D-Limonene: Promising and Sustainable Natural Bioactive Compound

Abstract

The discovery of antibiotics and pesticides has greatly contributed to the social and economic development of human society but, due to the long-term irrational application, it has led to drug-resistant microorganisms, environmental damage, and other hazards, so the selection of alternative natural, safe, and non-hazardous bioactive substances is an effective solution for this problem. D-limonene is a bioactive compound widely present in various plant essential oils, exhibiting excellent broad-spectrum bioactivity and promising prospects for development and clinical application. This review provides a detailed overview of the biological activities of D-limonene, emphasizing its antimicrobial, anthelmintic, insecticidal, and medicinal potential. While nanoencapsulation technology shows promise in improving the physicochemical properties of D-limonene and enhancing its practical applications, it is also crucial to comprehensively evaluate the potential side effects of D-limonene before use.

1. Introduction

Since their discovery, antibiotics and pesticides have been used to combat microbial contamination and crop production, but due to long-term uncontrolled misuse, they have led to serious hazards, such as the widespread spread of drug-resistant microorganisms, global environmental pollution, and incalculable losses to public health and safety [1,2,3]. Under this critical situation, the concept of green and sustainable development has been accepted by the public so researchers hope to find green methods to replace the long-term use of antibiotics and pesticides, among which, plant essential oils, due to their origin from natural plants, have reliable safety and excellent biological activity. This is one of the research directions that researchers have high hopes [4,5].

Plant essential oils are the main source of aromatic plant odors, which are mainly derived from the roots, stems, leaves, and other organs of plants and have long been used in culinary and medical applications in the historical record [6]. With deeper research, it has been found that plant essential oils have excellent antioxidant activity, antimicrobial activity, and other biological activities [7], which are mainly derived from the constituents of plant essential oils, which are dominated by terpenes and phenyl terpenoids, of which monoterpenoids are the most common [8] and, thus, natural compounds have been incorporated into the study of natural antimicrobial substances. Among the many natural compounds, limonene, due to its widespread presence in the essential oils of various plants, such as Citrus bergamia, Citrus deliciosa, Montanoa quadrangularis, Juniperus oxycedrus, Citrus unshiu, and Bursera graveolens [9,10], and excellent bioactivity has been considered by researchers as a potential sustainable natural active compound [11,12].

Limonene (1-methyl-4-isopropenylcyclohex-1-ene), also known as dipentene, is a natural monocyclic monoterpene that is a colorless liquid with two optical isomers: dextrorotatory D or (+) limonene and levorotatory L or (-) limonene and a racemic mixture (DL-limonene), which have different bioactivities, which may be attributed to the fact that different spatial structures result in different affinities for the active site [13,14]. Limonene in natural plant essential oils is mainly dominated by D-limonene, which is widely found in the peels of citrus and other fruits as a plant biomarker volatile organic compound with a content of up to 80% or more [15], whereas L-limonene is found in Cymbopogan nardus and Cymbopogan citratus, with a content of about 3% [16]; therefore, D-limonene has been extensively studied compared to L-limonene, so this paper will mainly describe the current status of D-limonene research but, in some studies, the authors did not specify the configuration of limonene. It is classified as “generally recognized as safe” (GRAS) by the U.S. Food and Drug Administration and is widely used in the food and cosmetic industries [13]. Additionally, D-limonene also has analgesic, anti-inflammatory [17,18], antioxidant [19], neuroprotective [20], and antimicrobial [21] effects and has hence received extensive attention from researchers.

In recent years, research on the practical applications of D-limonene has continued; however, despite its proven bioactivity, its hydrophobicity, volatility, and sensitivity to heat, light, oxygen, and moisture are affected by environmental factors (e.g., oxygen, light, and temperature) when applied directly to food systems and its oxidation gives off unpleasant odors, which, together with its own strong aromatic odor, can interfere with the sensory properties of the food to which they are added [22], which limits its application in food [23,24]. Currently, researchers are improving the physicochemical properties and bioactivity of D-limonene by producing new materials, such as nanoemulsions and nanofilms, through encapsulation and other techniques, and applying them in practical applications [25]. For instance, nanoemulsions of sodium caseinate and D-limonene in specific ratios were generated using high-pressure homogenization, which significantly improved preservation and thermal stability. The original particle size was maintained even after storage at 4 °C for 60 days and heating at 90 °C for one hour and the antibacterial activity was also significantly enhanced, with the minimum inhibition concentrations (MICs) reduced from 12.5, 7.8, and 7.8 μL/mL to 3.13, 3.13, and 1.56 μL/mL for E. coli, Bacillus subtilis, and S. aureus, respectively [23]; Shao et al. [26] homogenized Ulva fasciata polysaccharide with 0.15% w/w D-limonene into nanoemulsions and applied these to strawberries and they found that, although the elasticity of strawberries in the experimental group was slightly reduced compared to the control group, there were significant advantages in terms of color, morphology, texture, and weight loss.

Although studies have described the antibacterial and antifungal activity of D-limonene, detailed analyses of its mechanism of action are lacking and there is still a lack of research on other bioactivities, such as anthelmintic activity and pharmacological activity, possible modes and mechanisms of action, and enhancement of its biological activity by techniques such as nanoencapsulation. Therefore, this paper focuses on the antibacterial and antifungal mechanism of action of D-limonene; describes the anthelmintic activity, insecticidal activity, pharmacological activity, and antiviral activity of D-limonene; and discusses the current application of technologies, such as nanoencapsulation, for enhancement of the bioactivity and improvement of the physical properties of D-limonene.

In this review, we mainly searched for articles in English from databases of Web of Science (https://www.webofscience.com/wos/, accessed on 15 May 2024), NCBI (https://pubmed.ncbi.nlm.nih.gov/, accessed on 21 March 2024), and X-mol (https://www.x-mol.com/, accessed on 15 May 2024) and considered the most recent research. The keyword “Limonene” was used alone or in combination with, for example, “production”, “essential oil”, “antimicrobial activity”, “antibacterial activity”, “antifungal activity”, “antibiofilm activity”, “anthelmintic activity”, “insecticidal activity”, “antioxidant”, “anti-inflammatory” “antiviral activity”, and “application”.

2. Production of D-Limonene

Natural plant extraction and biotransformation are the main methods for D-limonene production [27,28,29].

2.1. Natural Plant Extraction: Adding Value to Waste

The fruit and vegetable industry produces a large amount of peel waste every year, causing great economic waste and environmental burden [30]. The extraction of high-value chemical substances from fruit peels can not only effectively alleviate this problem, but can also achieve sustainable development and promote economic cycles [31]. D-limonene is widely present in essential oils extracted from the peels and leaves of natural plants and its content depends on the plant species and cultivar [32,33,34,35]; notably, D-limonene is present in large amounts (up to 98.54%) in citrus (

Table 1. Source and composition of D-limonene.

Plant (Essential Oil)	Parts	Composition (%)	Ref.
Citrus × sinensis	Fruit peels	98.54	[45]
Citrus × aurantium	Fruit peels	93.70	[46]
Citrus reticulata	Fruit peels	91.65	[45]
Citrus deliciosa	Fruit peels	91.27	[45]
Tetradium daniellii	Leaves, Fruits	72.71	[47]
Hyptis Jacq	Leaves, Fine stems	72.60	[48]
Cymbopogon citratus	-	71.00	[49]
Citrus unshiu	Fruit peels	70.22	[50]
Citrus maxima	Fruit peels	67.58	[51]
Citrus × limon	Dry leaves, Fruit peels	62.97	[52]
Citrus × latifolia	Fruit peels	54.71	[53]
Bursera graveolens	Fruits	43.60	[54]
Citrus medica	Leaves, Fruits	39.77	[47]
Psidium guajava	Leaves	38.01	[55]
Citrus bergamia	-	21.47	[56]
Zanthoxylum schinifolium	-	21.24	[57]
Litsea cubeba	Fruits	14.30	[58]
Zanthoxylum armatum	Leaves, Fruits	10.70	[47]

). Natural plant extraction of D-limonene can be performed using organic solvent extraction, new bio-based solvent extraction, and natural deep eutectic solvent extraction [36,37,38]. These methods are safer than chemical pyrolysis, but the yield is affected by differences in the extraction methods, solvent dosages, and even the plant raw materials, which can be subject to seasonal, climatic, and geographical factors [36,39,40,41,42,43,44]. Therefore, the production of D-limonene via efficient bioconversion is an attractive strategy [29].

2.2. Biotransformation: A New Direction for Future Development

Biotransformation to high-value natural products is a direction to explore as an alternative to extraction from natural plants, usually using metabolic engineering and biosynthetic engineering to process microorganisms for sustainable production [59]. For example, using the methylerythritol 4-phosphate pathway or the mevalonate-dependent pathway [60], the introduction of a specific gene fragment into the test bacterium and induced expression for the synthesis of D-limonene resulted in D-limonene production up to 0.15 g/(L·h) under ideal conditions [61]. The yield of D-limonene obtained by metabolic engineering depends on the strain, the type of exogenous gene introduced, the incubation temperature, and other factors [29,62,63]. In addition to the methylerythritol 4-phosphate pathway and mevalonate-dependent pathways, the isopentenol utilization pathway and the isoprenoid alcohol pathway have also been used to produce D-limonene, with reported maximum yields of approximately 0.7 μg/(L·h) and 10.42 μg/(L·h), respectively [59,64,65,66]. Alternatively, the alcohol-dependent hemiterpene pathway has been suggested as a promising pathway for terpene production; however, its use for D-limonene production has not been reported to date [63]. Biotransformation as an alternative method to extract D-limonene from natural plants has the advantages of high concentration and yield and has great potential for expanding production. However, D-limonene has a certain degree of cytotoxicity, which greatly reduces the adaptability of engineered cells, and the specificity and inefficiency of the biosynthetic enzyme pathway in heterologous hosts hinder the large-scale production of D-limonene by microorganisms. In addition, because of public health, environmental protection, and other considerations, some regions, such as the European Union, have set the most stringent scrutiny standards for the application of genetically modified organisms, which has limited the promotion of genetically modified microorganisms, so plant essential oil extraction is still the main method to produce D-limonene [59,67,68].

3. Antimicrobial Activities of D-Limonene

3.1. Antibacterial and Antifungal Activity of D-Limonene

D-limonene is a monocyclic monoterpene with excellent antimicrobial properties and has received considerable attention from researchers. Numerous studies have reported the inhibitory activity and mechanisms of action of D-limonene against bacteria and fungi. The MICs and minimum bactericidal/fungicidal concentrations (MBCs/MFCs) of 11 bacteria and 14 fungi are listed in

Table 2. The antimicrobial activity of D-limonene.

Categories	Species	Strain	CFU/mL	MIC	MBC/MFC	Ref.
Bacteria	Aeromonas hydrophila	MF 372510	1 × 105	6.4 mg/mL	6.4 mg/mL	[21]
	Escherichia coli	ATTC 25922	1 × 106	16 μL/mL	32 μL/mL	[70]
			1 × 106	10 mg/mL	40 mg/mL	[71]
		ATCC 8739	1 × 108	1 μg/mL	-	[72]
			1 × 108	12.5 μL/mL	-	[23]
		CIP 54127	1 × 106	12.5 mg/mL	-	[73]
		O157:H7	-	50 μL/mL	-	[74]
	Staphylococcus aureus	CIP 4.83	1 × 106	12.5 mg/mL	-	[73]
		ATCC 6538	1 × 108	1 μg/mL	-	[72]
			1 × 108	7.81 μg/mL	-	[23]
		ATCC 43300	5 × 105	3 mg/mL	8 mg/mL	[75]
		ATCC 25923	5 × 105	3 mg/mL	3 mg/mL	[75]
			5 × 105	10 mg/mL	-	[76]
		ST30-t019	5 × 105	15 mg/mL	-	[76]
		ST5-t311	5 × 105	20 mg/mL	-	[76]
		-	5 × 105	7.9 mg/mL	12.9 mg/mL	[75]
	Mycobacterium tuberculosis	H37Ra	-	64 μg/mL	-	[77]
		ATCC 25177	1.5 × 108	32 μg/mL	-	[78]
	Enterococcus faecalis	-	1 × 106	12.5 mg/mL	-	[73]
	Listeria monocytogenes	ATCC 35152	1 × 106	12.5 mg/mL	-	[73]
		FSCC 178006	106–107	20 μL/mL		[79]
	Streptococcus uberis	-	1 × 106	3.3 mg/mL	210 mg/mL	[80]
	Streptococcus mutans	UA 159	1 × 108	21 mg/mL	-	[81]
	Lactobacillus acidophilus	-	1 × 106	40 mg/mL	80 mg/mL	[71]
	Salmonella	-	1 × 106	1.25 mg/mL	40 mg/mL	[71]
	Bacillus subtilis	ATCC 6633	1 × 108	7.81 μg/mL	-	[23]
			1 × 108	1 μg/mL	-	[72]
Fungi	Saccharomyces cerevisiae	ATCC 9763	1 × 108	0.5 μg/mL	-	[72]
	Fusarium sporotrichioides	ITEM 692	-	10 μL/mL	-	[82]
	Fusarium langsethiae	ITEM 11020	-	5 μL/mL	-	[82]
	Fusarium graminearum	ITEM 6477	-	5 μL/mL	-	[82]
	Candida albicans	ATCC 90028	-	0.31 mg/mL	0.62 mg/mL	[83]
			1 × 107	300 μg/mL	400 μg/mL	[84]
	Candida glabrata	ATCC 90030	-	0.31 mg/mL	1.25 mg/mL	[83]
	Candida parapsilosis	ATCC 27853	-	0.31 mg/mL	1.25 mg/mL	[83]
		URM 6404	-	256 μg/mL	-	[85]
		HAM 26	-	512 μg/mL	-	[85]
	Candida krusei	ATCC 6258	-	0.07 mg/mL	0.62 mg/mL	[83]
	Candida tropicalis	SH 1	1 × 107	20 μL/mL	40 μL/mL	[86]
	Bacillus cereus	ATCC 33018	1 × 107	2.5 mg/mL	>40 mg/mL	[87]
	Phytophthora capsici	LT 263	5 × 104	20 mg/L	-	[88]
	Trichophyton rubrum	KCTC 6345	1 × 105	5 μL/mL	-	[89]
	Trichophyton rubrum	MTCC 296	-	2 μL/mL	6 μL/mL	[90]
	Sclerotinia sclerotiorum	BRM 29673	-	200 μL/mL	-	[45]

(colony forming unit, CFU), indicating that D-limonene has a relatively broad antimicrobial spectrum, and it can be found that the antimicrobial activity of D-limonene is correlated with microbial species, number of viable microorganisms and strains, and D-limonene concentration. In addition, a study found that D-limonene reduced the D-value (Decimal reduction Times) of L. monocytogenes in carrot juice, but lipids and fibers led to significant changes in the D-value, suggesting that the food matrix interfered with the antimicrobial activity of D-limonene, but the mechanism is still unclear [69]. Other than the antimicrobial activity studies listed in Table 2, the inhibitory effect of D-limonene on multiple drug resistance (MDR) strains and its synergistic effect with antimicrobial drugs are also important.

3.2. Inhibition of MDR Strains by D-Limonene

Trials have revealed the potential of D-limonene to resist MDR bacterial and fungal contamination and exhibited synergistic effects with antimicrobials. For clinically isolated MDR Mycobacterium tuberculosis strains T1 1558, H1 47, and Beijing 1, D-limonene at concentrations of 128, 128, and 256 μg/mL, respectively, inhibited their growth; potentiated the inhibitory effects of antimicrobial drugs ethambutol, rifampicin, and isoniazid; and reduced the MICs by two-fold at subinhibitory concentrations and by eight-fold for the non-resistant strains [78]. In another study, D-limonene was found to have a synergistic effect with gentamicin, reducing the MIC values of resistant S. aureus and E. coli from 13.71 μg/mL and 30 μg/mL to 4 μg/mL and 20.1 μg/mL, respectively [91]; for MDR fungi, such as Candida tropicalis, Candida parapsilosis, Candida metapsilosis, Candida krusei, Candida lusitaniae, D-limonene in the concentration range of 16–64 μg/mL can inhibit their growth [92]; and D-limonene acts synergistically with fluconazole to resensitize fluconazole-resistant strains of C. albicans and inhibit their growth and biofilm formation [93]. This may be due to the increased permeability of the antimicrobial agent by D-limonene or to D-limonene’s lipophilic properties, which allow D-limonene to cross the cell wall and alter the permeability of the cell membrane [75,94]; another view also suggests that this may be due to the different targets of natural antimicrobial compounds and clinical drugs [95].

In addition, researchers have suggested that the microbial efflux pump (EP) is associated with microbial drug resistance and allows the excretion of antimicrobial substances, thereby reducing their intracellular concentrations [96,97]; one study found that D-limonene could act as an inhibitor of the efflux pump (EP) and synergistically with ciprofloxacin to inhibit drug-resistant S. aureus, reducing the MIC from 32 μg/mL to 3.17 μg/mL, better than carbonyl cyanide m-chlorophenylhydrazone from 32 μg/mL to 10.07 μg/mL, and molecular docking showed that this could be due to competition or non-competitive inhibition caused by D-limonene [98]. Several experiments have shown that D-limonene and antimicrobials have different synergistic effects against gram-positive and gram-negative bacteria, which may be due to the difference in cell wall structure between the two bacteria [99].

Although D-limonene is considered to be a potential substance against MDR strains, unfortunately, D-limonene was found to have no synergistic effect with penicillin in experiments, whereas D-limonene had an antagonistic effect with norfloxacin and elevated MIC value against E. coli MDR strains in the experiments, which may be due to chelation resulting in reduced antimicrobial effect [91,94,100].

3.3. Antibiofilm Activity of D-Limonene

It has been widely reported that biofilms formed by fungi or bacteria are found on the surfaces of foods, medical equipment, soil, and other substrates, thus posing serious threats to public safety [101,102]. Biofilms are large numbers of microorganisms aggregated together, attached to the surface of organisms (e.g., meat tissue) or abiotic (e.g., processing equipment) [103] and enclosed in an extracellular matrix [104] (Figure 1). The biofilm matrix is generally composed of water and extracellular polymeric substances (EPSs), mainly composed of polysaccharides, proteins, nucleic acids, lipids, dead microbial cells [105,106]), flagella, and other adhesive fibers [103], thereby enhancing their resistance to drying, liquid flow, antimicrobial agents, disinfectants, and other methods [106]. Biofilm formation is an adaptation strategy of microorganisms to their environment and it also increases their virulence, resulting in greater pathogenicity [107]. Statistics have shown that more than 80% of recurrent microbial diseases and chronic infections are associated with biofilm formation [108]. Therefore, it is important to investigate biofilm inhibition and removal.

Table 3. The antibiofilm activity of D-limonene.

Species	Strain	CFU/mL	MBIC	Inhibition Rate	Ref.
Aeromonas hydrophila	MF 372510	1 × 105	51.2 mg/mL	-	[21]
Escherichia coli	O157:H7	-	25 μL/mL	92%	[74]
Streptococcus pyogenes	SF 370	2 × 103	400 μg/mL	83%	[109]
	St 38	2 × 103	400 μg/mL	95%	[109]
Streptococcus uberis	-	1 × 106	3.3 mg/mL	88.25%	[80]
Streptococcus mutans	UA 159	1 × 108	10.5 mg/mL	94.88%	[81]
Candida albicans	ATCC 90028	1 × 107	300 μg/mL	87%	[84]

lists some of the reports on the D-limonene inhibition of microbial biofilm formation, indicating that the inhibitory activity on biofilm is related to the microbial species, number of viable microorganisms and strains, and D-limonene concentration. The biofilm inhibition of Streptococcus mutans has been reported to be 94.88% and 46.62% at D-limonene concentrations of 10.5 mg/mL and 2.625 mg/mL [81]. However, for Streptococcus uberis, the inhibition rate was 88.25% at a concentration of 3.3 mg/mL, thus indicating that the biofilm inhibitory activity of D-limonene on different microorganisms is variable [80].

4. Antimicrobial Mechanism of D-Limonene

4.1. Antibacterial and Antifungal Mechanism of D-Limonene

4.1.1. Damage to Cell Membranes and Cell Walls

The most intuitive and obvious effect of D-limonene on microorganisms is the disruption of cell membranes and cell walls (Figure 2). In several experiments, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) showed that the overall shape, cytoplasmic homogeneity, and cell wall thickness of bacterial and fungal cells were significantly changed after D-limonene treatment. In addition, it includes increased cell membrane permeability, altered membrane potential, and decreased heat resistance; leakage of intracellular substances, such as proteins, lipids, and nucleic acids, was also observed and even cell lysis at high concentrations, which may be due to the lipophilic and hydrophobic properties of D-limonene that promote lipid solubilization in the plasma membrane [70,77,79,86,110,111,112,113,114]. However, unlike bacteria, D-limonene affects ergosterol and β-1,3-glucan, which are unique to fungi. Ergosterol in fungal cell membranes is responsible for maintaining cell function and integrity and a decrease in its level can lead to cell death [115]; in one study, D-limonene without cosolvent had no effect on the ergosterol content in yeast cell membranes [116] but, in another study, D-limonene-Tween 80 cosolvent could reduce the ergosterol content in the Saccharomyces cerevisiae cell membrane, which may be due to the fact that Tween 80 improved D-limonene permeability [117]. Interestingly, 107 mg/L D-limonene ceased the growth of yeast but D-limonene-Tween 80 cosolvent did not cause a growth disturbance for reasons that are not clear [116]. In addition, preventing biofilm formation by inhibiting the quorum sensing (QS) system and EP system is also one of the properties of D-limonene in inhibiting microbial growth, which will be described in the biofilm section.

4.1.2. Effects on Lipids, Proteins, and Nucleic Acids

Studies at the subcellular level, including lipids, proteins, and nucleic acids, similarly revealed the antimicrobial activity of D-limonene. A study shows that D-limonene prevents lipid molecules from being tightly packed and alters the properties of the condensed phase, resulting in smaller and fewer structural domains, which causes membrane fluidization in a bacterial model [111] and, in another, the normal modification of nascent proteins by the endoplasmic reticulum of C. albicans was affected by 20 μL/mL of D-limonene treatment [118]. Notably, SDS-PAGE results showed a reduction in one plasmid band in D-limonene-treated E. coli compared to the control group, presumably due to a change in the E. coli DNA helix conformation, which may affect the stability and subsequent degradation susceptibility of E. coli [70].

4.1.3. Disturbance of Energy Metabolism

Moreover, modulations of energy metabolism pathways are also ways for D-limonene to inhibit microorganism growth. According to transcriptomics studies, D-limonene at a MIC of 20 μL/mL interfered with the transcription of key enzyme genes of the glycolysis pathway, tricarboxylic acid cycle, and oxidative phosphorylation pathway in C. tropicalis [118] and, in another study, the ATP concentration of L. monocytogenes decreased significantly after exposure to D-limonene, the activity of the respiratory chain I-V complex decreased, and the protein units were downregulated, which indicated that D-limonene could inhibit the synthesis of the respiratory chain complex proteins in L. monocytogenes, interfering with its respiratory function and energy metabolism, which led to the death of the cells [79]. D-limonene can also interfere with the synthesis of cellular ATP by inhibiting the respiratory complex and the ATPase in C. albicans, thereby affecting the respiratory intensity and energy metabolism [114].

4.1.4. Interference with Gene Expression

As the research got deeper, the mechanism of action of D-limonene at the gene level was revealed. After D-limonene treatment, the expression of dpeE1, a gene downstream of the cell wall synthesis, and clgR, a gene that protects cell membrane integrity in M. tuberculosis, thereby disrupted cell integrity, as well as Streptococcus pyogenes virulence-associated genes covR and sepB by 53% and 16%, respectively, and covS and mga being downregulated by 26% and 57%, respectively, with no significant difference in the expression levels of srv and luxS, indicating that D-limonene reduced the surface-mediated virulence factors of S. pyogenes [109,119]. And for fungi, it was shown that D-limonene induced disruption of the specific cell differentiation program of C. parapsilosis, leading to apoptosis, and also arrested the cell cycle of C. albicans in the G1 phase and the abundancies of 52 proteins were significantly changed (≥two-fold), 33 of which were upregulated and 19 downregulated. Furthermore, qPCR demonstrated that C. albicans cell wall and cell membrane damage stress genes (KRE 9, ERG 11), oxidative stress genes (TRR 1), nucleolus stress genes (PRL 11), and apoptosis-related genes (CaMCA 1) were overexpressed, indicating that D-limonene can induce apoptosis in C. albicans cells through multiple pathways [85,93].

4.2. Antibiofilm Mechanism of D-Limonene

4.2.1. The Effects of D-Limonene on EPS Secretion and Gene Regulation

Curli is recognized as a key factor in biofilm formation and D-limonene concentrations at 12.5 μL/mL and 25 μL/mL inhibited E. coli biofilm formation by 55% and 92%, respectively, EPS production was reduced by 35% and 60%, and no curli were observed after 25 μL/mL D-limonene treatment, which is probably due to inhibition of the transcription of E. coli curli production genes (csgA, csgB, csgC, and csgD) [74]. The MIC of D-limonene against C. albicans was reported to be 300 μg/mL, at which concentration the secretion of proteinases and phospholipases, adhesion ability, and biofilm formation were significantly reduced by 73%, 53%, 91%, and 87%, respectively, and, furthermore, a good docking score was obtained in the molecular docking analysis with virulence-associated proteins (Plb1 and Tec1), indicating that D-limonene may inhibit the adhesion ability, biofilm-forming ability, and morphological transition of Candida by associating with key Candida pathogenicity proteins [84].

4.2.2. The Inhibition of D-Limonene on Quorum Sensing and Efflux Pump Systems

The quorum sensing and efflux pump systems are important for biofilm formation and virulence expression [120,121]. The QS system is a process of microbial communication through small, diffuse chemical signaling molecules (i.e., QS molecules), which can be involved in the coordination of certain behaviors, such as biofilm formation, virulence, and antibiotic resistance [122,123], and the EP system is responsible for secreting toxins, proteins, and polysaccharides formed by the cell, as well as the efflux of toxic compounds found in the microbial context, such as antibiotics [124]; D-limonene has an inhibitory effect on both systems (Figure 3).

Autoinducer-2 (AI-2) is a QS signaling molecule commonly shared by gram-positive and gram-negative bacteria that is synthesized by the enzyme LuxS [125] and D-limonene may inhibit E. coli QS by interfering with AI-2 communication; the expression of AI-2-related genes (lsrA, lsrB, lsrC, and lsrF) was decreased [74]. Similarly, Luciardi et al. [126] also reported the inhibitory effect of D-limonene in the production of an autoinducer (AI) in P. aeruginosa, whereby D-limonene at 0.8–4 mg/mL reduced the AI production by 17–30%. Pseudomonas psychrophila is one of the main causes of the spoilage of frozen foods [127] and another study treated P. aeruginosa with 135, 65, and 35 μL/mL of D-limonene and showed that bacterial biofilm formation was inhibited and that proteolytic activity and exopolysaccharide synthesis were reduced by 35%, 29%, and 28%, and 58%, 32%, and 41%, respectively. Additionally, the synthesis of QS autoinducers (AHL and alkyl quinolone molecules) was also reduced below detectable levels, indicating that D-limonene effectively inhibited the normal process of QS, and it was found that D-limonene binds to QS receptor proteins (LasR, RhlR, TraR, and PqsR), which may result in inhibition of the normal functioning of the QS system through competitive or non-competitive binding [128]. Inhibition of the EP system significantly inhibited microbial biofilm formation, indicating that the EP system is important for microbial biofilm formation [129]. In the study, it was found that D-limonene suppresses the expression of the EP system-related genes MrsA and TetK of S. aureus [130], D-limonene also binds to EP proteins (MuxB, MexB, and Mfs) on the P. psychrophila cell membrane, and it inhibits the expression of the EP system transcriptional regulatory genes MarR and TetR [128].

5. Anthelmintic Activity and Insecticidal Activity of D-Limonene

Pesticides have caused extremely serious harm to the environment due to the unregulated use of pesticides in agricultural production over a long period of time and, with deeper research, it has also been found that pesticides not only contaminate the local soil but also spread to the world with the atmospheric cycle and enter the human body along with the food chain, which threatens public safety in many ways. In order to cope with this serious problem, researchers are searching for safe degradation of pesticides and safe and reliable alternatives to pesticides [131,132,133]. In addition to its antibacterial and antifungal properties, D-limonene has anthelmintic and insecticidal activities [134,135] and D-limonene has been the most used botanical insecticide in California in the past decade (averaging >20,000 kg per year) [136]. Research has shown that D-limonene is cytotoxic to some harmful insects and can have a repellent and toxic effect [137,138].

5.1. Anthelmintic and Insecticidal Activity

It is reported that low concentrations of D-limonene can kill Aonidiella aurantii [139], the LC₅₀ for adult Sitophilus oryzae was 36.85 μL/L [140], and the percentage of death in Rhipicephalus sanguineus treated with D-limonene at 0.1 μL/L was 82.6% [141]. The use of oil-in-water nanoemulsions made of D-limonene (8.9% w/w), water (30.2% w/w), Tween 20 (36.9% w/w), propylene glycol (15.1% w/w), and ethanol (8.9% w/w) were highly toxic to larvae of Culex pipiens molestus and Aedes albopictus and the toxicity effect was stronger than that of the unencapsulated D-limonene solution; this is probably due to the increased surface to volume ratio of nanoencapsulated D-limonene, which is beneficial to penetration, making it easier to pass through the insect’s exoskeleton and protect the active substances within it from inactivation and degradation. After treatment, the larvae have a fragile appearance, with a wrinkled body surface and changes to the head, thorax, siphon, and abdominal cuticle, indicating potential for application in integrated pest control [142], and for Aedes aegypti and Aedes albopictus eggs, D-limonene causes morphological damage to the outer villous cuticle and blocks stomata, leading to respiratory distress [143]. Of note, D-limonene exhibited low toxicity to the natural enemies of this mosquito, Poecilia latipinna and Poecilia reticulata, suggesting that it has potential for biological control.

Showler et al. [144] showed that D-limonene directly affected the development of the adults, larvae, and pupae of Haematobia irritans irritans and that D-limonene at a concentration of 5.8% rendered H. irritans irritans immobile and significantly interfered with the fecundity and egg hatchability of the insect, but the mechanism was not clear. Interestingly, D-limonene was present in salivary gland extracts of Ceratitis capitata [145] and D-limonene had an attractive effect on H. irritans irritans at concentrations below 0.1%, indicating that D-limonene has potential application in trapping these insects [144]. In addition, D-limonene not only inhibited the fecundity activity of Bactrocera dorsalis but also showed a significant toxic effect on adults and pupae and exhibited a dose-dependent killing effect on larvae [146]. However, Papanastasiou et al. [147] found that D-limonene exhibited acute toxicity to the insect at high concentrations but promoted its survival and reproduction at low concentrations, probably due to the hormetic-like effect of D-limonene.

5.2. Antiparastic Activity

D-limonene has also been used to treat parasitic infections [135]. Researchers have discovered that D-limonene exhibits cytotoxicity against parasites, such as Leishmania, causing increased plasma membrane fluidity and cell lysis, thus killing the parasite and acting as a treatment for leishmaniasis caused by the parasite [148,149]. In addition, D-limonene reduces the isoprenylation of Ras- and Rap-related proteins, thereby causing developmental arrest in Plasmodium falciparum [150], possibly due to the inhibition of dolichol and ubiquinone synthesis by D-limonene [151]. In a study, D-limonene was found to have an LC₅₀ of 245 μL/mL against Haemonchus contortus and exhibited extremely strong repellent activity (97.5%), probably because D-limonene has acetylcholinesterase inhibitory activity [152], and Moreno et al. [153] found that D-limonene was cytotoxic to Trypanosoma cruzi, causing altered cell morphology, impaired membrane potential, reduced cytoplasmic volume, absence of flagella, phosphatidylserine externalization, nuclear chromosome condensation, and DNA fragmentation, and it induced apoptosis by inhibiting the PIP3/Akt pathway, oxidative stress stimulation, and caspase activation.

6. Pharmacological Activity of D-Limonene: A Potential Natural Medicine

6.1. Antioxidant Activity

D-limonene is a potential antioxidant natural active substance that has been applied to counteract endoplasmic reticulum stress and reactive oxygen species release caused by methylglyoxal, etc. [154,155]. The ferric-reducing antioxidant power test showed that C. sinensis essential oil, which is rich in D-limonene (88.9%), has excellent antioxidant activity [156]. By in vitro assay, D-limonene at 16–64 mg/mL had significant ABTS radical scavenging ability, but the DPPH scavenging activity was weak [71]. In addition, in the same vitro assay, D-limonene inhibited low reactive oxygen species (ROS) production and accumulation induced by Aβ_1-42 oligomers [157], as well as inhibiting the activity of NADPH oxidase subunits and increasing the levels of antioxidant enzymes superoxide dismutase and heme oxygenase-1 [158]. In vivo tests of rats by AlSaffar et al. [159] showed that D-limonene prevented lipid oxidation and reduced catalase, superoxide dismutase, and glutathione peroxidase activities induced by carbon tetrachloride.

6.2. Anti-Inflammatory Activity

A number of studies have reported that D-limonene exerts anti-inflammatory and analgesic effects [160,161] because it blocks the release of inflammatory mediators, inhibits vascular permeability, and reduces neutrophil migration [20]. Huang et al. [162] found that D-limonene significantly inhibited formalin-injection-induced paw swelling and oxidative-stress-induced pain in mice and it inhibited RAW265.7 cell migration, suggesting that D-limonene has potential anti-inflammatory activity. In animal trials, D-limonene was shown to exert anti-inflammatory effects by reducing the production of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and myeloperoxidase, thereby increasing the concentration of antioxidant substances, such as glutathione peroxidase and superoxide dismutase, and maintaining the integrity of the inflammation site [17,163]. In addition, D-limonene can inhibit inflammation induced by exogenous substances [158], such as inhibition of the increased activity of inflammatory markers triggered by carbon tetrachloride, including high-sensitivity corticotropin-releasing factor, IL-6, and TNF-α [159].

Moreover, D-limonene is cardioprotective and gastroprotective [20,164], in addition to inhibiting the activity of cardiotoxicity biomarkers, such as alanine aminotransferase, lactate dehydrogenase, creatine kinase, creatine kinase MB, and the MAPK/NF-κB pathway; to reduce the damage of myocardial infarction, it also prevents cardiac histopathological changes [159,165]. Furthermore, oral D-limonene can increase local gastric mucosal defense mechanisms, such as mucus secretion, regulation of oxidative stress and inflammatory responses, and inhibition of the NF-κB pathway, to exert gastroprotective effects [17]. Aside from inhibiting oxazolone-induced colitis and histopathological changes, it also reduces pain caused by oxidative stress and prevents gastric ulcers caused by ethanol [166].

It has been demonstrated that D-limonene plays an active role in inhibiting the spread of cancer cells, acting on molecules such as Bcl-2, Bax, and caspases involved in various pathways, such as apoptosis pathways, PI3K/Akt signaling, autophagy, and vascular endothelial growth factor activity against cancer [167]. In addition, D-limonene has the ability to inhibit cancer cell growth; its combination with docetaxel induced more ROS production, thereby significantly enhancing the ability of docetaxel to induce apoptosis in cancer cells [168]; and D-limonene can induce apoptosis in cancer cells by inducing the mitochondrial death pathway and by inhibiting the PI3K/Akt pathway [169]. Other researchers have experimentally demonstrated that D-limonene has antifibrosis activity [170], prevents ischemia-induced brain injury, and alleviates acute kidney injury owing to its antioxidant and anti-inflammatory activities [171,172].

6.3. Neuroprotective Activity

Neuroprotective effects are other non-negligible biological activities of D-limonene, which can be used against neurodegenerative diseases [20]. D-limonene has been reported to inhibit acetylcholinesterase activity and protect neurons from cellular damage by blocking the decrease in mitochondrial dehydrogenase activity, ROS production, and KV3.4 channel hyperfunction triggered by Aβ_1-42 oligomers, thereby delaying the development of Alzheimer’s disease [157]. Tang et al. [158] showed that corticosterone triggers oxidative stress and inflammatory responses, ultimately leading to apoptosis and neural cell damage, but D-limonene was able to reduce the damage caused by corticosterone by activating the AMPKα signaling pathway. In addition, D-limonene reduced hyperalgesia and astrocytosis and improved the neuronal regeneration and recovery of sensorimotor function in peripheral nerve injury mice by reducing the inflammatory response and upregulating neurotrophic processes [173]. In another study, D-limonene acts as an antidepressant by inhibiting neuroinflammation and nitrite levels in the hippocampus and relieves anxiety through A2A receptor-mediated DAergic and GABAergic neuronal activity, both of which have been demonstrated in animal studies [174,175].

Although D-limonene has a variety of biological activities, some researchers have pointed out that while D-limonene has been shown to have low toxicity in humans [168], it still exhibits various adverse side effects at high concentrations, most commonly skin sensitization [176]. Therefore, its medical application needs to be further evaluated for safety.

6.4. Antiviral Activity

The antiviral efficacy of plant essential oils and their constituents has attracted the interest of researchers due to their safety and reliability. Dozens of herbs and hundreds of natural compounds have been reported to exhibit antiviral effects by modifying the immune system and inhibiting viral replication [177,178].

D-limonene has been considered to have antiviral properties. Nagy et al. [179] speculated that the antiviral activity of D-limonene may be attributed to the presence of cyclohexenyl. According to the paper, D-limonene has an inhibitory effect on the replication of herpes simplex virus type I; treatment with the maximum non-cytotoxic concentration of D-limonene before infection reduced the infection rate by 100%, but the treatment only slightly reduced plaque formation after the virus penetrated the cells, suggesting that D-limonene primarily targets the free herpes simplex virus type I. This result indicates the potential medicinal value of D-limonene in treating recurrent herpes labialis [180].

In addition, the influenza A virus H1N1 inhibition assay demonstrated that D-limonene achieved a virucidal activity log reduction that was 4.32 and 3.94 after 250 and 125 μg/mL (0.025% and 0.0125%), respectively, effectively killing 99.99% of the virus. This efficacy is comparable to that of household disinfectant sodium hypochlorite used at 0.21% [46]. Minari et al. [181] utilized molecular docking analysis to show that the inhibitory effect of D-limonene on Lassa virus L polymerase, an enzyme essential for viral transcription and replication, was comparable to ribavirin, which is currently the most effective drug for treating Lassa virus fever.

COVID-19, caused by SARS-CoV-2, remains a significant public health concern. ACE₂ is the human host factor or cell entry receptor for SARS-CoV-2 [182]. Molecular docking revealed that D-limonene has structural similarity to thymine in the SARS-CoV-2 viral genome and can potentially inhibit the receptor binding domain of the SARS-CoV-2 spike protein from fusing with the ACE2 protein. This could reduce SARS-CoV-2 host cell binding, cellular internalization, and pathogen release [53,183]. Moreover, cytokine storm is a major cause of death in COVID-19 patients. D-limonene, by mediating multiple inflammatory pathways and mediators, might inhibit cytokine storm [27] and regulate the expression of various signaling pathways, such as PI3K/Akt/IKK-α/NF-κB P65, making it a potential candidate for preventing or treating COVID-19-related pulmonary fibrosis [184].

Current research shows the potential of D-limonene in the development of novel antiviral drugs. However, as different viruses cause varied infection symptoms, further in-depth studies are necessary to understand the mechanisms and efficacy of D-limonene against different viral infections.

7. Application: Nanotechnology and D-Limonene

Although D-limonene has great potential in the fight against bacteria and fungi, its large-scale application is limited because of its highly hydrophobic and oxidative degradation properties [185]; volatilization under light, air, moisture, and high temperature; and sensitivity to oxidation and chemical transformation [186]. To develop D-limonene products with high stability, controlled release, and long-term effectiveness, D-limonene is currently loaded using polymer encapsulation technology and the possibility of its practical application is being investigated (Figure 4) [22].

7.1. Nanotechnology: Improving Properties and Enhancing Bioactivity

Different encapsulation techniques, such as spray drying, thin-layer drying, freeze-drying, emulsification, coalescence, and ionic polymerization, have been used to improve the physicochemical properties and bioactivity of D-limonene to form polymeric materials, such as nanoemulsions, nanoparticles, nanogels, and microcapsules, whereas gum arabic, chitosan, alginate, and maltodextrins are common loading materials [25,186,193,194,195,196,197,198].

Castel et al. [186] used brea gum as an encapsulating wall material to produce D-limonene microcapsules and they found that brea gum allowed good encapsulation and protection of D-limonene. Microcapsules made from 20% w/w brea gum solution had the best stability and there was no significant difference in the average particle size after 7 days of storage at room temperature. In addition, using high-viscosity sodium alginate and gelatin (type A) as wall materials and fabricated composite coalescent microcapsules, we encapsulated D-limonene by a spray drying and in situ composite coalescence method, which had 75% ± 6% of the degree of reconsolidation and retained up to 82.7% of the D-limonene during drying and 80% of the D-limonene when stored at room temperature for more than 72 days [194]. In a study, D-limonene, lipoic acid, and 1-ethyl-3-methylimidazole sulfate were used to prepare a D-limonene-lipoic acid-ionic liquid polymer that exhibited long-term stability and moderate strain, high bond strength to various substrates, good self-healing ability due to the presence of disulfide and hydrogen bonds, and excellent antibacterial ability [190] and the electrostatic spinning technology can be used to prepare a new antibacterial active packaging fiber of polyvinyl alcohol/D-limonene with excellent tensile strength and elongation at break, which exhibited optimal degradability, antibacterial properties, and the lowest oxygen permeability at a polyvinyl alcohol:D-limonene ratio of 7:3 [191]. In another study, zein-based microcapsules loaded with D-limonene were prepared using an antisolvent method, which resulted in good stability and lower initial release compared to whey protein isolate (WPI)-stabilized emulsions, and it exhibited better retention as the duration phase tended to be progressive [192].

This shows that nanotechnology can not only improve the physicochemical properties of D-limonene and make it applicable to more fields but can also enhance its biological activity, which is valuable for in-depth research.

7.2. Applications

7.2.1. Food

As a natural compound with excellent antimicrobial activity, D-limonene has the potential to be used in food preservation but, if it is added directly, it will affect the quality of the food itself; nanoencapsulation is one of the feasible ways to overcome this problem. Nanoencapsulation not only improves the physicochemical properties of the active substance and prevents them from interacting with the food matrix but also increases the passive cellular absorption mechanism, reducing mass transfer resistance and improving antimicrobial activity, possibly due to its subcellular size. [199].

Nanoencapsulated D-limonene can improve the shelf life of food. For example, the nanoemulsion coating prepared using deionized water, propylene glycol, D-limonene, and sodium alginate had good particle size uniformity (3–5 nm) and resulted in better inhibition of L. monocytogenes, S. enterica, and E. coli than unencapsulated D-limonene, with 50%, 90%, and 87.5% reductions in the MBCs, respectively, and it reduced the rate of water and weight loss during storage of bananas, inhibited pectinase activity, and extended shelf life [200] and Umagiliyage et al. [201] found the materials made from D-limonene encapsulated in liposomes exhibited good inhibitory activity against both bacteria and fungi and it significantly extended the shelf life of blueberries.

In addition, the biobased composites made of D-limonene and β-cyclodextrin have excellent heat resistance, which could prevent the loss of D-limonene during high-temperature processing and have potential applications in the preparation of reactive food packaging films [202]. Edible biopolymer (pullulan/carrageenan) functional composite films prepared by Roy et al. [203], with the addition of copper sulfide nanoparticles and D-limonene, exhibited enhanced tensile strength and UV-blocking function, as well as a degree of antibacterial activity. The new fragrant starch-based films prepared with D-limonene exhibited significantly lower hygroscopicity and water solubility, as well as significantly higher tensile strength and, because of the addition of D-limonene, they exhibited effective antimicrobial activity and a pleasant aromatic odor, thus favoring their use for the preservation of some foods with special odors [204]; Lan et al. [205] prepared a composite film made of polyvinyl alcohol/chitosan combined with D-limonene that had good biodegradability and light transmission and the addition of D-limonene (5% w/w) enhanced the barrier capacity and mechanical properties of polyvinyl alcohol/chitosan films and improved their antibacterial activity against E. coli and S. aureus, which prolonged the shelf life of mangoes at room temperature. In addition, fish-gelatin–chitosan-edible films supplemented with D-limonene exhibited better antimicrobial activity and D-limonene addition effectively improved their ductility, as well as their water vapor and light barrier properties [206].

Although the above-mentioned D-limonene-loaded nanocarriers overcome the defects in the physicochemical properties of D-limonene, there is still a lack of research on the possible modes and mechanisms of their antimicrobial action.

7.2.2. Agriculture

Along with the increasing concern for environmental protection, the search for a non-hazardous natural compound to replace chemical pesticides has attracted the interest of researchers. As a potential alternative to chemical pesticides, a large number of studies have demonstrated D-limonene’s ability to combat a wide range of pathogenic bacteria and pests, but its use has been challenged by properties such as high volatility, low solubility, and thermal instability. It has been found that nanoencapsulation can overcome these issues, providing better stability, protection, release control, and bioavailability [207].

Feng et al. [188] reported greater antifungal activity of D-limonene nanoemulsions against Pyricularia oryzae, Rhizoctonia solani, Colletortrichum gloeosporiodes, and Phomopsis amygdali compared to free D-limonene at the concentration of EC₅₀s, with an increase in inhibition from 22.2%, 30.4%, 24.4%, and 32.5% to 48.7%, 50.9%, 47.4%, and 51.7%, respectively, which could be attributed to the increased permeability and water solubility of the nanoemulsions.

Anopheles stephensi and Culex quinquefasciatus are vectors of several diseases. Alireza et al. [208] found that, compared to D-limonene, nanoliposomes of D-limonene had a stronger larvicidal effect on these two mosquitoes, with the LC₅₀s decreasing from 20.12 and 16.36 μg/mL to 13.6 and 6.41 μg/mL, respectively, and the LC₉₀s decreasing from 80.05 and 31.29 μg/mL to 25.08 and 12.71 μg/mL, respectively, and the D-limonene nanoemulsions prepared by Ioanna et al. [142] showed larvicidal properties against the third- to fourth-instar larvae of Aedes albopictus and Culex pipiens molestus, with LC₅₀s and LC₉₀s showing the same decreasing trend compared to D-limonene; this may be due to the fact that nanoencapsulation enhances the physical stabilization, permeation, and propagation of D-limonene.

Columbicola columbae is the main cause of ectoparasitic infections in pigeons, which can lead to symptoms such as anemia, dermatitis, decreased egg production, and slow weight gain, and the general method of counteracting this is the use of deltamethrin but this not only leads to environmental contamination but also negatively affects the pigeons and their products. Therefore, Gadelhaq et al. [24] proposed the use of D-limonene instead of deltamethrin and showed that 30 mg/mL of D-limonene and its nanoemulsion exhibited equivalent lousicidal activity to 0.025 mg/mL of deltamethrin without affecting pigeons, both through the neuromuscular inhibition of AchE and contact distortion of the body wall, with the difference that D-limonene and its nanoemulsion did not affect the pigeons and, remarkably, the D-limonene nanoemulsion remained stable after 50 days of storage and exhibited significant insecticidal activity.

Although studies have shown that nanoencapsulated D-limonene has enhanced physical stability and bioactivity, there is a need for more extensive research and evaluation as nanoencapsulated D-limonene has been studied to control fewer pest species and has not been validated in practical applications.

7.2.3. Medicine

A large number of experimental results have shown that D-limonene has excellent clinical pharmacological activity and antimicrobial activity against many clinically pathogenic microorganisms, but its use and efficacy are hampered by reasons such as its solubility and stability so the use of nanoencapsulation technology to improve its properties for clinical application is the direction of the investigation.

Doxycycline is an important antineoplastic anthracycline chemotherapeutic drug, but it has cumulative cardiotoxicity. In a study, nanodelivery systems (nanoemulsion, niosomes, and polylactic nanoparticles) of doxycycline and D-limonene were prepared with a maximum loading rate of up to 75.8%, which had desirable stability and antioxidant properties, and exhibited enhanced anticancer activity against liver cancer cells as well as lower cytotoxicity against normal liver cells [209].

In the study, D-limonene was found to have anticancer activity against both melanoma cell line A-375 and breast cancer cell line MDA-MB-468 with IC₅₀s of 246.05 and 2118.94 μg/mL, respectively, which was significantly improved by chitosan nanoencapsulation, with IC₅₀s reduced to 30.24 and 650.7 μg/mL, respectively, which may be attributed to the nanoencapsulation improving the hydrophobicity of D-limonene or possibly of the use of its lipophilicity to improve permeability [210,211].

Researchers believe that D-limonene can act as a permeation enhancer, fluidizing or disrupting the integrity of the stratum corneum of the skin for the purpose of transdermal drug delivery, decreasing systemic adverse effects or increasing bioavailability [212]. The study showed that benzocaine-loaded poly(D,L-lactide-co-glycolide) nanoparticles with added D-limonene significantly enhanced permeation rate and prolonged anesthetic efficacy and reduced cytotoxicity [211], whereas another study showed that D-limonene-containing nanovesicles showed higher encapsulation efficiency and transdermal delivery of asenapine maleate compared to cineole and hydromiscible cosolvent (transcutol^®). In addition, this was probably due to the effect of structural activity, which allowed better transdermal delivery of lipophilic molecules of hydrocarbon terpenes (D-limonene) than ketone terpenes (cineole), and a significant increase in the bioavailability of transdermal administration compared to oral administration of 3% to 54.5% [213].

Through nanoencapsulation, the physicochemical properties of D-limonene can be effectively improved and the available range can be expanded, but the current research mainly stays focused on in vitro cellular experiments or animal experiments and putting into application in the clinic needs more review studies.

8. Necessity of a Security Assessment

Exposure to D-limonene is very common because it is a volatile flavor component widely present in various fruits or plants. Food is considered the main source, accounting for 96% of exposure, followed by ambient air at 4% [214].

Previous reports have identified D-limonene peroxide as a serious skin contact allergen. The widespread use of D-limonene in food, detergents, and cosmetics significantly increases the risk of skin exposure for consumers, leading to reported cases of skin allergies in multiple regions [215]. According to one study, dermal exposure to high concentrations of D-limonene resulted in irritation or a purpuric rash, which was related to the degree of oxidation of D-limonene [216]. To mitigate this risk, some researchers suggest producing and transporting D-limonene at low temperatures or adding antioxidants ensuring the peroxide content remains less than 20 mmol/L [215,216].

In addition, clinical trials have shown that non-peroxidized D-limonene did not exhibit genotoxicity, neurotoxicity, and reproductive toxicity in humans but did exhibit skin irritation [217]. In animal experiments, D-limonene showed carcinogenicity, respiratory sensitization, and nephrotoxicity in rats [214]. However, this finding is not applicable to humans due to the absence of the related protein α_2u-globulin in humans [217].

It is worth noting that there have been some reports of sensitization or other adverse reactions to products containing D-limonene, such as perfumes, cosmetics, and household products, but none of these reports have conclusively identified D-limonene as the direct cause [218,219,220].

Currently, the daily intake of D-limonene is set at “not specified”, but Ravichandran et al. point out that the metabolic pathway, safe levels, and risk assessment of D-limonene are necessary; as the consumption of citrus juices increases, the risk of consumers’ exposure rises, potentially leading to toxicity under prolonged exposure [214].

9. Conclusions and Outlook

In this review, we present the preparation of D-limonene and its antimicrobial, anthelmintic, and medicinal potentials at cellular and molecular levels. However, the properties of volatility, high hydrophobicity, and oxidative degradation have limited its expansion in many application scenarios. With the development of nanoencapsulation technology, making D-limonene into novel materials, such as nanoemulsions and microcapsules, not only improves the undesired physicochemical properties but also effectively enhances its biological activity.

Although D-limonene as a natural active substance has promising applications in many fields, the current research on it is mainly in the laboratory environment and the feasibility of extending its application in real-life production still needs to be verified in practice. Moreover, D-limonene peroxide is a well-known skin-contact allergen that has been shown to cause allergic reactions at high concentrations in clinical trials. It has also demonstrated carcinogenic and other effects in animal studies; although some of the results may not apply to humans, a more detailed re-examination of the safety of D-limonene is necessary.