20 mg/mL, by the DPPH method [55]. In another work, the antioxidant activity of MCEO was studied by DPPH and β-carotene/linoleic acid methods, and the IC 50 value was determined as 5.63 ± 0.20 mg/mL [56]. In the BCB assay, MCEO gave the best inhibition result, with an inhibition rate of 82.5%, supporting the antioxidant activity of MCEO [57]. The antioxidant activity of MCEO was attributed also to their terpenes, such as α-pinene, β-pinene, β-myrcene and γ-terpinene, which are known to have good antioxidant properties [43,58,59]. According to these results, the combination between M. chamomilla honey and MCEO presented the best antioxidant capacity and also had the capacity to inhibit lipid peroxidation, which may be due to the synergistic effects of their compounds. These results give this mixture an important potential in combating oxidant damage and can be potentially used as a safer alternative to synthetic antioxidants in the pharmaceutical and food industries.

Anti-Diabetic Activity
α-Glucosidase and α-amylase are key digestive enzymes that are implicated in the intestinal metabolism of carbohydrates and lipids [60]. Therefore, inhibition and suppression of these enzymes is a promising therapeutic strategy in the management of type 2 diabetes mellitus (T2DM). In this regard, the inhibitory capacity of α-glucosidase and α-amylase of MCEO, M. chamomilla honey and their mixture was evaluated ( Table 4). The results showed that the EO presented the best values against α-glucosidase (265.57 ± 0.03 µg/mL) and α-amylase (121.44 ± 0.05 µg/mL), compared to the honey sample (1351.02 ± 0.01 µg/mL and 845.31 ± 0.02 µg/mL, respectively) and their mixture (981.44 ± 0.05 µg/mL and 757.23 ± 0.02 µg/mL, respectively). However, its activity remained lower than that of acarbose as a standard reference for α-glucosidase (199.53 ±1.12 µg/mL) and more important than that of acarbose for α-amylase (396.42 ± 5.16 µg/mL). Moreover, our results are in agreement with those obtained in several works (in vivo and in vitro) that evaluated the antidiabetic effect of chamomilla plant extracts. Indeed, M. chamomilla ethanolic extract demonstrated anti-glycation properties with an IC 50 of 264.2 µg/mL for lipase inhibition activity [61]. Moreover, Villa-Rodriguez et al. [62] showed that the M. chamomilla extract and isolated apigenin, apigenin-7-O-glucoside, cis and trans-2-hydroxy-4-methoxy cinnamic acid glucosides exhibited concentration-dependent inhibition of α-amylase and maltase [62]. On the other hand, Najla and collaborators revealed that oral administration of M. chamomilla leaves' water extract at a dose of 200 mg/kg/day significantly reduced blood glucose levels and increased plasma insulin in streptozotocin-diabetic rats [63]. Using the same animal model, the same results were recorded by Ramadan et al., [64] which have demonstrated that the administration of the water extract of M. chamomilla leaves at a concentration of 100 mg/kg body weight significantly reduced blood glucose levels in diabetic animals and elevated the levels of serum insulin and C-peptide. The antidiabetic activity of EOs was certainly due to their terpenes, such as α-pinene, β-myrcene, α-thujone and γ-terpinene [43,59] that are known to have good antidiabetic activity. Indeed, α-pinene was found to be able to reduce fasting blood glucose levels in alloxan-induced diabetic mice after 2 and 24 h of treatment [65]. Moreover, oral administration of α-thujone (60 mg/kg/day) has been shown to be able to decrease plasma glucose levels in STZinduced diabetic rats [66]. From this, and the antioxidant properties already confirmed in our work, it can be deduced that M. chamomilla can be used for controlling blood glucose levels and the oxidative stress that accompanies diabetes.

In Vivo Anti-Inflammatory Activity
Immunomodulators are natural or synthetic substances that regulate the type, duration, and intensity of innate or adaptive immune responses. Because of excessive side effects of anti-inflammatory synthetic drugs on human health, high cost, and drug resistance of chemical immunomodulators, researchers have now focused on naturally originated agents as a specific, safe, and inexpensive treatment [67]. Indeed, in this work, we evaluated the anti-inflammatory activity of MCEO, M. chamomilla honey and their mixture on carrageenan-induced acute inflammation by measuring the increased paw volume of the rats at different time periods (1, 3 and 6 h) ( Table 5). Our results showed that the MCEO and honey mixture at a dose of 100 mg/kg exhibited maximum anti-inflammatory activity, with 63.75% and 77% edema inhibition after 3 h and 6 h, respectively. The results were comparable with the reduction produced by 10 mg/kg of indomethacin, a standard drug, at 3 h and 6 h, which presented inflammation inhibition values of 56.25% and 69%, respectively. Our data are similar to previous studies that demonstrate the anti-inflammatory activity of the extracts and MCEO. For instance, Wu et al. [68] showed that MCEO and aqueous extract could significantly inhibit pedal swelling induced by carrageenan in rats by decreasing the concentration of PGE2 and NO. Elmallh et al. [69] revealed that the aqueous chamomile extracts significantly decreased the paw thickness of rats after induction of pedal inflammation, as compared to the positive group. Ortiz et al. [70] demonstrated also that M. chamomilla ethanolic extract (MCE) was able to inhibit the cyclooxygenase (COX) enzyme in silico. This activity could be attributed to the presence of chemical constituents, such as flavonoids in honey, which have been widely reported to inhibit the COX and lipooxygenase pathways of arachidonate metabolism [71]. They have been shown also to inhibit the release of proinflammatory cytokines TNF-α and IL-1β, as well as down-regulate the expressions of inducible nitric oxide synthase (iNOS) and the production of reactive oxygen species (ROS). Moreover, several other mechanisms are involved, including the activation of the nuclear factor erythroid 2-related factor 2/antioxidant response element pathway and down-regulation of the nuclear factor kappa B pathway (NF-kB). On the other hand, flavonoids can also modulate gene expression of numerous inflammatory factors via down-regulation of epigenetic transcriptional control of these genes [72,73]. It is also due to MCEO's constituents, such as α-pinene, β-pinene, β-myrcene, and γ-terpinene [43,58,59] that have been widely investigated for their anti-inflammatory effects [74,75] [76].

In Vitro Anti-Inflammatory and Dermatoprotective Activity
The inhibition of tyrosinase activity is an important strategy for skin protection. Indeed, the tyrosinase inhibitory method is the most used in vitro method today for revealing the dermatoprotective activity of medicinal plant products. On the other hand, the inhibition of lipoxygenase is the most used test to evaluate the anti-inflammatory activity in vitro. In this work, the ability of MCEO, honey and their mixture to protect the skin was assessed by its tyrosinase inhibitory property and anti-inflammatory effect by the 5-LOX inhibition assay. The results of tyrosinase and 5-LOX inhibition by all samples are expressed as IC 50 (Table 6). As listed, MCEO presented the best activity, with an IC 50 value of 41.11 ± 0.03 µg/mL compared to honey (81.53 ± 0.01 µg/mL) and their mixture (57.32 ± 0.03 µg/mL) but was lower than that of the standard, quercetin (IC 50 = 39.28 ± 0.02 µg/mL). Moreover, the anti-inflammatory effect of EO (IC 50 = 1.58 ± 0.02 µg/mL) was also higher than that of the other samples and lower compared with quercetin (IC 50 = 1.02 ± 0.01 µg/mL). Our results are in agreement with those obtained by Jo and colleagues [77], who examined, in vitro, the whitening effect of M. chamomilla L. extract using tyrosinase inhibitory assay. The authors recorded important anti-tyrosinase activity in a concentration-dependent manner of the ethanol extract. Furthermore, Danciu et al. [78] showed that methanolic extract of M. chamomilla showed good inhibition of lipoxygenase, with an EC 50 value of 166.32 mg/mL. From this, it can be deduced that preparations based on M. chamomilla can be used as sunscreen formulations to protect the skin against sunburn and to slow down skin aging and inflammation diseases.

Antimicrobial Activity
In vitro, the Moroccan MCEO, honey and their mixture together were screened for their antimicrobial activity using disk diffusion assay against Gram-positive, Gram-negative pathogenic bacteria, yeast and fungus and the reference species and the results are presented in Table 7. The obtained inhibitory diameters, expressed in millimeters, showed a variation in results in terms of an examined sample of M. chamomilla. As confirmed, the EO of the study species exhibited an excellent antimicrobial, antifungal and anti-yeast effect against all the tested strains, compared to the M. chamomilla honey samples. The measured inhibition zones of MCEO registered very potent activity, in which Staphylococcus aureus ATCC 29213 (22.97 ± 0.16 mm), Listeria monocytogenes ATCC 13,932 (22.10 ± 0.13 mm), Candida albicans (21.07 ± 0.24 mm) and Proteus mirabilis ATCC 25933 (19.33 ± 0.16 mm) were the most sensitive strains, followed by Aspergillus niger (18.13 ± 0.18 mm), Escherichia coli ATCC 25922 (16.87 ± 0.16 mm), Salmonella Typhimurium ATCC 700408 (16.23 ± 0.09 mm) and Trichophyton rubrum (15.87 ± 0.16 mm). Moreover, our findings indicated a mild antimicrobial activity of M. chamomilla honey against L. monocytogenes, S. aureus, P. mirabilis, C. albicans and E. coli corresponding to inhibition diameters ranging from 11.97 ± 0.11 mm to 13.90 ± 0.13 mm, respectively. One must note that the honey was not active on P. aeruginosa and T. rubrum and A. niger were represented by the smallest inhibitory zone of M. chamomilla honey of 8.10 ± 0.01 mm, respectively. It is worthwhile noting that the mixture exhibited an additive effect between both samples of this plant. All examined bacteria, fungus and yeast were responsive and sensitive to chloramphenicol and nystatin, respectively (Table 7).
Using broth microdilution and gradient plate methods, the measurements of MIC and MBC of MCEO and honey and their mixture were performed for bacterial, yeast and fungal strains. As recapitulated in Table 8, the values of inhibitory concentrations registered for MCEO showed higher MICs than those obtained in MCEO honey and their association in all tested microorganisms. Indeed, S. aureus and L. monocytogenes indicated the lowest MIC values of MCEO (0.25% (v/v)), followed by E. coli, P. mirabilis and C. albicans, with an MIC of M. chamomilla 0.5% (v/v). An MIC of 1% (v/v) of MCEO was observed in S. Typhimurium and A. niger, whereas 2% (v/v) was the MIC marked in T. rubrum inoculums in the same aforementioned sample type ( Table 8). The highest MICs identified were in E. coli, P. mirabilis, S. Typhimurium, A. niger for an MIC of 4% (v/v), pursued by T. rubrum and P. aeruginosa for an MIC that ranged between 4 and 8% (v/v) in M. chamomilla honey samples. Similar to the disk diffusion test, the findings of MIC in the combination with honey and MCEO showed mutual interaction against the investigated pathogens. The recorded MIC values indicate that a bactericidal effect of MCEO was only detected in Gram-positive bacterial suspensions of S. aureus and L. monocytogenes (MIC = MBC = 0.25% (v/v)) and S. Typhimurium (MIC = MBC = 1% (v/v)). However, a double quantity of MIC was the efficient dose to kill other bacteria, in which the MBC was equal to 1% and > 8% in E. coli, P. mirabilis and P. aeruginosa by MCEO, respectively. Likewise, the same doses were measured in M. chamomilla honey and the combination between MCEO and M. chamomilla honey (Table 8).
Numerous investigations on aromatic and medicinal plants agreed that EOs exhibited strong inhibitory action on pathogenic microbes [24,79,80]. Nonetheless, the antimicrobial activity findings of the present work revealed a variation in response degree according to the strains' origin (bacteria, fungus and yeast) and type of tested specimen of M. chamomilla (EOs, honey and their mixture). This is the first research paper in vitro about Moroccan M. chamomilla honey and its association between EOs and honey of M. chamomilla. In reality, a very powerful antimicrobial effect from MCEOs by both disk diffusion and broth microdilution techniques were cited, where the measured diameter was between 22.97 ± 0.16 mm and 15.87 ± 0.16 mm, and MIC values ranged from 0.25% (v/v) to 2% (v/v), respectively, against the studied microorganisms, except P. aeruginosa. Moderate activity was observed in the M. chamomilla mixture of honey and EOs (inhibitory diameter was between 13.90 ± 0.13 mm and 8.10 ± 0.01 mm vs. MIC ranging from 1% (v/v) to >8% (v/v)). However, a weak antimicrobial impact was displayed in M. chamomilla honey (inhibitory diameter was between 13.90 ± 0.13 mm and 8.10 ± 0.01 mm vs. MIC ranging from 2% (v/v) to >8% (v/v)).
Few studies were consistent with our findings, such as the outcomes of the Brazilian team conducted by Silva et al. [81]. These researchers confirmed, in their comparative antibacterial and phytochemical analysis of crude extracts, that MCEOs showed the best antimicrobial agents, in which S. aureus (MIC 90% = 1.2 mg/mL) was significantly (p ≤ 0.05) more susceptible compared to E. coli (MIC 90% = 28.2 mg/mL) [81]. Das et al. [82] assessed antimicrobial activity by the formulation of a pickering nanoemulsion of MCEO and confirmed the great antibacterial action on P. aeruginosa PMC 103, S. aureus ATCC 29,213 and E. coli PMC 201 for an MIC 90 equal to 1.02 µg/mL, 1.06 µg/mL and 2.19 µg/mL, respectively. Moreover, the extraction and valorization of the antibacterial activity of wild chamomile from the Taounate province, Morocco, stated comparable relevant inhibitory diameters and MICs [83].
Contrary to the impressive antimicrobial action exposed in this investigation, most published data have reported the powerless antimicrobial activity of MCEOs across the entire world. The disc-diffusion and broth microdilution tests carried out by Soković et al. [84] demonstrated that MCEOs extracted from the aerial plant in Serbia were ineffective against the human strain of P. mirabilis and reference strains of P. aeruginosa ATCC 27,853 (0 mm vs. MIC = MBC = 10.0 and 15.0 µg/mL, respectively). In the same way, no effects were recorded in S. aureus ATCC 25923, E. coli ATCC 0157:H7, S. typhimurium ATCC 13,311 or L. monocytogenes inoculums, where the inhibitory diameter, MIC and bactericidal doses ranged from 8.0 mm to 10.0 mm, and from 5.0 µg/mL to 10.0 µg/mL [84]. As proven by Herman et al., [85] and Mekonnen et al. [86], and Niknam et al. [87] through the use of the agar diffusion method, the methanol fraction of M. chamomilla flower extract (50 mg/mL) showed weak antibacterial effect against S. aureus ATCC 6538p (1.3 ± 0.3 mm) and P. aeruginosa ATCC 9027 (0.3 ± 0.3 mm). A higher MIC was noticed toward the methanol fraction of flower M. chamomilla extract on S. aureus ATCC 6538p (62.5 µg/mL) and (500 µg/mL) P. aeruginosa ATCC 9027.
Based on the evaluated antifungal activity from the M. chamomilla flower EOs on A. niger, Tolouee et al. [88] marked a dose-dependent effect with maximum growth inhibition of approximately 92.50% of A. niger. In addition to this, the authors described other morphological changes, such as retardation in conidial production of the fungus species, observed by transmission electron microscopy due to direct penetration of MCEOs through the fungal plasma membrane [88]. However, Mekonnen et al. [86] have proven that Trichophyton spp., and Aspergillus spp., donated by the Ethiopian Public Health Institute, were resistant to EOs extracted from matured flower heads of M. chamomilla. In addition, several common molds found in cakes and bakery products, including A. niger, were significantly killed after treating these with chamomile EO at 0.15% [89]. Their findings allowed them to increase the shelf-life of food products without the use of synthetic agents [89]. Das et al. [82] concluded that the nanoemulsion of chamomile EOs demonstrated relevant antifungal activity, especially on C. albicans ATCC 1001 for an MIC 90 equal to 2.65 µg/mL. Concerning the data about the antimicrobial effect of M. chamomilla honey or their association with EOs, no published paper was found while reporting this work. Instead, the additive effect shown in the combination of MCEO and honey confirmed the promising antimicrobial activity of MCEO against the most abundant pathogens assayed in this report.

Conclusions
Chamomile is a highly requested plant in the worldwide market, due to its high medicinal benefits and irreproachable pharmacological characteristics. In addition, there is an emerging trend of using more natural ingredients rather than chemical synthetics, since many herbal remedies are safe, easily available, healthful, and cost-effective. In this current work, we have focused on the chemical composition and identification of different pharmacological properties of M. chamomilla EOs, honey, and their mixture. Regarding the analysis of the phytochemicals of MCEO, among the 49 molecules identified, camphor was the major compound of our EO, followed by 3-carene, β-myrcene, and chamazulene (6.54%), as evidenced by the GC-MS method. In these results, we have also concentrated on exploring the pharmacological properties of the extracts obtained from this plant, especially their antimicrobial, antidiabetic, antioxidant, and anti-inflammatory effects. As demonstrated by the MIC and MBc methods, the EOs of the study species showed an excellent antimicrobial (Staphylococcus aureus ATCC 29213), antifungal (Aspergillus niger), and anti-yeast (Candida albicans) effect against all the tested strains. In terms of antidiabetic effect, the EOs exhibited the most effective levels against α-glucosidase and α-amylase. Furthermore, M. chamomilla EOs, honey, and its mixture exhibited antioxidant activity against the DPPH assay. The mixture was found to exhibit the best radical scavenging activity. Moreover, EOs, honey, and their mixture at a dose of 100 mg/kg exerted a high anti-inflammatory effect. We can suggest that this biological diversity of M. chamomilla could be ascribed to its chemical composition, containing several types of biochemical compounds. In addition, from the obtained outcomes, it is evident that the most prominent area of application of M. chamomilla is in the medicinal field, as demonstrated in the in vivo and in vitro models. Accordingly, the EO and extracts of the studied plant materials could be a suitable source of drug materials for the preparation of new antimicrobial, antidiabetic, antioxidant, and anti-inflammatory agents. However, the major bioactive molecules identified in the EOs of chamomile need to be investigated further, in terms of their toxicity to verify their safety and also to determine the underlying mechanisms of action. Nevertheless, despite this type of validation, we could not use these EOs because they are volatile compounds. Moreover, there is another very important perspective that requires the encapsulation of these EOs in order to provide a product in powder form that could be applied in the field of cosmetics, pharmaceutical, or food industries.

Conflicts of Interest:
The authors declare no conflict of interest.


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Physiological and morphological response of tomato plants to nano-chitosan used against bio-stress induced by root-knot nematode (Meloidogyne incognita) and Tobacco mosaic tobamovirus (TMV)

Root-knot nematodes (Meloidogyne spp.) have been reported to be responsible for large economic losses of agricultural crops due to their wide host range and variety of suitable climates. The control measures of these parasitic nematodes depend upon synthetic nematicides and a small number bio-based products. Chemical nematicides are eliciting adverse effects on the environment and human health. In the present study, an alternative tool, nano-chitosan was tested for the control the root-knot nematodes, Meloidogyne incognita, and Tobacco mosaic tobamovirus (TMV) in greenhouse-cultivated tomato. The effect of nano-chitosan on morphological (weight and length of shoot and root systems) and biochemical responses (Polyphenol oxidase, Peroxides, Total soluble phenol and Total protein) was assessed. The obtained results indicated that densities of Meloidogyne incognita alone or in the presence of TMV were decreased by nano-chitosan at a range of 45.89 to 66.61%, while root gall desntiy was reduced between 10.63 and 67.87%. Moreover, the density of TMV on tomato leaves singly or in the presence of M. incognita was suppressed at range of 10.26 to 65.00% after 20 days of infection, and reached up to 58.00% after 40 days of infection. However, soil application of nano-chitosan pre infection reduced TMV density only by 5.48%. Morphogenesis of tomato plants such as shoot and root systems were significantly improved. The impacts of nano-Chitosan applications on total soluble phenol, total protein, polyphenol oxidase and peroxides after 20 and 40 days of infections varied.

Introduction
In Egypt, tomatoes (Solanum lycopersicum, Mill) are very popular vegetables which are utilized for both local consumption and exportation (Rakha, 2014). Tomato is one of the richest sources of minerals, vitamins and carbohydrates (Ibrahim et al., 2010). There are about 159 countries globally that produce tomatoes commercially. China is one of the biggest producers of tomato with over 59 million tons in 2017 followed by Turkey, USA, Egypt, Italy, Spain, Mexico, Nigeria, Brazil and Russia (Ong et al., 2020).
Root-knot nematodes (Meloidogyne spp.) are a serious disease/pest which parasitize the roots of tomato plants causing damages leading to significant yield losses (Abd El-Monem et al., 2016;Niu et al., 2020).
Farmers depend on synthetic nematicides to manage nematodes. Such nematicides can impact on human healthy and the environment. Nanotechnology is increasingly being investigated for use in agriculture, food industry, pharmaceuticals and medicine (Giongo et al., 2016;Alfy et al., 2020). A Nanoparticle has a size range from 1 to 100 nm and many are investigated for their ability to manage pests and diseases (Sun et al., 2014;Alfy et al., 2020).
Chitosan is a natural biopolymer which is formed by chitin deacetylation that exists in the outer shell of shrimp, shellfish, lobster or crab, as well as infungal cell walls. It was discovered in 1811 by Henri Braconnot (Chakraborty et al., 2020). Chitosan is an eco-friendly and biodegradable material (Park et al., 2008;Khalil, 2016), is cheap and easy to work with (Anitha et al., 2014;Malerba & Cerana, 2016). Moreover, chitosan is able to control viruses, bacteria, fungi, insects, plant nematodes and other pests locally and systemically (Abd El-Aziz & Khalil, 2020;Alfy et al., 2020).
Tobacco mosaic tobamovirus (TMV) is a positivesense single stranded RNA virus in the genus Tobamovirus and known as one of the most stable viruses (Silva et al., 2011;Hashemi et al., 2014;Ong et al., 2020). TMV causes severe economic losses in several crops included tomato (Abo-Zaid et al., 2020). The management of TMV is difficult because it is easily dispersed, it is transmitted mechanically, and symptoms are appearing 7 to 14 days after infction (Ara et al., 2012).
Therefore, in this study we aimed to investigate the impact of nano-chitosan on the presence of root-knot nematode (Meloidogyne incognita) and Tobacco mosaic tobamovirus (TMV) or their combination. We also studied their effect on morphological and physiological characteristics of tomato plants under greenhouse conditions.

Nematode inoculum
The egg inoculum of root-knot nematode (Meloidogyne incognita) was isolated from eggplant roots (cv. Balady). The roots were cut into small segments (1-2 cm long), and shaken for 3 min in sodium hypochlorite (5%). The suspension was passed through 200 and 400 mesh sieves to obtain free eggs (Hussey & Barker, 1973). The eggs were washed several times with water and then counted under a stereo-microscope. The species of root-knot nematode (M. incognita) was identified by using the perineal patterns method according to Taylor and Nelscher (1974).

Source of tobacco mosaic virus inoculum
Leaf samples of tomato plants showing mosaic symptoms (Fegla et al., 2000;Aghamohammadi et al., 2011) were collected separately in plastic bags from locations in Alexandria governorate during the 2019-2020 growing seasons. Virus inoculum was prepared by grinding infected leaf tissues 1:10 (w/v) with a mortar and pestle in 0.1 M phosphate buffer (pH 7.0), containing 0.5% of 2-mercaptoethanol. To identify TMV, the leaves of healthy Nicotiana glutinosa plants in the seedling stage were first dusted with carborundum (600 mesh) and then inoculated with a freshly prepared viral inoculum using the forefinger method, and kept in an insect proof greenhouse (Hamza et al., 2018).

Diagnostics of tobacco mosaic virus
Hosts and symptomology test Chenopodium amaranticolor, Datura metal, Nicotiana rustica, N. glutinosa and Solanum lycopersicum (Abd E l -A z i z , 2 0 0 0 ) and S o l a n u m m e l o n g e n a (Aghamohammadi et al., 2011) were used to confirm the identification of isolated virus because they give distinct symptoms. Plantlets (five replicates for each host) of each plant species were mechanically inoculated with TMV and kept under greenhouse conditions. Plants were assessed daily for about 4 weeks for symptoms expression. Inoculated plants which didn't show any symptoms were checked for latent infection by back-inoculation to the indicator host N. glutinosa (Fegla et al., 2001).

Serological diagnostic test
Serological diagnostics was used to confirm the identification of TMV by indirect ELISA according to Abd El-Aziz and Younes (2019). Antiserum of Tobacco mosaic tobamovirus (TMV) used in the serological diagnosis was supplied by the Lab. of Prof. Fath-Allah Mervat (Plant Pathology Institute, Agricultural Research Center). This Antiserum was used to discriminate Cucumber mosaic cucumovirus (CMV) and TMV (Abd El-Aziz & Younes, 2019).
Enzyme linked immunosorbent assay (indirect ELISA) was carried out as described by Abd El-Aziz, 2019 and Abd El-Aziz and Younes (2019). The infected and healthy leaves of tomato plants, 20 and 40 days of infection, were extracted to detect the infection by TMV.
The obtained values of ELISA (Sunrise ELISA plat reader™ instrument) were expressed as absorbance at 405 nm. The absorbance values considered positive when the value was at least twice that in uninfected plants. In each set of tests, wells lacking antigen (coating buffer only) were included as controls (Abd El-Aziz, 2019).
Nano-chitosan source, preparation and SEM Chitosan was obtained from ChitoLytic, Canada with deacetylation of 88.3% and a viscosity 70 (at 20°C). The nanoparticles of chitosan were prepared according to the method described by Qi et al. (2004). The SEM of nano-chitosan was carried out in the electron microscopy unit of the Faculty of Science, Alexandria University. The solution of nano-chitosan was prepared by dissolving 0.225 g nano-chitosan in 150 ml distilled water blended with 0.1% glacial acetic acid (Khan et al., 2021).
The efficacy of nano-chitosan on some parameters related to induced resistance against Meloidogyne incognita, Tobacco mosaic tobamovirus (TMV) and their mixture in the tissue of tomato plants

Total soluble phenol contents determination
The total soluble phenol content of tomato leaves, was extracted as described by Slinkard and Singleton (1997). The absorbance was carried out on 765 nm by using a spectrophotometer (Tuner, model 390). Total soluble phenol content was standardized against tannic acid and absorbance values were converted to μg of phenols per gram fresh weight of tomato leaves. Each value reported was the average of three replicates. The results were expressed as tannic equivalents according to the following equation: μg tannic acid/g fresh weight = (((OD/K) * (10/ 0.2)/0.5)) Where: OD = absorbance at 765 nm, K = the extension coefficient = 0.016898 μg-1 .

Protein content
Total soluble protein was extracted from tomato leaves according to Dixon (1985) and determined according to Lowry et al. (1951). The blue color was measured at 700 nm by using a spectrophotometer (Tuner, model 390). The reading was related to standard curve prepared from known concentration of bovine serum albumin (BSA) protein.

Polyphenol oxidase (PPO)
Polyphenol oxidase activity (expressed as OD/mg protein), was determined according to Gauillard et al. (1993). The samples were repeated three times and absorbed on 420 nm by using a spectrophotometer (Tuner, model 390).

Peroxidase (POD)
Peroxides activity (expressed as OD / mg protein), was determine according to Murage and Masuda (1997) with some modification. The reaction mixture contained 200 μl of enzyme extract, 0.1 M sodium phosphate buffer (pH 6.1, 1.5 ml of 20% H 2 O 2 and 1.5 ml of catechol 0.04 M). The initial rate of increase in absorbance was measured within 1 min at 470 nm determined by using a spectrophotometer (Tuner, model 390).

The experimental design
A pot experiment was carried out under greenhouse conditions to evaluate the performance of nanochitosan against M. incognita, Tobacco mosaic tobamovirus (TMV) and their combination on tomato plants. All plastic pots (15 cm diameter) were filled with 1 kg of autoclaved loamy sand soil. A single plantlet of S. lycopersicum L. (cv. 086) of 40 days old was transplanted in each pot. The nano-chitosan was applied as foliar and soil application (drench) 5 days before infection as well as at the same time of infection. Each pot received 5000 nematode eggs by pouring the nematode suspension into holes made 2-4 cm below the soil surface around the base of the plants. All pots including controls (inoculated and uninoculated plants) were replicated four times and arranged in a completely randomized design on a bench under greenhouse conditions. During the course of the experiment, irrigation and fertilization were applied as needed. After 47 days, plants were uprooted and the roots were washed free of soil. The shoot and root lengths, in addition to their fresh weights were recorded. Furthermore, the number of galls / root system and number of J2 / 250 g soil were estimated at the end of the experiment. The second stage juveniles (J2) were extracted from the soil by using sieving and the Baermann plate technique (Ayoub, 1980) and then counted. Meanwhile, to inoculate TMV, the leaves of the tomato plants were dusted with carborundum (600 mesh) and then inoculated with a freshly prepared viral inoculum using forefinger method (Hamza et al., 2018).

Statistical analysis
The statistical analysis of the data was carried out using Costat (2005) version 6.303. Statistically significant differences between means were compared using analysis of variance (ANOVA) with the least significant differences (LSD) and P-values at 0.05 probability.

Results
Nematicidal impact of nano-chitosan on Meloidogyne incognita, alone or in the combination with TMV on tomatoes Application of nano-chitosan (ranged from 19.11 upto 22.79 nm, Fig. 1) was effective against the root-knot nematode (M. incognita) singly or in the coexist of Tobacco mosaic tobamovirus (TMV) as soil and foliar application on tomato plants under greenhouse conditions (Table 1).
The results indicate that nano-chitosan as soil drench suppressed the second stage juveniles (J 2 ) in the soil by 64.50 to 58.28%. While, foliar applicatios showed a reduction of 55.23 and 46.62%, respectively, without significant differences. For the infection with M. incognita and TMV the soil application of nanochitosan decreased J 2 density by 66.61 and 45.89%, and while foliar application 61.61 and 57.50% when applied after or before infection, respectively. Root gall density was reduced with soil application of nano-chitosan by 67.87 and 56.40% pre and post infection, respectively, without significance difference. The foliar application showed a significant reduction of galls by 40.45 and 26.07% post and pre infections, respectively (Table 1). In dual infections (M. incognita + TMV), nanochitosan decreased root gall density by 50.79 and 30.71% as soil application pre and post infection, respectively. Foliar application showed a reduction by 29.53 and 10.63% post and pre infections, respectively.

Serological detection of tobacco mosaic tobamovirus (TMV)
The TMV used in this study was isolated from naturally infested tomato plants collected from various locations in the Governorate of Alexandria. TMV was isolated from tomato plants showing mosaic, severe mosaic and yellowing with vein clearing (Fig. 2 b ) and yellowing mosaic on Nicotiana gluca (Fig. 2 a ). Necrotic local lesions induced after mechanical inoculations with TMV on S. melongena, C. amaranticolor, Datura metal and N. glutinosa were shown (Fig. 3). To identify the infection of TMV serologically specific antisera and indirect ELISA tests were utilized ( Table 2). The obtained results showed that with antiserum of TMV the optical density of infected plants was 0.554 while healthy plants had an optical density of 0.196. Meanwhile, with antiserum of CMV, the optical density was 0.319 and healthy plant gave 0.280, this means that the recognized virus was TMV only and there is no CMV contamination.
Efficacy of nano-chitosan on TMV alone or in the presence of M. incognita Table 3 shows the impact of nano-chitosan as soil and foliar applications before and 5 days after infection in the presence of TMV alone or combined with the rootknot nematode (M. incognita). Healthy plants ( After 20 days of infections, in the case of dual infection results showed that nano-chitosan as soil application recorded reduction in virus densities by 50 and 10%, while foliar application recorded 16.67 and 21.79% with and before infections, respectively. However, soil application of nano-chitosan against TMV alone decreased the virus densities in leaves tissues by 45.00 and 65.00% with and before infections, respectively. Suppression in virus densities were noticed in foliar application by 12.50 and 40.00% with and before infections, respectively.
In respect to the dual infection after 40 days, results indicated that application of nano-chitosan with infection as soil drench minimized the density of virus by 42.47%, while application before infection recorded increasing in virus density by 5.48%. Meanwhile, foliar application with infection recorded a reduction by 13.70%, whereas the application prior infection showed no suppression. In the presence of TMV only, soil application showed reductions by 28.77 and 58.90%, with and prior infections, consecutively. The implementation of nano-chitosan as foliar achieved reductions in virus densities estimated by 15.07 and 21.92% with and prior infections, respectively.
Impact of nano-chitosan on the morphogenesis of tomato plants Data shown in Table 4 revealed the impact of soil (drench) and foliar applications of nano-chitosan with The effect of nano-chitosan on some parameters related to induced resistance The efficacy of Nano-chitosan on total soluble protein and phenol content of tomato leaves The efficacy of nano-chitosan as soil and foliar application on total soluble protein was estimated in fresh  (Table 5). Soil application of nano-chitosan showed a mean increase in the total soluble protein (TSP) by 3.65 and 9.97% after 20 days of infection with M. incognita and TMV alone, respectively, while the mixture of M. incognita + TMV reduced the level of TSP by 0.71%. Also, the foliar application of nano-chitosan increased the levels of TSP by 0.56 and 9.88%, in the presence of TMV alone and M. incognita + TMV, respectively, whilst TSP decreased by 4.05% in the presence of M. incognita alone. Similar trends hold for the infection after 40 days (Table 4).
Total soluble phenol (TSN) content of the tissue of tomato leaves was affected by the application of nanochitosan (Table 5). The soil application of nano-chitosan after 20 days of infection gave general mean increasing in the levels of TSN estimated by 6.51 and 6.27% in the presence of M. incognita + TMV and TMV alone, respectively, while a reduction by 8.41% was recorded in the presence of M. incognita alone. Also, the foliar application of nano-chitosan increased the levels of TSN by 9.19 and 5.67%, in the presence of M. incognita + TMV and M. incognita alone, respectively. Application of nano-chitosan in the presence of TMV alone diminished TSN level by 11.67% in tomato leaves. Similar trends hold for the infaction after 40 days (Table 5).

The effect of nano-chitosan on polyphenol oxidase and peroxidase activity
The impact of nano-chitosan on the levels of Polyphenol oxidase (PPO) after 20 and 40 days of infection with M. incognita, TMV and their mixture was shown in Table 6. The soil application of nano-chitosan after 20 days of infection by M. incognita alone and M. incognita + TMV increased the levels of PPO by 11.61 and 4.67%, respectively, while a reduction (6.16%) was noticed in the presence of TMV alone. Foliar application of nano-chitosan showed an increase by 15.31 and 20.79% in the presence of M. incognita and TMV alone, respectively, while a decrease was recorded in PPO levels of 1.56% in the presence of The experiment was repeated twice and ELISA optical density at 405 nm is average of three replicates      M. incognita + TMV. Similar trends hold for the infection after 40 days (Table 6). The levels of Peroxides (POD) were affected by application of nano-chitosan after 20 and 40 days of infection in the presence of M. incognita, TMV or their mixture ( Table 6). The soil application of nano-chitosan after 20 days of infection augmented the levels of POD by 6.76 and 23.17% in the presence of M. incognita alone and M. incognita + TMV, respectively, while in the presence of TMV alone the POD level was decreased by 16.59%. Meanwhile, applied nano-chitosan as foliar increased the levels of POD by 10.17 and 8.71% in the presence of both M. incognita and TMV alone, respectively. Nano-chitosan in the presence of binary mixture of M. incognita + TMV minimized the level of POD by 8.42%. Similar trends were found for the infection after 40 days (Table 6).

DISSCUSION
Our results indicate that nano-chitosan was very effective against the plant parasitic nematode (Meloidogyne incognita) singly or in the presence of TMV. The results of the present study are in agreement with a recent study by Alfy et al. (2020) who found that nano-chitosan at 500 to 2000 mg/l induced a mortality in juveniles of the rootknot nematode (M. incognita) ranging from 85.2 to 97.5% after 72 hours of exposure, and inhibited egg hatching by 73 to 95.3%. Meanwhile, the pot experiment under greenhouse conditions revealed that nano-chitosan at 2000 mg/l recorded suppression in galls, egg masses and juveniles/ 250 g soil by 90, 78 and 98%, respectively. The low, medium and high molecular weights of chitosan decreased the soil population at range of 37.67 to 68.60%, and from 70.30 to 83.33% in root galls (Khalil & Badawy, 2012). Similalry, high and low molecular weights of chitosan were minimized root galls by 90 and 93%, respectively (El-Sayed & Mahdy, 2015). da Silva et al. (2014) found that using chitosan in different molecular weights suppressed the severity of the pine wilt disease caused by Bursaphelenchus xylophilus.
The action of chitosan as soil application against plant parasitic nematodes (PPNs) are assumed to be linked with its stimulation of the reduplication of chitinolytic microorganisms which degrade chitin in organs of the PPNs, in addition to suppressing egg hatching and the viability of larvae and adults for both root-knot and cyst nematodes (Kalaiarasan et al., 2006;El-Sayed & Mahdy, 2015;Westerdahl et al., 1992). The parasitism by Pochonia chlamydosporia of the eggs of Meloidogyne javanica was increased by chitosan (El-Sayed & Mahdy, 2015). Meanwhile, chitosan has a high nitrogen content which releases ammonia causing toxicity to PPNs (Fan et al., 2020). Chitosan and its derivatives induce systemic acquired resistance in plants against a range of pathogens, in addition to root-knot nematode M. incognita (Chakraborty et al., 2020). Also, several reports mentioned that glucanase, chitinase, peroxidase, phenylalanine ammonia-lyase, polyphenol oxidase, superoxide dismutase and catalase were triggered with the application of chitosan or its derivatives in certain crops (Burkhanova et al., 2007;Eilenberg et al., 2009;Yin et al., 2010;Chang & Kim, 2012;Orzali et al., 2014;Xing et al., 2015;Li et al., 2016). The levels of protein and some secondary metabolites linked to the protection from pathogens, such as phenolics and phytoalexins, were increased in treated plants (Lin et al., 2005;Hadwiger, 2013;Zhang et al., 2015). The positive charge of chitosan molecules may be linked with negative charges on the surfaces of wide array pathogens and causing damage in cell structure (Chakraborty et al., 2020). Viral diseases such as; alfalfa mosaic alfamovirus, peanut stunt cucumovirus, potato virus X potexvirus, tobacco mosaic tobamovirus, Tobacco necrosis alphanecrovirus and cucumber mosaic cucumovirus are affected by chitosan applications (Nagorskaya et al., 2014;Jia et al., 2016). TMV has a range of effects on fruit crops including the fall of flowers or failure of flowering leading to decreased production (El Shafie et al., 2005). The efficacy of chitosan as anti-viral agent may be due to chitosan being able to inducing hypersensitive response in infected plants (Chirkov, 2002). In addition, the antiviral activity of chitosan depends on the average polymerization frequency, degree of N-deacetylation, quality of positive charge, and character of the molecule's chemical modifications (Chakraborty et al., 2020).
Our study revealed that application of nano-chitosan in the presence of root-knot nematode or TMV alone and in combination improved plant growth (shoot and root heights and weights) significantly which is in agreement with previous studies of Alfy et al. (2020) and Khalil and Badawy (2012). The enhancement in plant vigor with nano-chitosan or its derivatives may be linking to certain necessary nutrients, trace elements and improved index of photosynthesis (Chakraborty et al., 2020). However, chitosan can invigorate cell elongation, the division of root cells via stimulation plant hormones including auxin and cytokinin (John et al., 1997;Amin et al., 2007;Dzung et al., 2011). The positive charge of nano-chitosan can easily penetrate plant cell walls' or adhere to plant surfaces and enhance seed germination and biophysical properties. Also, crop yield was increased substantially by enhancing stomatal function and chlorophyll content (Chakraborty et al., 2020).
Foliar applications of chitosan enhanced plant growth parameters, yield and improved the production of some important metabolites e.g. polyphenolics, flavonoids, lignin and phytoalexins (Emami et al., 2017;Xoca-Orozco et al., 2017). Chitosan and its derivatives induced the activity of polyphenol oxidase (PPO), chitinase, phenylalanine ammonia-lyase (PAL), tyrosine ammonialyase (TAL) and catalase (CAT), superoxide dismutase in orange, tomato, cucumber, table grapes, strawberries, sweet cherries and pears (Romanazzi, 2010;Chang & Kim, 2012;Xing et al., 2015), in addition to sugar and proline concentration (Guan et al., 2009). Furthermore, the level of peroxidase (POD) which is responsible for scavenging of reactive oxygen species in plants was enhanced significantly with chitosan application (Ahmad et al., 2013). The structural barriers in root tissues of tomato plants as defense mechanism were induced with chitosan application (Benhamou et al., 1998).
Funding Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).

Declarations
Disclosure of potential conflicts of interest We have no potential conflicts of interest.
Research involving human participants and / or animals No human participants or animals were involved in this study.

Informed consent Not applicable.
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