Estragole: A review of its pharmacology, effect on animal health and performance, toxicology, and market regulatory issues

1. Estragole has several medicinal uses, including antioxidant, anti-inflammatory, antibacterial, and antiviral properties.
2. Estragole's biological actions might be ascribed to its high antioxidant capability and anti-inflammatory activity through boosting cytokine release.
3. There is still debate about the carcinogenic effect of Estragole

Introduction

Essential oils are complex substances with a strong odor and are volatile. Terpenes, oxidizing chemicals, and fatty compounds in essential oils impact their qualities, which are widely utilized in food, perfumes, cosmetics, pesticides, herbicides, traditional remedies, sedatives, diuretics, expectorants, and digestion (1). Estragole is one of the relatively nontoxic terpenoid ethers and has broad applications for aromatherapy and treatment compared to other essential oils (2). In addition, Estragole is a eugenol derivative widely used through chemical synthesis pathways for new alternative drugs (3). Estragole is a monoterpene extracted from natural ingredients such as basil, star anise, anise, and fennel. Additionally, plant environmental conditions, harvest time, storage, and extraction affect the amount of Estragole obtained (4), and Estragole exhibits anesthetic, sedative, antimicrobial, antioxidant, anticonvulsant, and macrophage phagocytic and leukocyte activity (5). Research on Estragole has been done before (6,7). However, in research, it is necessary to distinguish between Estragole as a single substance or in the complex herbal extract form (8). Therefore this study aims to determine the therapeutic properties of Estragole by observing its sources, chemistry, extraction, bioavailability, absorption, distribution, and effect on animal health toxicity and carcinogenicity either singly or extracted from plants.

Sourcesand chemistry of estragole

Estragole is a volatile phenylpropanoid that belongs to the alkylbenzene group. This group includes myristicin, isosafrole, methyl eugenol, anethole, eugenol, safrole, isoeugenol, apiole, and elemicin (9). Estragole compounds could be extracted from plants (Figure 1), especially basil (Ocimum basilicum), ravensara (Ravensara anisata), fennel (Foeniculum vulgare), tarragon (Artemisia dracunculus), Korean mint (Agastache rugosa), Illiciaceae (Illicium anisatum), Canela de cunhã (Croton zehntneri), and Apiaceae (Pimpinella anisum) which are widely processed for the food, beverage and flavoring industries, detergents, soaps, and perfumes (5,10). Estragole is a colorless compound, smells of anise, and is insoluble in water. The chemical class of Estragole is alkylbenzenes, with a molecular weight of 148.20. Its molecular formula is C₁₀H₁₂O, and its Chemical Abstracts Service (CAS) is 140-67-0. Its flash point is 81˚C, and its density is 0.965 g/L, with a boiling point of 215-216˚C. Furthermore, it has many synonyms such as esdragon, esdragol, Estragole, tarragon, isoanethole, p-allylanisole, 4-allylanisole, 1-allyl-4-methoxybenzene, 4-allyl methyl benzene, 1-methoxy-4-prop-2-enyl benzene, 1-methoxy-4-(2-propenyl) benzene, 3-(p-methoxyphenyl)propane, p-methoxy allylbenzene, methyl chavicol, chavicyl methyl ether, o-methyl chavicol, chavicol methyl ether, dan NCI-C60946 (11).

Figure 1: Natural sources of Estragole

In anethole, the double bond of the propenyl side chain is conjugated to the aromatic ring, but in Estragole, it is unconjugated (12). Another difference is that Estragole could form homobenzylate radical cations, while trans-anethole could form conjugated radical cations. In addition, trans-anethole has dimerization by forming an intermediate radical cation, whereas Estragole does not (13-15).

Estragole extraction

Estragole is commonly obtained by extracting plants containing a compound in the form of phenylpropene consisting of anethole, monocyclic monoterpene hydrocarbon limonene, and bicyclic oxygenated monoterpene fenchone (16). Espagnole could be extracted from plants such as fennel by 2 methods, namely Soxhlet or Accelerated Solvent Extraction (ASE), in which the Soxhlet method provides better and more abundant extraction results compared to ASE. The solvents used in the extraction are polar protic solvents such as methanol and ethanol, in which methanol gives a higher extraction yield than ethanol. For example, in fennel extraction, methanol solvent could produce 87-89% estragole, while ethanol produces 86-87% estragole (17).

Estragole may be extracted from Agastache rugosa using Headspace Solid Phase Microextraction (HS-SPME) or gas chromatography-mass spectrometry (GC-MS) methods, which could provide 95 percent estragole extraction (10). Moreover, in basil, the method for extracting Estragole could use Microwave-Assisted Hydro-Distillation (MAHD), which is then improved by Response Surface Methodology (RSM) and analyzed by GC-MS. The yield of estragole extraction could reach 87.869% depending on size, water ratio, time, and wave power used in basil (18). Another technique used to increase the phytochemical and antioxidant activity of Estragole in basil is gamma-radiation (19-21). Commonly, irradiation is used as a treatment technique such as heating, cooling, high pressure, and fumigation (21).

Figure 2: Estragole metabolism and carcinogenesis-related pathways

Estragole bioavailability, absorption, and distribution

When Estragole enters the digestive tract, its metabolism occurs, especially within the liver (22). Estragole's allylic side chain is broken down by mixed-function oxidase into three compounds: estragole 2',3'-oxide, 1'-hydroxyestragole, and 4-methoxycinnamate alcohol. After a spontaneous breakdown, SULTs convert 1'-hydroxyestragole to 1'-sulfooxyestragole by releasing carbocations that can bind to proteins and DNA (Figure 2.). However, it has a negative effect by inducing carcinogenesis (9). Meanwhile, 4-methoxycinnamyl alcohol is oxidized to 4-methoxycinnamic acid and 4-methoxy benzoic acid. In the compound Estragole-2',3'-oxide, it is converted to 2',3'-dihydroxy-4-propylanisole, followed up by epoxide hydrolase, then further oxidized to 4-methoxyphenylacetic acid and 4-methoxyphenyllactic acid (11). In addition, in rodents and humans, there is another estragole metabolic pathway, namely O-Demethylation, in which the O-demethylation pathway shifts to 1'-hydroxylation (23).

Effect of estragole on the animal production

During animal growth, estragole supplementation could increase body weight (24,25). Previous study reported that supplementing Ocimum gratissimum leaf extracts containing Estragole as the main ingredient in broiler chickens increases growth, preserves blood components, and reduces harmful microorganisms in the intestine that could stimulate inflammation for finishing broilers (26). The birds without the extracts had higher microbial loads, indicating that Ocimum had antimicrobial properties. Moreover, experiments El-Naggar and El-Tahawy (27) used broilers treated with basil extract (0.1, 5, and 20 g/Kg body weight) for 39 days, indicating that body weight (BW) increased significantly. Furthermore, supplementation of basil seeds and leaves (0.8% estragole in the herb) might enhance feed intake (FI), feed efficiency, and average daily gains (ADG) (28-30). The increased FRC in herbal-based feeds is due to increased protein digestibility in the small intestine (31). Meanwhile, supplementation with dried basil leaves could increase the relative weight of broiler organs such as the liver, gizzard, and heart (29). In sheep, supplementing a 6% fennel by-product containing Estragole (9.79g/100g) could increase FCR and ADG (30). Lately, in rabbits, the administration of fennel (3.5-12.0% estragole in essential oil) could improve their nutrient digestibility, nutritive values, ADG, FCR, and increase antioxidant enzymr production (31). Estragole also exhibited a notable antistress effect against stress (32). Estragole supplementation in rats of 50 - 200 mg/kg body weight increased urinary VMA and ascorbic acid excretion. VMA and ascorbic acid levels in mice might be used to assess antistress activity. As a result, Estragole may work as an antistress agent (33). However, a study by Riyazi et al. (34) indicated that supplementation with the O. gratissimum in the starter and grower diets (200, 400, and 600 ppm) of broilers had a non-significant influence on the FI, ADG, and FCR. Meanwhile, the experiment conducted by Abbas (30) showed that 3 g/kg bacillus sp. dietary did not affect carcass train and viscera organ. Also, the effects of fennel seeds (F. Vulgare Mill.) (1.5-5.0% estragole in essential oil) supplementation on the production performance of Japanese quail (Coturnix japonica) exhibited no significant differences in initial body weights (35). While according to Abbas et al. (28), Basil essential oil may enhance total protein (4.21 mg/Kg), globulin (2.62 mg/Kg), albumin (4.21 mg/Kg), and reduce total E. coli bacteria (4.07 x 10³) and total fungi (4.46×10³). In summary, Estragole could improve broiler performance by enhancing growth performance through increasing BW, ADG, and FI and providing favorable FCR. However, further studies need to be conducted on other animals to determine the effect of Estragole on animal performance.

Antioxidant effect of estragole

Several studies show that Estragole has antioxidant, anti-inflammation, antibacterial, and antiviral properties that benefit animal health (Figure 3 and Table 1). Junior et al. (36) observed that estragole supplementation positively influenced antioxidant status by preserving GSH and GPx and could reduce MPO and MDA activity, which may minimize lipid peroxidation products and reactive oxygen species, which could also reduce gastrointestinal damage (37,38). Meanwhile, MDA indicates oxidative stress and lipid peroxidation that could accumulate in gastrointestinal inflammation and ulcerated lesions (37-39). Another free-radical builder is MPO which is a marker of neutrophil infiltration. Besides, chloramine is associated with pro-inflammatory responses and oxidative damage (40,41).

The antioxidant activity of fennel extracts in the aqueous-alcoholic mixture is attributed to the presence of Estragole, which may block the peroxidation of the linoleic acid system (42,43). Moreover, fennel also can increase SOD, HDL, and catalase activity and decrease MDA (44). DPPH radical scavenging activity was remarkable in F. vulgare var. Azoricum (12% estragole in essential oil), even greater than ascorbic acid and BHT. Meanwhile, F. Vulgare var. dulce (6% estragole in essential oil)had scavenging efficacy comparable to ascorbic acid and BHT. Furthermore, the cultivar F. Vulgare var. Vulgare exhibits 37 - 44 times the radical scavenging activity of azoricum or dulce cultivars (13).

The source of free radical scavengers in basil (O. basilicum) comes from Estragole which could reach 87.869%. Basil supplementation could improve antioxidant status by increasing GSH, SOD, and CAT and decreasing lipid peroxidation (MDA) (45). Arya et al. (46) discovered that bacillicum might restore the activity of GPx, SOD, and CAT in cardiac tissue after myocardial necrosis while lowering MDA levels. O. basilicum also dramatically restored the GSH content changes caused by global ischemia and reperfusion. (19). But, in extract form, basil significantly decreased MDA, SOD, GPX, and CAT concentration (47).

In rats, gamma irradiation of basil water extract could reduce oxidative stress initiated by arsenic (45). The antioxidant effect of basil in mice could also increase MDA levels in brain tissue and protect brain cells from harmful effects (47,48).

In addition, Kahlo et al. (49) concluded that feeding O. basilicum leaves powder to broilers was found to have good antioxidant and immunostimulant effects against gibberellic acid and auxin through the antioxidant effect of SOD, CAT, GSH, GPx, and GST. In summary, supplementing Estragole as a single substance and from plant extracts could positively affect animal antioxidant capacity by increasing GSH, GPx, SOD, and CAT, also reducing MDA and MPO.

Anti-inflammatory and immunomodulatory effect of estragole

Estragole is a significant chemical component of Ocimum basilicum, increasing anti-inflammatory (50). In an in vitro chemotaxis test, Silva-Comar et al. (5) discovered that Estragole might prevent neutrophil migration toward fMLP. Meanwhile, fMLP is a chemotactic drug that promotes the production of cytokines, including IL-1 𝛽, IL-8, and TNF-𝛼 (51). Moreover, Estragole could decrease the number of attached or rolling leukocytes going to the perivascular tissue in an in vivo microcirculation test. When inflammation occurs, leukocytes will quickly go to the site of the lesion through the postcapillary venules through transmigration, rolling, and adhesion (52,53). However, when leukocytes migrate in a persistent state, it could initiate tissue damage because they could release nitrogen and reactive oxygen metabolites and proteolytic enzymes (54). Furthermore, Silva-Comar et al. (5) demonstrated that Estragole possessed potential anti-inflammatory properties, most likely by blocking leukocyte migration and increasing macrophage phagocytosis.

The previous research by Ponte et al. (55) concluded that estragole supplementation in rats could reduce paw oedema due to the presence of TNF-𝛼. According to Oliveira et al. (56), Estragole has an anti-inflammatory activity that could decrease paw oedema activity. Júnior et al. (36) also showed that levels of pro-inflammatory cytokines could be lowered by estragole supplementation and could also increase IL-10. Estragole could also affect other substances, such as nitric oxide (NO), P, cytokines, bradykinin, serotonin, and histamine (55). Júnior et al. (36) reported that Estragole could also play a role in gastroprotective activity through the NO pathway, which could be inhibited by NOS, thereby reducing gastroprotective activity. Estragole could also decrease the immunostaining activity of iNOS, which indicates that it has immunomodulatory activity. Furthermore, Estragole has been demonstrated to exhibit antinociceptive and anticonvulsant effects in vivo, which have been linked to GABAAR regulation (57,58).

According to Farida and Nahid (59), adding Ocimum basilicum leaves powder to broiler chicken feed improves the innate immune response, as seen by considerable improvements in cells' phagocytic activity of peripheral blood monocyte, lymphoid organs, and lysozyme concentrations. According to ELnaggar and El-Tahawy (60), Adding Ocimum basilicum leaves powder or seed oils to the broiler basal diet boosted the phagocytic index and activity of the macrophages, allowing it to be administered effectively as a natural growth promoter to improve broiler chick development and immunological response. In addition, oral treatment of aqueous and ethanolic leaf extracts of O. basilicum in mice resulted in a considerable rise in the carbon clearance index in mononuclear macrophages, indicating an improvement in phagocytic function and nonspecific immunity (61). Additionally, O. basilicum essential oil has been shown to lower the overall number of leukocytes, monocytes, granulocytes, and lymphocytes (50).

Supplementation of Estragole as a single substance and from plant extraction could positively affect anti-inflammatory and immunomodulatory agents in animals by increasing IL-10, preventing neutrophils, decreasing leukocyte activity, and the immunostaining activity of iNOS. Also, preserve gastroprotective activity through the NO pathway and improve the phagocytic activity of peripheral blood monocyte, lymphoid organs, and lysozyme concentrations.

Antimicrobial and anti-virus activity

Estragole also has antibacterial activity but is relatively minor compared to cinnamaldehyde (13). The antibacterial activity of Estragole compared to cinnamaldehyde was more robust in the liquid culture test but lower in the vapor diffusion test. Estragole was also reported to have the effect of lowering the antibiotic MIC and positively influencing the action of erythromycin RN4220 S. aureus strain (62). Coêlho et al. (63) showed that combined at sub-inhibitory concentrations. Estragole could decrease norfloxacin's MIC, which can induce SA1199-B S. aureus infection by pumping Nora. Accordingly, Estragole does not directly affect bacterial growth but could reduce MIC when associated with erythromycin, which exhibits an antibiotic effect.

Mohamad and Abasali (64) found that O. basilicum extracts in a diet mixture enhanced lysozyme activity in common carp and significantly augmented fish's immune systems to avoid and control microbial diseases. Therefore, E. coli growth could also be reduced by supplementation of basil essential oil (35). Meanwhile, Naksang et al. (65) discovered that O. basilicum leaves powder had high antibacterial action towards foodborne infectious Gram-positive bacteria, specifically Bacillus cereus and Listeria monocytogenes, as well as S. aureus. Therefore, E.coli infection could also be reduced by supplementing basil essential oil and in S. Typhimurium and C. perfringens (66-68).

Moreover, Ivanov et al. (69) showed that the Agastache foeniculum composed of 93.45% estragole produced a MIC of 1.25 L/ml on the growth of S. aureus (ATCC 25923) and other gram-positive bacteria. Estragole, the main volatile ingredient in A. rugosa essential oils, may be responsible for their phytotoxic and antibacterial properties (70).

Figure 3: Biomedical applications of estragole.

Estragole is likewise a significant component of the subspecies Vulgare, which has a practical antibacterial impact on methanogenic bacteria development (17). For example, Shahat et al. (13) showed that oil from F. Vulgare azoricum was more efficient than ampicillin against Gram-negative bacteria, including E.coli and Pseudomonas aeruginosa, by 25% and 7%, respectively. Meanwhile, compared to ampicillin, the essential oil of F. vulgare Vulgare could combat Gram-positive bacteria such as S. aureus and Bacillus subtilis by 58.3 percent and 114 percent, respectively.

The effectiveness of Estragole against virus infection was reported by Farida and Abdulaziz (59). They found that supplementation of O. basilicum seeds in broiler diets significantly increased the serum hemagglutination inhibition antibody titers and eliminated Infectious Bronchitis Viruses (IBV) and Newcastle Disease (ND). Meanwhile, Haiyan (62) showed antiviral activity of F. Vulgare essential oil against parainfluenza virus type-3 (PI-3) RNA and Herpes simplex virus type-1 (HSV-1) DNA. In conclusion, supplementing Estragole as a single substance and from plant extracts may improve antiviral and antimicrobial activity by lowering serum hemagglutination, or MIC, of gram-positive and detrimental bacteria. MIC is the least amount of a chemical, usually a drug, that prevents the growth of bacteria or visible bacteria.

Toxicity and carcinogenicity of estragole

Estragole's carcinogenicity is caused by a genotoxic mechanism (11). Estragole may operate as a genotoxic hepatocarcinogen in rodent liver by producing DNA adducts responsible for Estragole's genotoxicity (71). Genotoxicity occurs due to estragole metabolism in the liver by cytochrome P450, which results in 1'-hydroxy Estragole and certain DNA-reactive epoxides (72). Moreover, due to its subsequent activation to 1′ -sulfoxyestragole by sulfotransferases, 1′ -hydroxyestragole was primarily responsible for the observed genotoxic impact (73). The spontaneous cleavage of sulfate produces an allylic carbocation, rapidly forming covalent connections with proteins and DNA (74).

Estragole epoxides, namely estragole-2',3'-oxide and 1'-hydroxy estragole-2',3'-oxide, have also been demonstrated in vitro to be hepatocarcinogenic and to form DNA adducts (72-75). On the other hand, Estragole epoxide has a modest function in tumorigenicity, and in vivo, estragole administration tests have not discovered the related DNA adduct (76). Meanwhile, epoxide hydrolase enzymes and glutathione S-transferases are more effective and quicker at removing glutathione (77).

When converted to the carcinogenic 10-sulfoxymetabolite, estragol isolated from basil might be carcinogenic and genotoxic (78). Most testing for genotoxicity has yielded negative findings. Martins et al. (79) discovered that Estragole's genotoxicity could impact cell death since Estragole only triggers apoptosis after extended incubation durations (more than 18 hours) and only at the maximum dose evaluated (2000 M).

Estragole's toxic impact and negative geotaxis assays on adult D. melanogaster flies have also been reported to alter mortality, with the flies experiencing damage to their locomotor system and being unable to move in the vertical plane (80,81). Furthermore, the concentration of Estragole in the essential oil of the variety O. basilicum varied from 52 to 100 percent and was poisonous to Rhyzopertha Dominica, Sitophilus oryzae, and Cephalotes pusillus, indicating that Estragole might limit growth (82,83).

On the other hand, Estragole did not generate chromosomal aberrations (CAs) in V79 Chinese hamster cells, whether exogenous biotransformation systems were present or not. Estragole shows no toxicity in mice consuming low doses of food-containing compounds (84,85). Moreover, in the study by Schulte-Hubbert et al. (71), incubation of Estragole for 24 hours did not result in significant cytotoxicity. Various literature shows that Estragole has toxicity and carcinogenicity effects, but further studies need to be done to determine the dose that could initiate toxicity and carcinogenicity.

Market and regulatory issues of estragole

Estragole is an essential oil ingredient and a position isomer of monoterpenes utilized in medicinal formulations, flavorings, and alcoholic drinks (86). Estragole has been certified safe by the United States Food and Drug Administration (FDA-US)(87). Estragole and essential oils from fennel, anise, tarragon, basil, and bay leaves are listed as safe for eating by the US Department of Food and Agriculture (88). Meanwhile, the Flavor and Extract Manufacturers Association (FEMA) Expert Panel maintains that intake of estragole and estragole extract from plants is safe and does not initiate cancer risk based on evidence of nonlinear interactions, covalent bonding, metabolism, and metabolic activity (89).

In 1990, 1994, and 1998 it was reported that the production of Estragole in the US reached one million pounds per year (90). Estragole also plays a role in promoting economic development by increasing the production of chemicals (91). However, the Committee of Experts on Flavouring Substances (CEFS) identified Estragole as a naturally found genotoxic carcinogen in experimental animals following either chronic or a few repeated exposures in 2000 (89). Furthermore, the Scientific Committee on Food recommends limiting the supplementation of Estragole because it is known to have carcinogenic and genotoxic effects (92). Estragole has been widely used for various products for both human and animal needs, but its direct use needs to be taken into account due to the effects of toxicity and carcinogenicity.

Table 1: Effect of Estragole on animal

Conclusion

This review explains natural sources, chemistry, extraction, bioavailability, absorption, distribution, toxicity, and biological effects, also market and regulatory issues of Estragole. Estragole has several medicinal uses, including antioxidant, anti-inflammatory, antibacterial, and antiviral properties. Estragole's biological actions might be ascribed to its high antioxidant capability and anti-inflammatory activity through boosting cytokine release. In addition, there is still debate about the carcinogenic effect of Estragole. Therefore, further detailed research is required to understand the safety of Estragole.

Conflict of interest

There is no conflict of interest in this study.

Acknowledgment

This study was supported by Faculty of Vocational Studies, Airlangga University.