Usage of Natural Volatile Organic Compounds as Biological Modulators of Disease

Plants produce a wide variety of natural volatile organic compounds (NVOCs), many of which are unique to each species. These compounds serve many purposes, such as fending off herbivores and adapting to changes in temperature and water supply. Interestingly, although NVOCs are synthesized to deter herbivores, many of these compounds have been found to possess several therapeutic qualities, such as promoting nerve stability, enhancing sleep, and suppressing hyperresponsiveness, in addition to acting as antioxidants and anti-inflammatory agents. Therefore, many NVOCs are promising drug candidates for disease treatment and prevention. Given their volatile nature, these compounds can be administered to patients through inhalation, which is often more comfortable and convenient than other administration routes. However, the development of NVOC-based drug candidates requires a careful evaluation of the molecular mechanisms that drive their therapeutic properties to avoid potential adverse effects. Furthermore, even compounds that appear generally safe might have toxic effects depending on their dose, and therefore their toxicological assessment is also critical. In order to enhance the usage of NVOCs this short review focuses not only on the biological activities and therapeutic mode of action of representative NVOCs but also their toxic effects.


Introduction
Natural volatile organic compounds (NVOCs), also known as biogenic volatile organic compounds, are compounds that derive from living organisms such as plants. Further, volatile organic compounds (VOCs) are organic compounds that are volatile at environmental temperature; however, this definition may vary between countries and jurisdictions. For example, Canada defines VOCs as organic compounds with boiling points between 50 and 250 • C [1], whereas the European Union defines them as organic compounds with an initial boiling point less than or equal to 250 • C measured at a standard atmospheric pressure of 101.3 kPa [2]. In India [3] and the United States of America [4], these compounds tend to be defined from an environmental pollutant standpoint.

Camphene
Camphene is a monoterpene that is also referred to as comphene, 79-92-5, and 2,2dimethyl-3-methylenenorbornane [45]. Camphene inhibits the growth of Paracoccidioides lutzii, a fungus that causes paracoccidioidomycosis [46], through protease inhibition and the dysregulation of important biological pathways [47]. Further, this compound induced the death of the old-world bollworm (Helicoverpa armigera) the eggs of which were dipped [48]. Regarding its therapeutic properties, this compound decreases the level of serum lipids such as cholesterol and triglycerides by upregulating sterol regulating binding protein-1 (SREBP-1) and downregulating MTP expression [21] while leaving the expression of 3-dydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase largely unaffected [49]. Further, this compound decreased the oxidative stress on respiratory macrophages by preventing the upregulation of superoxide dismutase (SOD) and reducing glutathione (GSH) levels, in addition to decreasing the levels of lipid peroxidation and nitric oxide (NO) [50], thus stimulating tumor cell death via endoplasmic reticulum stress ( Figure 2) [22].

Camphor
Camphor is a monoterpene that is also referred to as camphor gum, (1R)-camphor, and 464-49-3 [51]. Many tropical diseases such as malaria, dengue, and elephantiasis are caused by mosquito bites. A total of 434,000 mosquito-borne diseases were reported worldwide in 2015, thus highlighting the serious threat that these insects pose to human health [52]. Camphor has been used as a mosquito repellent in topical application, cosmetics, incense, fumigants, or sprays [53] and to inhibit the growth of some pathogenic microorganisms such as Candida albicans, a representative opportunistic pathogenic yeast that causes severe health complications, particularly in immunodeficient or cancer patients [54]. This compound also inhibits the growth of Staphylococcus aureus, a Gram-posi-  [22]). Camphene stimulates Ca 2+ efflux, downregulates membrane potential to induce mitochondrial dysfunction, induces endoplasmic reticulum stress, increases caspase 3 activation, HmgB2, and calreticulin, and finally results in apoptosis. ↑: increasing; ↓: decreasing.

Camphor
Camphor is a monoterpene that is also referred to as camphor gum, (1R)-camphor, and 464-49-3 [51]. Many tropical diseases such as malaria, dengue, and elephantiasis are caused by mosquito bites. A total of 434,000 mosquito-borne diseases were reported worldwide in 2015, thus highlighting the serious threat that these insects pose to human health [52]. Camphor has been used as a mosquito repellent in topical application, cosmetics, incense, fumigants, or sprays [53] and to inhibit the growth of some pathogenic microorganisms such as Candida albicans, a representative opportunistic pathogenic yeast that causes severe health complications, particularly in immunodeficient or cancer patients [54]. This compound also inhibits the growth of Staphylococcus aureus, a Gram-positive bacterium linked to skin abscesses and food poisoning [55], and Pseudomonas aeruginosa, an opportunistic Gram-negative pathogenic bacterium that infects plants and animals including humans [56] and has reportedly acquired mulita-antibiotic resistance [57]. Camphor alters cold and heat perception by modulating blood flow in the skin and muscles [58]. Particularly, cold perception is regulated by transient receptor potential melastatin 8 (TRPM8) [59], whereas heat perception is modulated by transient receptor potential vanilloid 3 (TRPV3) (Figure 3) [60].
ginosa, an opportunistic Gram-negative pathogenic bacterium that infects plants and animals including humans [56] and has reportedly acquired mulita-antibiotic resistance [57]. Camphor alters cold and heat perception by modulating blood flow in the skin and muscles [58]. Particularly, cold perception is regulated by transient receptor potential melastatin 8 (TRPM8) [59], whereas heat perception is modulated by transient receptor potential vanilloid 3 (TRPV3) (Figure 3) [60].  [60]). When camphor is applied onto the skin, ○ 1 the stimulus affluxes via the dorsal root ganglion and nucleus tractus solitaries and ○ 2 up to the somatosensory cortex. Once the response to the stimulus is defined in the brain ○ 3 vasoconstriction occurs if the stimulus was associated to cold temperature, otherwise ○ 4 the blood vessels dilate if a hot temperature is sensed. DRG, dorsal root ganglion; NTS, nucleus tractus solitaries; TRPM8, transient receptor potential melastatin 8; TRPV3, transient receptor potential vanilloid 3. The blue line indicates the conduction of the nervous stimuli.

1,8-cineol
1,8-cineol is a monoterpene and there are many synonyms for this compound including eucalyptol, zineol, trepan, and zedoary oil, among others [61]. In 1870, Cloez reported that 1,8-cineol accounted for 90% of the total composition of Eucalyptus globulus oil [62]. This compound has many therapeutic properties, such as protective effects against the influenza A virus, which induces pneumonia through cytokine modulation and the NF-κB pathway [63], as well as anti-cancer effects via G0/G1 arrest [64], anti-asthmatic effect through mucolysis, downregulation of TNF-α and IL-1β, inactivation of the NF-κB and TLR4 pathways [64][65][66][67], and anti-inflammatory and anti-oxidative effects via inhibition of NF-κB translocation and the JNK pathway ( Figure 4) [68].  [60]). When camphor is applied onto the skin, 1 the stimulus affluxes via the dorsal root ganglion and nucleus tractus solitaries and 2 up to the somatosensory cortex. Once the response to the stimulus is defined in the brain 3 vasoconstriction occurs if the stimulus was associated to cold temperature, otherwise 4 the blood vessels dilate if a hot temperature is sensed. DRG, dorsal root ganglion; NTS, nucleus tractus solitaries; TRPM8, transient receptor potential melastatin 8; TRPV3, transient receptor potential vanilloid 3. The blue line indicates the conduction of the nervous stimuli.
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Limonene
Limonene is another monoterpene whose synonyms include eulimen, dipentene, nesol, goldflush II, cajeputene, and dipanol [76]. This compound effectively controls Listeria monocytogenes, a food poisoning-associated microbe, by disrupting its cell membrane and decreasing ATP activity [77]. Further, it prevents Aβ42-induced neurotoxicity in a Drosophila Alzheimer's disease model by eliminating H 2 O 2 and nitric oxide-induced inflammation and cell death [26]. This compound also prevents reactive oxygen species (ROS)-induced gastritis by both downregulating proinflammatory cytokines such as TNF-α, IL-1β, and IL-6 and upregulating the anti-inflammatory cytokine IL-10 [78]. Limonene has also been reported to modulate depressive behaviors by suppressing psychostimulant and monoamine neurotransmitters, in addition to inhibiting both neurotrophic factor release and its receptor activation [79], as well as the proliferation of cancer cells via the stimulation of apoptosis ( Figure 6) [80].
of mechanical hyperalgesia and nociception in the somatosensory cortex (modified from Santos et al. 2019 [75]). When p-cymene is subcutaneously administered, ○ 1 there is a decrease in Ca 2+ channel currents and ○ 2 the level of Fos expression decreases in the spinal cord. However, ○ 3 Fos expression in the pons, particularly in the nucleus raphe magnus (NRM) and in the periaqueductal gray (PAG), decreases, thus resulting in ○ 4 a decrease in both mechanical hyperalgesia and nociception through the somatosensory cortex, thus attenuating cancer-associated pain. ↑ : increasing; ↓ : decreasing

Limonene
Limonene is another monoterpene whose synonyms include eulimen, dipentene, nesol, goldflush II, cajeputene, and dipanol [76]. This compound effectively controls Listeria monocytogenes, a food poisoning-associated microbe, by disrupting its cell membrane and decreasing ATP activity [77]. Further, it prevents Aβ42-induced neurotoxicity in a Drosophila Alzheimer's disease model by eliminating H2O2 and nitric oxide-induced inflammation and cell death [26]. This compound also prevents reactive oxygen species (ROS)-induced gastritis by both downregulating proinflammatory cytokines such as TNF-α, IL-1β, and IL-6 and upregulating the anti-inflammatory cytokine IL-10 [78]. Limonene has also been reported to modulate depressive behaviors by suppressing psychostimulant and monoamine neurotransmitters, in addition to inhibiting both neurotrophic factor release and its receptor activation [79], as well as the proliferation of cancer cells via the stimulation of apoptosis ( Figure 6) [80].  [80]). Limonene not only induces apoptosis by modulating the bcl-2 gene family and upregulating pro-apoptotic proteins such as BAD and down-regulating anti-apoptotic proteins such as BCL2, but also by G1 arrest through SMAD regulation and by inhibiting metastasis through suppression of Myc caused by the vascular endothelial growth factor (VEGF) receptor/Akt pathway.
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Linalool
Linalool is a monoterpene that is also widely known as coriandrol, howood oil, alloocimenol, caswell No. 526A, and phantol [81]. It has antifungal effects against dermato- Figure 6. Anti-proliferation mechanism of limonene against cancer cells (modified from Shojaei et al. 2014 [80]). Limonene not only induces apoptosis by modulating the bcl-2 gene family and up-regulating pro-apoptotic proteins such as BAD and down-regulating anti-apoptotic proteins such as BCL2, but also by G1 arrest through SMAD regulation and by inhibiting metastasis through suppression of Myc caused by the vascular endothelial growth factor (VEGF) receptor/Akt pathway.
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Linalool
Linalool is a monoterpene that is also widely known as coriandrol, howood oil, allo-ocimenol, caswell No. 526A, and phantol [81]. It has antifungal effects against dermatophytes such as Microsporum spp. and Trichophyton spp. and has synergic effects when combined with azole [82]. Moreover, this compound promotes operant behavior through the GABA receptor [83], is used as an ingredient for perfume and cosmetics with antioxidative effects and low cytotoxicity [84], prevents ovalbumin-induced asthma occurrence by inhibiting airway remodeling [85], and decreases oxidative stress-induced cell death by regulating glutamate metabolism in the cornu ammonis 1 and 3 and dentate gyrus in the hippocampal regions of the brain (Figure 7) [27].
GABA receptor [83], is used as an ingredient for perfume and cosmetics w tive effects and low cytotoxicity [84], prevents ovalbumin-induced asthma inhibiting airway remodeling [85], and decreases oxidative stress-induce regulating glutamate metabolism in the cornu ammonis 1 and 3 and denta hippocampal regions of the brain (Figure 7) [27].  [27]). In several n ditions such as Alzheimer's disease and neuroinflammation, glutamate induces cy the cytoplasm and stimulates oxidative stress, thus inducing apoptosis. Linalool pr eration and Ca 2+ activation in the mitochondria in the cornu ammonis 1 and 3 and the hippocampal regions of the brain.

p-cymene
There are many synonyms for p cimene, 4-isopropyltoluene, and camp effects, such as downregulating of bo ylococcus aureus, Streptococcus mutans Gram-negative bacteria such as Esch monella enterica [70]. Particularly, thi tors such as thiobarbituric acid react activity [71]; inhibits inflammation v the GABAergic pathway [74]; and su pain (  cene not only blocks collagen degradation by protecting UVB-induced ROS but also stimulate lagen synthesis through TGF-β1 activation. UVB-induced ROS finally induces collagen degrada which is caused by MMP-1, MMP-3, and MMP-9 that are made by transcription factors such a 1, cFos, and cJun. UVB radiation inhibits TGF-β1 activation, which stimulates collagen synthe : to inhibit the follow action.

Pinene
Pinene consists of two isomers including α-pinene and β-pinene [94]. α-pinene and β-pinene have many therapeutic properties such as antimicrobial activity, anti-proliferation effects against cancer cells, antioxidation, and anti-inflammation [95], in addition to gastroprotective activity by modulating gastrointestinal transitional time [96], as well as synergistic effects against the proliferation of non-small cell lung carcinoma (NSCLC) when co-administered with paclitaxel ( Figure 10) [97]. . α-phellandrene promotes wound healing by inhibiting oxidative stress and inflammation, in addition to stimulating wound healing via potent fibroblast activation (modified from de Christo Scherer et al. 2019 [93]). ↑ : increasing; ↓ : decreasing; : to inhibit the follow action

Pinene
Pinene consists of two isomers including α-pinene and β-pinene [94]. α-pinene and β-pinene have many therapeutic properties such as antimicrobial activity, anti-proliferation effects against cancer cells, antioxidation, and anti-inflammation [95], in addition to gastroprotective activity by modulating gastrointestinal transitional time [96], as well as synergistic effects against the proliferation of non-small cell lung carcinoma (NSCLC) when co-administered with paclitaxel ( Figure 10) [97]. α-pinene has many therapeutic properties, such as antiviral effects against herpes simplex virus type 1 (HSV-1) [98], modulation of antibiotic resistance in Campylobacter jejuni through antibiotic efflux down-regulation [99], apoptosis induction in cancer cells via ROS production, mitochondrial malfunction, caspase cascade activation, and inhibition of metastasis [100]. Additionally, this compound mediates sleep-enhancement-induced hypnosis through GABAA-benzodiazepine receptor modulation [101], and preventive and therapeutic effects against allergic rhinitis via the regulation of disease-related factors such as IgE, IL-4, NF-κB, and receptor-interacting protein 2 (RIP2), as well as eosinophils infiltration in the lungs [102].

β-pinene
Although the chemical formula of β-pinene is the same as that of its isomer α-pinene, the former possesses unique biological properties. For instance, β-pinene attenuates Crinduced phytotoxicity due to its antioxidant properties [103] and possesses antifungal and anti-biofilm properties against Candida spp. [104]. This compound also suppresses hypertension via Ca 2+ influx inhibition-mediated vasorelaxation [105] and has antiviral effects against herpes simplex virus type 1 (HSV-1) [106]. 2.10.1. α-Pinene α-pinene has many therapeutic properties, such as antiviral effects against herpes simplex virus type 1 (HSV-1) [98], modulation of antibiotic resistance in Campylobacter jejuni through antibiotic efflux down-regulation [99], apoptosis induction in cancer cells via ROS production, mitochondrial malfunction, caspase cascade activation, and inhibition of metastasis [100]. Additionally, this compound mediates sleep-enhancement-induced hypnosis through GABA A -benzodiazepine receptor modulation [101], and preventive and therapeutic effects against allergic rhinitis via the regulation of disease-related factors such as IgE, IL-4, NF-κB, and receptor-interacting protein 2 (RIP2), as well as eosinophils infiltration in the lungs [102].

β-Pinene
Although the chemical formula of β-pinene is the same as that of its isomer α-pinene, the former possesses unique biological properties. For instance, β-pinene attenuates Crinduced phytotoxicity due to its antioxidant properties [103] and possesses antifungal and anti-biofilm properties against Candida spp. [104]. This compound also suppresses hypertension via Ca 2+ influx inhibition-mediated vasorelaxation [105] and has antiviral effects against herpes simplex virus type 1 (HSV-1) [106].

Perspectives
NVOCs are promising modulators of disease, as each NVOC has a potency to control diseases such as insomnia via sleep-enhancing by (+)-3-carene [20], cancer proliferation via induction of cancer cells' apoptosis by camphene [22], microbial infection by camphor [54], chronic disease through suppression of oxidative stress and inflammation and asthma/COPD via modulation of NF-κB activation and TNF-α secretion by 1,8cineol [64][65][66][67], cancer proliferation via cancer cells' death and cancer pain by p-cymene [75], mental disorder via antidepressant-like effects of limonene [79], allergy through controlling MAPKs/NF-κB pathway by linalool [85], pain via TRPV1 regulation by myrcene [88], skin damage through controlling inflammation by α-phellandrene [93], allergic rhinitis through NF-κB controlling and caspase pathway by α-pinene [100], hypertension via modulation of Ca2+ influx by β-pinene [105], antibiotic-resistance of Staphylococcus aureus through NorA efflux pump inactivation by α-terpinene [109], and Microcystis aeruginosa propagation through induction oxidative stress and carbonyl stress by terpinolene [115]. However, characterizing the mechanisms by which NVOCs inhibit disease progression is critical for their wide application in clinical contexts. NVOCs can be administered either directly (e.g., oral administration, inhalation) or indirectly using a syringe, nebulizer, or patch. However, oral administration and inhalation are more convenient to the patients and caregivers (e.g., nurses, doctors). Most NVOCs have relatively low boiling points, thus facilitating their administration via inhalation. This is an important advantage, as it enables the administration of therapeutic compounds without the need for highly specialized equipment.
Nevertheless, the safety of NVOCs must also be carefully evaluated to ensure their safety. Paracelsus [126] is credited for stating that "the dose makes the poison," meaning that virtually all compounds can be toxic if administered at high enough doses.
Currently, most drugs and therapeutic compounds can be chemically synthesized [127], and NVOCs are no exception. However, artificially synthesized and natural NVOCs could have different physiological effects. For example, myrcene has been long used as a food additive and a flavoring agent or adjuvant due to its pleasant aroma; however, its safety has been debated for several years. In 2010 the National Toxicology Program (NTP) reported that β-myrcene could induce renal carcinogenesis in mice and rats [128]. Nevertheless, the FDA ultimately determined that β-myrcene did not induce genotoxicity. Moreover, although this compound was linked to renal carcinogenesis in laboratory animals, some speculated that the compound was safe for human use as a synthetic food additive. Nevertheless, the FDA prohibited the use of synthetic β-myrcene as a food additive in 2018 [129]. This case serves as a precedent for the use of NVOCs (particularly those of natural origin) in medications and food products. Nevertheless, the current changes in climate conditions could induce an increase in NVOC concentrations in plants as a stress response mechanism, which might increase the likelihood of toxic effects. Therefore, future studies should focus on the dose-dependent toxicity of NVOCs.