Journey of Limonene as an Antimicrobial Agent

Injudicious consumption of antibiotics in the past few decades has arisen the problem of resistance in pathogenic organisms against most antibiotics and antimicrobial agents. Scenarios of treatment failure are becoming more common in hospitals. This situation demands the frequent need for new antimicrobial compounds which may have other mechanisms of action from those which are in current use. Limonene can be utilized as one of the solutions to the problem of antimicrobial resistance. Limonene is a naturally occurring monoterpene with a lemon-like odor, which mainly present in the peels of citrus plants like lemon, orange, grapefruit, etc. The study aimed to enlighten the antimicrobial properties of limonene as per previous literature. Advantageous contributions have been made by various research groups in the study of the antimicrobial properties of limonene. Previous studies have shown that limonene not only inhibits disease-causing pathogenic microbes, however, it also protects various food products from potential contaminants. This review article contains information about the effectiveness of limonene as an antimicrobial agent. Apart from antimicrobial property, some other uses of limonene are also discussed such as its role as fragrance and flavor additive, as in the formation of nonalcoholic beverages, as solvent and cleaner in the petroleum industry, and as a pesticide. Antibacterial, antifungal, antiviral, and anti-biofilm properties of limonene may help it to be used in the future as a potential antimicrobial agent with minimal adverse effects. Some of the recent studies also showed the action of limonene against COVID-19 (Corona virus). However, additional studies are requisite to scrutinize the possible mechanism of antimicrobial action of limonene.


InTRODuCTIOn
Antibiotic resistance is emerging very rapidly because of the inappropriate or misuse of antimicrobials which is facilitated because of their accessibility over the counter, without prescription, and through unregulated delivery chains 1 . Antimicrobial agents also comprise adverse side effects along with microbial resistance. Hence, the interest of researchers is turned towards ethnopharmacology. Bioactive compounds are natural and ubiquitous in most plants and easily accessible to humans. Plant extracts provide immeasurable occasions for novel drug findings due to the exuberance of multifarious chemicals. Hence, thousands of obtainable medicinal phytochemicals are safe and more effective alternatives, for this reason, coupled with advancing microbial resistance to synthetic drugs; ethnopharmacology is swiftly gaining world acknowledgment 2, 3 . In countries like Japan forest bathing trips are very popular and consider natural chemotherapy. Forest bathing trips include a trip to a forest for recreation and relaxation while breathing in volatile substances which are antimicrobial compounds derived from trees 4 . One of such components is limonene, which is one of the main constituents present in aromatic plants and the most common terpene present in nature. It is mainly found to be present in essential oils of peels of citrus spp. like lemon, orange, grapefruit, lime, and mandarin, etc. D-limonene is generally recognized as safe (GRAS) to be utilized as a flavoring agent. Due to its citrus fragrance, it is extensively used as a flavor and fragrance additive in soaps, perfumes, and chewing gums 5 . Limonene is also found to be present in many fruits, vegetables, meats, spices, and other food items 6 . Limonene is also popular for its various properties like antibacterial, antifungal, antiviral, and anti-biofilm. In the present study, the abovementioned properties of limonene discussed and proved experimentally in various studies are included and reviewed. what is limonene?
Limonene is also known by some other synonyms like D-(+)-limonene, (+)-limonene, (R)-limonene, (R)-(+)-limonene, cajeputene, carvene, cinene, (+)-dipentene, etc. The molecular formula of limonene is C 10 H 16 and molecular weight is 136.24. At room temperature limonene is a colorless liquid having lemon-like odor with a melting point temperature of 74.3°C, boiling point temperature of 175.5-176°C, and density of 0.8411g/ml (at 20°C) and 0.8402g/ml (at 25°C). It is slightly soluble in water (13.8mg/L at 25°C) and easily soluble in acetone, dimethyl sulfoxide, ethanol, benzene, carbon tetrachloride, diethyl ether, and petroleum ether. Limonene also gets oxidized when comes in contact with air and forms various oxidation products like carvone, limonene oxide, carveol, and limonene hydroperoxides hence it should be stored away from light and air. It is found to be present in peels of various citrus fruit species like orange, grape, lemon, and can be produced commercially from alkali treatment and steam distillation of citrus peel and pulp which remain after juice and cold-pressed oil production [7][8][9][10] .

uses of Limonene
Limonene is used as a flavor and fragrance additive in perfumes (0.005% and 1%), soaps, beverages, food, and household cleaning products for nearly 50 years. It is also used in the formation of nonalcoholic beverages (31ppm), ice creams (68ppm), candy (49ppm), baked goods (120ppm), sweets, gelatin, puddings (48-400ppm), and chewing gums (2300ppm). In the petroleum industry, it has been used as solvent and cleaner, in the transdermal application of medicines it is used as an additive to increase the penetration of active substance, it is also used as a degreasing agent before the lacquering of industrial products (30%), in the electronic industry for cleaning of printed circuits (50-100%) and for cleaning print cylinders in printing work. Pesticide, insect repellent, and dog/cat repellent are also well-known uses of limonene 6,7,9,10 .

Antimicrobial activities of Limonene Antiviral
Many viral diseases e.g. caused by Herpes simplex virus (HSV), influenza virus, and HIV are known as life-threatening. However various antiviral drugs against HIV, HSV, influenza, and other pathogenic viruses have been developed but the antecedence of these antiviral drugs is the potential and side effects of these drugs. There is still an urgent need for the development of new anti-viral agents which can fulfill all the drawbacks possessed by different antiviral drugs. Limonene can be used as a suitable anti-viral agent as some of the previous studies have emphasized its antiviral properties.
Anti-viral properties of limonene were studied against human pathogenic (Herpes simplex and influenza virus) as well as plant pathogenic viruses (tobacco mosaic virus). The anti-viral property of limonene against the Herpes simplex type 1 virus was found as complete inhibition of HSV1 at 25μg/ml concentration of limonene was observed. Limonene inactivated the virus in the early phase of virus multiplication 11 . Cytotoxic concentration value (CC 50 ) of limonene at 1155μg/ ml against HSV1 was also observed in another study 12 . Inhibition of the herpes simplex virus was observed by the formation of plaque on the kidney Vero cells 13 . On the other hand, the anti-viral property of Citrus deliciosa 14 and Citrus reshni 15 essential oils which contain limonene as the major component was also observed against the H5N1 virus. Anti-viral property of limonene against plant pathogenic virus, Tobacco mosaic virus (TMV) was also observed and it was found that TMV was inhibited by more than 45% after limonene treatment 16 .
Apart from influenza and other viruses limonene also identified as the inhibitor of the SARS corona virus as described in many studies. The SARS corona virus has protein S a specific binding site for the angiotensin-converting enzyme 2 (ACE2) which serves as an entry point into the host cell. Limonene can act as the inhibitor for ACE2 target which is verified by performing docking studies 17 . Mechanism of action of herbal essential oils against viruses is either they coat the glycol proteins (potent of viral sites) of viruses or they bind with the human cell receptors (respiratory cells ACE-2) which lead to the nonspecific and nonproductive binding of the virus particle to host cells which hinder the virus from infection. Some of the herbal essential oils which have antiviral or virucidal properties such as lavender oil, peppermint oil, and eucalyptus oil have limonene as the component 18 . ACE-2 inhibition property of limonene as a receptor for SARS-CoV-2 can prevent the invasion of SARS-CoV-2/COVID-19 into the human body 19,20 . Immunomodulatory, anti-inflammatory, and anti-viral properties of limonene can help limonene to limit the severity and succession of the COVID-19. Hence, limonene can act as a possible candidate against infection, immunity, and inflammation in COVID-19 21 . An oil blend was synthesized for the investigation of the effectiveness of the oil for the treatment in patients having COVID-19 like symptoms. The oil consisted of limonene as one of the chief components which provided anti-viral property to the oil. Limonene is known to restrain exclusively the viral DNA polymerase during the reproduction cycle when new viral DNA is synthesized. Hence, the oil was found as an effective remedial measure for COVID-19 22 . Limonene was also used for the quantitative evaluation of olfactory dysfunction in asymptomatic COVID-19 carriers 23 .

Antibacterial
Antibacterial properties of limonene are well known and discussed in several reports against various bacterial species. Limonene is known to be active against many pathogenic bacteria involved in different diseases such as respiratory or skin diseases whereas some are known as contaminants causing contamination in the food industry. Synergistic activity of the combination of limonene enantiomers (D and R) and with other compounds has also been very well documented in many studies. Some of the studies have also highlighted the evaluation of chemical compositions of various essential oils from different plant materials and observed limonene as a major constituent and the reason for antibacterial activity shown by that essential oil. On the other hand, several methods were also reported in various studies to design nanoemulsions of limonene to be utilized in drug delivery, etc. Antibacterial actions of limonene against various bacterial species in various studies are summed up in Table 1.
Apart from the study on the antibacterial action of limonene, the mechanism of antibacterial action of limonene was also explained in some of the studies. A β-barrel protein (LptD) is an essential protein of the outer membrane present in lipopolysaccharide (LPS) assembly, depletion of this protein leads to the increase in membrane permeability of the bacterial cell. Attenuated total reflectance infrared microspectroscopy results after the treatment of E. coli cells with limonene showed the damage of LPS and altered outer membrane permeability was also observed in the study. It was concluded that the damage of LPS is the mechanism of inactivation by  It is evident by above-mentioned studies that limonene causes damage to the cell membrane of Gram-positive as well as Gramnegative bacterial cells which initiates the leakage of intracellular materials and ultimately leads to cell death. However, the efficacy of limonene or any other antibacterial agent may differ in Grampositive and Gram-negative cells. The cytoplasmic membrane is known as the primary target in Grampositive bacterial cells while in Gram-negative bacteria outer membrane is known as the primary target for antibacterial agents 29 . Gram-negative cells are more efficient than Gram-positive bacterial cells in maintaining their membrane homeostasis, which is why there is a difference between antimicrobial agents towards the bactericidal activity 30 . It was also explained that the outer membrane of Gram-negative bacteria, which is composed of lipopolysaccharide molecules establishs a hydrophilic permeability barrier that protects against the effect of highly hydrophobic drugs 31 . This also explains the reason for the low sensitivity of Gram-negative bacterial cells to the lethal effects of lipophilic monoterpenes like limonene.
Most of the studies mentioned aboveobserved cell membrane damage and alteration in the membrane permeability as the mechanism of action of limonene. However, the precise mechanism of limonene as an antibacterial drug is largely unknown as the series of events which leads to the cell death by the action of limonene are not studied. In a recent study, the effects of limonene on protein expressions related to respiratory chain complex in L. monocytogenes were studied. Limonene treatment to bacterial cells was observed to down-regulate different respiratory chain-related complexes 32 . Moreover, additional studies are required to decipher the exact molecular mechanisms of limonene against various other pathogenic Gram-positive or Gramnegative bacteria.

Antifungal
Limonene is also known for its antifungal properties. It has been observed that limonene is effective against various yeasts and molds which are known as the major contaminant of food products (pudding), dairy (yogurt, cheese, and dairy caramels), fruits (Wine grapes, satsumas, apples, strawberries), grains (wheat flour, corn, peanuts, coffee, cocoa powder, cereal), flavored water and chocolate 88 . Some of the yeasts are known to cause contamination in other food products such as meat, vegetables, nonalcoholic beverages, alcoholic beverages, and bakery products 89 . While some of the fungal species are known to be pathogenic to humans. Antifungal properties of limonene have been explored against various fungal species. Various antifungal properties of limonene are summarized in Table  2.
The antifungal mechanism of action of limonene was also studied against few fungal isolates. Treatment of limonene to yeast cells arise  isolates, as a low fungal burden in mice was observed after the treatment with limonene in comparison to untreated mice. Electron microscopy also revealed that limonene treatment caused dramatic structural changes in fungal cells including cell wall rupture 92 . Limonene is also found to inhibit the intracellular and extracellular enzymes such as cellulase and pectin methyl esterase present in some fungal isolates such as A. niger, P. digitatum, F. oxysporium and R. solani 93 .
All the above-mentioned studies explained a few of the antifungal mechanisms of action of limonene against various fungal isolates. However, exact mechanisms of the antifungal actions of limonene are not yet unrevealed hence; further studies are required to explore the antifungal mechanisms of limonene.

Anti-biofilm production property of Limonene
Resistance in microorganisms against various antimicrobial agents has become the main concern in the medical industry nowadays. The formation of biofilm by microorganisms is one of the mechanisms to acquire resistance against antimicrobial agents. Hence, it is important to find alternative therapeutic agents with anti-biofilm properties. Many plant-based compounds are being examined for their therapeutic properties of which only a few are reported to exhibit antibiofilm activity 119 . Limonene is one the plant-based compounds which possess anti-biofilm property. The anti-biofilm property of limonene is also investigated and proved in various studies by many researchers. The anti-biofilm property of limonene was examined against B. cereus, E. coli, P. anomala and P. putida and significant inhibition of biofilm of B. cereus, E. coli, and P. anomala by limonene was observed 48 . About 75-95% biofilm inhibition was observed against S. pyogenes, S. mutans, and S. mitis at 400μg/ml concentration of limonene 120 . Limonene was also found to reduce the biofilm mass production up to 90% after 8h of incubation at the concentration of 2000μl/L in different strains of S. aureus 121 . In another study, effective biofilm inhibition by limonene against P. aeruginosa, C. albicans, and C. parapsilosis was also observed 55 .
In silico determination of anti-biofilm property of limonene against S. mutans showed that limonene can act as a good candidate for the inhibition of development in biofilm formation 122 .

Concluding Remarks and Future Perspectives
As discussed above, limonene possesses antimicrobial properties such as antibacterial, antifungal, and antiviral. Limonene was also found to hinder biofilm formation and most importantly limonene also shows the inhibitory action against COVID-19. Limonene is suggested to be safe as it is derived from natural sources e.g. citrus plants. Hence, the use of limonene in the future for therapeutic purposes in clinical settings may be considered. Moreover, researches on combinatorial studies of limonene with other drugs are also inevitable to achieve better outcomes in breaking the therapeutic resistance of microbes. Delineation of the mechanistic approach of limonene towards antimicrobial activities is also desirable. Therefore, it may be suggested that there is still a wide lacuna of knowledge to be acquired to venture limonene as a future potential drug molecule.

ACKnOwLEDgmEnTS
The authors gratefully acknowledge the support extended by Sam Higginbottom University of Agriculture, Technology and Sciences, Prayagraj, towards this piece of work.