Plant Monoterpenes and Essential Oils as Potential Anti-Ageing Agents: Insights from Preclinical Data

Ageing is a natural process characterized by a time-dependent decline of physiological integrity that compromises functionality and inevitably leads to death. This decline is also quite relevant in major human pathologies, being a primary risk factor in neurodegenerative diseases, metabolic disorders, cardiovascular diseases and musculoskeletal disorders. Bearing this in mind, it is not surprising that research aiming at improving human health during this process has burst in the last decades. Importantly, major hallmarks of the ageing process and phenotype have been identified, this knowledge being quite relevant for future studies towards the identification of putative pharmaceutical targets, enabling the development of preventive/therapeutic strategies to improve health and longevity. In this context, aromatic plants have emerged as a source of potential bioactive volatile molecules, mainly monoterpenes, with many studies referring to their anti-ageing potential. Nevertheless, an integrated review on the current knowledge is lacking, with several research approaches studying isolated ageing hallmarks or referring to an overall anti-ageing effect, without depicting possible mechanisms of action. Herein, we aim to provide an updated systematization of the bioactive potential of volatile monoterpenes on recently proposed ageing hallmarks, and highlight the main mechanisms of action already identified, as well as possible chemical entity–activity relations. By gathering and categorizing the available scattered information, we also aim to identify important research gaps that could help pave the way for future research in the field.


Introduction
The number and proportion of people aged 60 years or older is increasing at an unprecedented rate and, by 2050, will reach up to 2.1 billion, as stated by WHO [1].Indeed, population ageing is the most global demographic trend, conveying several public challenges primarily in the health sector.The ageing process is associated with an impairment of several body functions and an increase in age-related disorders, including neurodegenerative, cardiovascular, cancer and metabolic diseases [2].Several factors including genetics, lifestyle and environmental features are key in this inevitable gradual process, and interventions that promote health and longevity, thus increasing lifespan, are a matter of current interest and research.
In the last years, several strategies have been proposed to delay ageing, namely caloric restriction, nutritional interventions and microbiota transplantation [3,4].In addition, Biomedicines 2024, 12, 365 2 of 31 specific treatments including depletion of senescent cells, stem cell therapy, antioxidant and anti-inflammatory treatments, and hormone replacement therapy have been used in order to promote healthy ageing and longevity [5].The identification of potential drugs for age-related diseases such as metformin, rapamycin, resveratrol and senolytics is also a hot topic in the field, although several recognized limitations and side effects raise some concerns that need further clarification [2].
The main cellular and molecular mechanisms involved in biological ageing enabled the identification, in 2013, of ageing hallmarks [6].Later, in 2022, a research symposium held in Copenhagen considered the addition of new hallmarks to the existing list, bearing in mind their role in the ageing process [7].The accepted current list now includes 12 ageing hallmarks that comprise genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, compromised autophagy, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, altered intercellular communication, chronic inflammation and dysbiosis [8].These hallmarks have proven to be very useful in the identification of intervention targets, primarily in age-related diseases with promising results in preclinical models [9,10] and validations in clinical trials [11].Among the tested compounds, natural compounds have attracted researchers' interest, with recent studies pointing out their effect on critical ageing pathways such as mammalian target of rapamycin (mTOR), adenosine monophosphate-activated kinase (AMPK), Sirtuin 1 (SIRT1) and tumor protein p53, as reviewed elsewhere [12].Nevertheless, the majority of the studies resort to in vitro approaches, lacking in-depth analysis of individual compounds and specific effects on these pathways [12].Bearing this in mind, we sought to review the potential of volatile monoterpenes, a group of compounds highly prevalent in aromatic plants.These compounds are valued by several industries due to their bioactive potential, namely antimicrobial, antioxidant, anti-inflammatory, analgesic, antinociceptive and anticancer effects, as reviewed elsewhere [13].Importantly, many monoterpenes are considered safe for consumer use and have a generally recognized as safe (GRAS) status by the Food and Drug Administration (FDA), being included in many foods and beverages.
In the present review, an up-to-date overview of the effect of monoterpenes is provided, considering the current hallmarks of ageing.In addition, whenever known, the main mechanisms of action underlying the observed effects are stated, and a possible chemical entity-bioactivity relation is attempted.Importantly, the main gaps in the current knowledge are pointed out to help guide future research in the field.

Aromatic Plants and Volatile Compounds
The term aromatic plants refers to plants that produce volatile compounds.These compounds can be extracted from the plant source as a volatile mixture called essential oil.To comply with regulatory requirements, in particular those from the International Standard Organization on Essential Oils [14] and the European Pharmacopoeia [15], essential oils are obtained from the plant raw material by hydrodistillation, steam distillation or dry distillation, or by a suitable mechanical process in the case of Citrus fruits.Several monographs on essential oils are available, for example, in the European Pharmacopoeia [15] or the European Medicine Agency [16], which highlights their importance in the pharmaceutical industry.The main types of compounds present in essential oils are monoterpenes and sesquiterpenes.Monoterpenes are isoprene dimers with carbon and hydrogen atoms (C 10 H 16 ), such as α-pinene, limonene, camphene, myrcene and p-cymene.Monoterpenoids include in their chemical structure functional groups like alcohols (e.g., linalool, geraniol, menthol), esters (e.g., linalyl acetate, geranyl acetate, neryl acetate), ketones (e.g., camphor, menthone, carvone), aldehydes (e.g., geranial, neral), ethers (e.g., 1,8-cineole) or phenols (e.g., carvacrol, thymol) [17].Monoterpenes are synthetized from geranyl pyrophosphate by monoterpene synthases and, according to their structure, can be classified as acyclic, monocyclic or bicyclic.These volatile compounds generally present a pleasant aroma and can be found in fruits, vegetables, spices and herbs, as well as in different plant parts like leaves, flowers, seeds and roots.They are appreciated as aromatic and flavoring agents in cosmetic, pharmaceutical and food industries.Also, emerging evidence shows that monoterpenes present several biological properties including antimicrobial, antioxidant, anti-inflammatory and cardioprotective, as previously reviewed [18].These findings have led to the incorporation of monoterpenes in distinct products, such as diet supplementation, animal feed additives, food packaging and biopesticides, among others.Interestingly, modifications of these compounds have also been considered with growing evidence on the biological and medical application of monoterpene derivatives [19].

Ageing Hallmarks
Ageing is characterized by a progressive loss of physiological integrity and impaired function that increases vulnerability to pathology or age-associated diseases, such as neurodegenerative diseases, metabolic disorders, cardiovascular diseases and musculoskeletal disorders [6].Ageing per se does not cause these diseases but favours their clinical manifestation [20], since the mechanisms driving ageing and those driving age-associated diseases largely overlap [21].
In 2013, López-Otín and colleagues proposed nine hallmarks of ageing, namely genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion and altered intercellular communication.Accordingly, to be considered a hallmark, these processes should manifest during ageing; their aggravation, resorting to experimental models, should accelerate the ageing process, and experimental improvement should delay the ageing process [6].During the last years, extensive research in the ageing field and its hallmarks has been carried out, leading to an update of the initially proposed list [8].Thus, three new hallmarks were added, namely disabled macroautophagy, chronic inflammation and dysbiosis.Indeed, López-Otín and colleagues considered that macroautophagy does not only affect proteins but also targets organelles and non-proteinaceous macromolecules, thereby being separated from the hallmark loss of proteostasis.Moreover, the authors also suggested that altered cellular communication was a vast hallmark, so chronic inflammation and dysbiosis were separated from it [8].
Independently of the number of hallmarks considered, there is an established hierarchy among them [6,8].Thus, the hallmarks are categorized as: primary, which reflects the damage, inflicted to the genome, telomeres, epigenome, proteome and organelles; antagonistic, which reflects the response to the damage and includes deregulated nutrient sensing, mitochondrial dysfunction and cellular senescence; and integrative, which considers the end result, leading to stem cell exhaustion, intercellular communication alterations, chronic inflammation and dysbiosis.Consequently, the integrative hallmarks are responsible for the functional decline associated with ageing [6,8].
Interestingly, the hallmarks of ageing are not independent, implying that experimental accentuation or attenuation of one specific hallmark will consequently affect the others [8].This is an interesting aspect to bear in mind, regarding the discovery and/or development of new drugs to tackle ageing and related diseases.

Monoterpenes and Essential Oils in Ageing Hallmarks
In this section, a compilation of the main studies reporting the effect of volatile monoterpenes on ageing hallmarks is presented.The search was carried out in the PubMed database combining the words "monoterpene" and each hallmark of ageing, without date restrictions.The results obtained are presented in the following subsections.However, for inflammation, due to the high number of studies on this topic, a more targeted search was carried out, as detailed in Section 3.2.3.For each hierarchized hallmark, a brief overview is provided followed by a table summarizing the studies organized in alphabetic order of the compound name.Information on the in vitro or in vivo study model used and the main effects reported is shown.In addition, evidence regarding the effect of essential oils on these hallmarks is also provided.Cytokinesis failure [24] ↑-increase; ROS-reactive oxygen species.
Although the primary hallmarks of ageing are the primary causes of cell damage, only two in vitro studies reported the negative effects of monoterpenes on genomic instability, namely α-pinene and D-limonene.These monoterpenes are quite frequent in several essential oils, α-pinene being characteristic of Pinus spp.(pine) essential oils and Dlimonene highly prevalent in Citrus spp.essential oils.The results presented in Table 1 suggest that α-pinene and D-limonene favour genomic instability.Indeed, α-pinene induces genomic instability by promoting mitotic alterations and reactive oxygen species (ROS) production, which causes DNA damage [23].On the other hand, D-limonene affects the cell division by preventing the assembly of mitotic spindle microtubules, which then affect chromosome segregation and cytokinesis, consequently leading to aneuploidy [24].
Regarding other primary hallmarks, a study on the effect of Thymus vulgaris (thyme) essential oil on blood telomere attrition, in chronologically aged C57BL/6J mice, was carried out [25].Interestingly, the authors reported a beneficial effect and demonstrated that mice supplemented with this essential oil (0.2% w/w) showed higher survival rates and significantly longer blood telomere lengths than the control mice.Although the study did not assess isolated compounds, the authors suggest that the phenolic monoterpene thymol and the monoterpene hydrocarbon p-cymene, present in high amounts in the oil, may primarily contribute to the observed telomere-protecting effect, due to their antioxidant and anti-inflammatory potentials [25].Thus, it seems that the aromatic ring might be responsible for the different outcomes observed in these primary hallmarks, particularly genomic instability and telomere attrition.However, further studies are needed to verify this hypothesis.

Antagonistic Hallmarks Mitochondrial Dysfunction
Mitochondria are organelles that play a vital role in homeostasis maintenance [26].Deterioration in mitochondria function increases with ageing due to the occurrence of diverse mechanisms, namely accumulation of mitochondrial DNA mutations and changes in their dynamics.These alterations might compromise the mitochondria contribution to cellular bioenergetics, increasing the production of ROS and, consequently, promoting the mitochondria membranes permeabilisation.The latter phenomenon leads to inflammation and cell death [8].
Mitochondrial dysfunction can be assessed both in vitro and in vivo.For example, isolated mitochondria can be used to assess mitochondria respiratory capacity, defined as the increase in respiration rate in response to ADP.In cells, studies include the measurement of several parameters such as ATP production rate, proton leak rate, coupling efficiency, maximum respiratory rate, respiratory capacity ratio and spare respiratory capacity [27].Table 2 compiles both in vitro and in vivo studies on the effect of monoterpenes on mitochondrial dysfunction.Combining both in vitro and in vivo studies, 1,8-cineole (synonym of eucalyptol), carvacrol, geraniol and perillyl alcohol are the most studied compounds.These compounds can be found in the essential oils of several aromatic plants; for example, 1,8-cineole is very common in Eucalyptus globulus, while carvacrol is characteristic of Origanum species, geraniol is abundant in Rosa spp.and perillyl alcohol occurs in different Mentha species.A closer analysis of the information presented in Table 2 shows that the studied monoterpenes have a beneficial effect, thus inhibiting mitochondrial dysfunction in both in vitro and in vivo models.The majority of the tested compounds present in their structure a hydroxyl group or a phenol group that might contribute to the observed effects.In general, the tested monoterpenes decreased ROS production, thus showing a beneficial effect, by restoring the mitochondrial membrane potential as shown for 1,8-cineole [29,31] and geraniol [34].The increase in mitochondrial membrane potential is also beneficial as ATP synthase activity is enhanced [51].Indeed, most studies reporting an increase/restoration of mitochondrial membrane potential also demonstrate an increase in ATP production, such as geraniol [34] and carvacrol [33].The activity of the mitochondrial respiratory chain complexes can also contribute to the maintenance of the mitochondrial membrane potential through proton flux [51].These effects were reported, for example, for geraniol [34] and linalool [37].
Besides monoterpenes, several studies have reported the effects of essential oils on mitochondrial dysfunction, although in distinct contexts not directly related to ageing.Indeed, the majority of the studies are carried out in cancer models, thus showing an overall beneficial effect but through an enhancement of mitochondrial dysfunction.For example, Schisandrae semen essential oil induced ROS-and caspase-dependent cell death involving mitochondrial dysfunction and nuclear translocation of mitochondrial pro-apoptosis proteins in human leukaemia U937 cells [52].The essential oil of Alpinia officinarum promoted lung cancer regression by triggering mitochondrial membrane potential dysfunction and activating caspase-3 cleavage, inducing cell apoptosis [53].Cinnamomum cassia essential oil showed anti-oral cancer activity in HSC-3 cells, through a significant increase in ROS production [54].The cytotoxic potential of Croton heliotropiifolius on different human cancer cell lines, namely leukaemia, colon, melanoma and glioblastoma, was also related to oxidative stress, culminating in mitochondrial respiratory dysfunction and DNA damage, triggering cell death via apoptosis [55].In the context of cancer, some monoterpenes like carvacrol [56] and hinokitiol (synonym of β-thujaplicin) [57] are also able to potentiate mitochondrial dysfunction.
In addition to essential oils' anticancer effects, their antimicrobial properties have also been related to mitochondrial dysfunction, being the mitochondrial impairment referred to as the mechanism of action that compromises infection.These effects were shown, for example, in Rosmarinus officinalis essential oil against Candida albicans [58], Piper nigrum essential oil against Aspergillus flavus [59], and Anethum graveolens essential oil against both A. flavus [60] and C. albicans [61].Moreover, insecticidal properties of essential oils through mitochondrial impairment in insects have been reported, for example, for Eugenia uniflora essential oil in Drosophila melanogaster [62] and Melaleuca alternifolia, that prevented bioenergetics dysfunction in the spleen of silver catfish naturally infected with Ichthyophthirius multifiliis [63].

Cellular Senescence
Cellular senescence is a state of permanent cell cycle arrest occurring when telomere length decreases below a critical size, in response to different damaging stimuli, such as oxidative stress and DNA damage [64,65].This is an important barrier mechanism to tumorigenesis as it limits the growth of potentially oncogenic cells [65].Moreover, cellular senescence plays a key role in some physiological processes such as embryogenesis, tissue remodelling and repair [65].The senescent phenotype is characterized by chronic activation of the DNA damage response, upregulation of Cyclin-Dependent Kinase (CDK) inhibitors (e.g., p16INK4a, p15INK4b and p21CIP), apoptosis resistance, altered metabolic rates, endoplasmic reticulum stress and increasing secretion of pro-inflammatory and tissue remodelling factors, known as the Senescence-Associated Secretory Phenotype (SASP) or senescence-messaging secretome (SMS) [64].The SASP is quite important to ensure the efficient growth arrest by autocrine signalling, in particular immediately after senescence induction, to signal senescent cells for clearance by the immune system and, consequently, for tissue repair and remodelling [66,67].Moreover, the SASP is highly heterogeneous [64] and can include a wide range of cytokines, chemokines, proteases, growth factors [66] and non-macromolecular elements [65].Although various signalling pathways are involved in the regulation of SASP components, most of them converge to NF-κB activation [65,66].Morphologically, senescent cells show structural aberrations, including an enlarged and more flattened shape; modified composition of the plasma membrane due to an upregulation of caveolin-1; increased lysosomal content which is directly correlated with higher senescence-associated β-galactosidase activity (pH 6.0); accumulation of mitochondria and nuclear changes like loss of Lamin B1 and formation of senescence-associated heterochromatin foci [64].Excessive and anomalous accumulation of senescent cells in tissues has a negative impact on homeostasis mainly via SASP [67].This phenomenon can be detrimental to regenerative capacities and generate a disruptive pro-inflammatory environment that is favourable for the onset and progression of a variety of age-related diseases [64,67].The accumulation of these cells can occur during ageing [65].
González-Gualda and colleagues proposed that, at least, three different types of markers that confirm (i) cell cycle arrest, (ii) increased lysosomal compartment or another global senescent-related structural change and (iii) an additional trait specific for the particular type of senescence being assessed (such as SASP or DNA damage features) should be considered to assess senescence [68].Monoterpenes known to possess antisenescent effects are summarized in Table 3. Regarding the effect of monoterpenes on cellular senescence, only five compounds have been tested for their senolytic or senomorphic effects.Senolytic compounds are those that are able to effectively kill senescent cells, by modulating several associated pathways, such as p21/p53 and by inducing apoptosis, whereas senomorphic compounds are responsible for inhibiting SASP, through modulation of associated pathways, such as NF-κB, mTOR and JAK/STAT [73].For instance, α-pinene can be considered a senolytic compound due to its effect on the p21/p53 axis [71], thus increasing DNA repair, preventing the entrance in cell cycle arrest and, consequently, senescence [74].D-Limonene also acts as a senolytic, probably by inducing cell death by apoptosis as it prevents the nuclear translocation of p53 [35], which is known to induce mitochondria-dependent apoptosis [75].Myrcene, on the other hand, showed a senomorphic effect through a direct decrease of IL-6 secretion [39].Secretion of matrix-degrading mediators, such as metalloproteinases, is also characteristic of SASP [73].Having this in mind, α-pinene [72], camphor [69], myrcene [39] and hinokitiol [70] are all considered senomorphics due to their inhibitory effect in the synthesis or secretion of metalloproteinases.The compounds, referred to in Table 3, are present in several essential oils.Camphor is typically encountered in Cinnamomum camphora, hinokitiol is present in Chamaecyparis spp., D-limonene is characteristic of Citrus fruit peels, myrcene occurs in Cannabis sativa and in several other essential oils and α-pinene is one of the main compounds in Pinus spp.
Interestingly, other studies assessed the effect of monoterpenes on features related to senescence but in a cancer context, similar to that reported in the previous section.In these cases, an enhancement in cellular senescence is the beneficial effect as it negatively affects the cancer cell growth.For example, a nanoemulsion of carvacrol induced cell senescence leading to cell cycle arrest by reducing CDK2, CDK4, CDK6, Cyclin E, Cyclin D1 and enhancing p21 protein expressions.Also, an increase in SA-β-galactosidase activity was observed [56].Hinokitiol decreased cell proliferation and increased DNA damage and phase S cell cycle arrest in osteosarcoma cells U2OS and MG63 [76], while increasing γH2AX-positive cells in a xenograft mice model [77].Other monoterpenes assessed in the context of cancer include 1,8-cineole that was effective in increasing phase G0/G1 cell cycle arrest, while decreasing CDK2, CDK4 and CDK6 and cyclin A protein levels.This compound also increased p27 and cyclin D1 protein levels and SA-β-galactosidase activity [78].Geraniol increased phase sub G1 cell cycle arrest in prostate cancer cells PC3 [79].Menthol and α-phellandrene increased p53 levels in the leukaemia cell lines NB4 and Molt 4 [80] and nuclei levels of p53 while decreasing its cytoplasmic levels in the hepatocellular cell line J5, respectively [81].
Regarding essential oils, several studies have assessed their effect on cellular senescence.For example, Thymbra capitata decreased SA-β-galactosidase activity in fibroblasts treated with the senescence inducer etoposide [82].Similar results were reported for Santolina rosmarinifolia [83], Salvia aurea [84] and Ferulago lutea [85].For the latter, additional senescence features (p21/p53 protein levels and the nuclear accumulation of γH2AX) were also decreased following essential oil treatment [85].In addition, Eucalyptus globulus essential oil showed anti-senescent effects in both etoposide-stimulated keratinocytes (HaCaT) and fibroblasts (NIH/3T3).Thus, in the presence of the oil, the number of senescent cells decreased as well as the levels of p53 [86].Other essential oils showed beneficial properties in vitro, senescence being induced by distinct agents.For example, several Cistus spp.oils significantly attenuated UVB-induced cellular senescence in a keratinocyte cell line (HaCaT) [87], while Cymbopogon nardus and Cymbopogon citratus oils showed cytoprotective properties on doxorubicin-induced senescence in the fibroblast-like kidney cell line (Vero) and fibroblasts (NIH/3T3) [88].
An in vivo study was also carried out in diabetic mice with Alpiniae zerumbet administered orally, ameliorating vascular endothelial cell senescence by activating PPAR-γ signalling [89].

Integrative Hallmarks Disabled Macroautophagy
Autophagy is a physiological cellular mechanism whereby cytoplasmic material is delivered to the lysosome for degradation [90].Macroauthophagy is the most well characterized and involves the sequestration of cytoplasmic material, including soluble macromolecules and organelles, into a double-or multi-membrane-bound structure, named autophagosome.These structures will then fuse with the lysosome, promoting degradation and recycling of its content [90].This section will focus only on macroautophagy, henceforth referred to as autophagy.Under normal conditions, constitutive autophagy occurs as a housekeeping mechanism [91].However, autophagy can be activated during starvation and stress conditions such as hypoxia, increased ROS production, DNA damage, protein aggregates, damaged organelles or intracellular pathogens, in order to restore homeostasis [92].Moreover, it is well established that autophagy declines with age [6,93].
Several approaches have been developed to assess autophagy, namely immunoblotting of canonical markers like LC3 and p62, detection of autophagosomes by fluorescence microscopy or autophagosome maturation resorting to mRFP-GFP fluorescence microscopy, although a combined use of several methods is recommended [94].Table 4 includes studies on the effect of monoterpenes on autophagy.

Compound
Study Model Effect Ref.
Besides the studies highlighted in Table 4, the effect of monoterpenes on autophagy has also been reported in cancer cells.The majority of the compounds tested in this setting increase autophagy, thus suppressing tumorigenesis by inhibiting cell survival and inducing cell death.For example, hinokitiol was largely assessed and increased autophagy in vitro in hepatoblastoma cell line HepG2 [57], HeLa cervical cancer cells [107], U2OS and MG63 osteosarcoma cells [76] and H1975 lung cancer cells [77], through an increase in LC3 levels, Beclin-1 levels and/or autophagosome formation, among other autophagy markers.In addition, α-phellandrene induced autophagy in human hepatocellular carcinoma (J5) cells by regulating mTOR and LC3-II expression, as well as p53 signalling.Indeed, an increase in autophagic vacuoles formation was observed together with a decrease in PI3K, Akt and mTOR protein levels and an increase in Beclin-1 and LC3-II [81].In addition, αthujone induced oxidative stress and autophagy in a glioblastoma cell line (T98G), as shown by an increase in autophagolysosome formation, an increase in LC3-II/LC3-I ratio, as well as an increase in the levels of LC3-II, Beclin-1, Atg3, Atg5 and Atg7 [108].Furthermore, terpinen-4-ol induced an accumulation of LC3-I/II, Atg5 and Beclin-1, as well as regulatory proteins required for autophagy in human leukemic HL-60 cells [109].In vivo studies also pointed out the effects of hinokitiol on the inhibition of xenograft tumour growth in association with DNA damage and autophagy, as shown by the expression of γ-H2AX and LC3 in the tumour tissue [77].
Essential oils also affect autophagy, and have been assessed in distinct contexts, namely cancer and cell toxicity, among others.Concerning anticancer effects, for example, the essential oil of Origanum montana inhibited colony growth of human HT-29 colorectal cancer cells by inducing protective autophagy, associated with downregulation of the mTOR/p70S6K pathway and apoptotic cells death, via the activation of the p38 MAPK signalling pathway [110].In what concerns cell toxicity, the essential oil of Schisandra chinensis was able to attenuate acetaminophen-induced liver injury by alleviating oxidative stress and activating autophagy, showing an upregulation of hepatic LC3-II and a decrease in p62 in mice with an overdose of acetaminophen [111].Moreover, the essential oil of Acorus tatarinowii ameliorated Aβ-induced toxicity in Caenorhabditis elegans by maintaining protein homeostasis through the autophagy pathway regulated partly by hsf-1 and sir-2.1 genes [112].

Inflammation
According to Antonelli and Kushner (2017), inflammation is defined as "an innate immune response to harmful stimuli such as pathogens, injury and metabolic stress that aims to restore homeostasis" [113].In basal conditions, tissues are maintained in the homeostatic state with the aid of tissue-resident macrophages, when it is required.Nevertheless, in noxious conditions, tissues undergo stress and can malfunction.In the case of considerable changes, adaptation to these conditions requires the help of tissue-resident macrophages or the recruitment of other macrophages and may require small-scale delivery of additional leukocytes and plasma proteins.This response has characteristics that are intermediate between basal and inflammatory states and was termed para-inflammation [114].Parainflammation is not a classic form of inflammation triggered by exogenous tissue injury or infection, but it is switched on by tissue malfunction in order to promote its adaptation to a harmful environment and to maintain its adequate functionality.This state is characterized by a low-grade/subclinical immune reaction [114].Nevertheless, if the tissue malfunction is present for a sustained period, para-inflammation progresses into a chronic low-grade inflammation (pathophysiological para-inflammation) [115,116].Ageing and related diseases are characterized by the presence of chronic low-grade inflammation [8,114].Moreover, chronic inflammation might be a consequence of alterations in other hallmarks of ageing and vice-versa, representing a vicious cycle [8,117].This hallmark is, by far, the most addressed with a PubMed bibliographic search on "monoterpenes" and "inflammation" encountering more than 1500 studies.Therefore, only the studies performed in the last 4 years that assessed the mechanism of action associated to the observed effect were considered, being summarized in Table 5.The anti-inflammatory potential of monoterpenes is usually studied by evaluating the expression of pro-inflammatory mediators (e.g., IL-1β, IL-6, TNF-α, iNOS, NO and COX-2), anti-inflammatory mediators (e.g., IL-10) and the impact on signalling pathways relevant to the inflammation process, particularly NF-κB, MAPKs and Nrf2.

Compound Study Model Observed Effects
Ref.

Compound Study Model Observed Effects
Ref.

Compound Study Model Observed Effects
Ref.
Similar to monoterpenes, many studies also report the anti-inflammatory potential of essential oils.Indeed, the combined search of "essential oil" and "chronic inflammation" brings up 37 reviews in PubMed.Regarding original studies, the essential oil of Thymus vulgaris was assessed in ageing-induced brain inflammation in chronologically aged C57BL/6J mice and showed a significantly lower gene expression of the pro-inflammatory cytokine Il6 in the hippocampus and lower Il1b expression in the liver and cerebellum, [25].Many other studies have been performed not directly related to ageing, with some genera standing out as the most studied.These include essential oils from Cinnamomum spp.[179,180], Lavandula spp.[181,182], Thymus spp.[183,184] and Citrus spp.[185][186][187].

Dysbiosis
Microbiota includes the microorganisms (e.g., bacteria, fungi, viruses and parasites) that inhabit all parts of our body and can affect human health.The composition and tissue colonization by microbiota are affected by external and internal factors.The former include mainly infant-related factors, such as the mode of delivery, the gestation time and the feeding type, as well as the type of diet and use of antibiotics.The latter includes host internal factors, particularly genetics and the immune system [188].Importantly, alterations in the microbiome have been recently associated with the emergence of several chronic diseases such as cancer, diabetes, cardiovascular diseases, as well as some psychological disorders, for example, schizophrenia [189].Moreover, ageing also affects microbiota, particularly gut microbiota, and is associated with microbiome disturbance, named dysbiosis, which is characterized by a shift in microbiota populations and the loss of diversity [8,190].Indeed, ageing-induced dysbiosis has been associated with the development of several diseases such as lung diseases [191], cardiovascular diseases [192], glaucoma [193] and neurological disorders [194].
Microbiome can be assessed resorting to a plethora of technologies, particularly gene marker analysis, shotgun metagenomics, metabolomics, metaproteomics and metatranscriptomics [195].Although gene marker analysis that resorts to a targeted sequencing method encompassing the 16S ribosomal RNA gene for bacteria and internal transcribed spacer (ITS) region for fungi is the most common technique, it fails to include other microorganisms, such as virus.Having that in mind, shotgun metagenomics was developed, which resorts to untargeted sequencing methods that allow the capture of the full repertoire of genetic information from a sample, detecting the presence of several microbes.The remaining technologies rely on the detection of expressed microbiome-related genes in the sample (metatranscriptomics), on the small metabolites and their interaction with the host and vice versa (metabolomics) and on the proteins present in the sample (metaproteomics) [195].The keyword combination of "dysbiosis" and "monoterpenes" only gave back one result, as shown in Table 6.The authors of the study resorted to gene marker analysis by sequencing the 16S ribosomal gene in order to identify the main bacteria found in the mice belonging to the Bacteriodetes and Firmicutes divisions.The study herein reported shows that geraniol is able to prevent colitis-associated dysbiosis in the dextran sulphate sodium-induced colitis mouse model, when administrated orally or through enema [196].
Essential oils have also shown promising effects on dysbiosis.For example, several essential oils were tested against species of intestinal bacteria mostly found in the human gastrointestinal tract.Overall, Carum carvi, Lavandula angustifolia, Trachyspermum copticum and Citrus aurantium var.amara essential oils were the most effective in inhibiting pathogens growth without affecting beneficial bacteria [197].Another study showed that Zanthoxylum bungeanum essential oil showed a beneficial effect on chronic unpredictable stress-induced anxiety behaviour in rats, by restoring gut microbiota dysbiosis, namely by increasing the Sobs and Chao indexes, inhibiting Lachnospiraceae, facilitating Bacteroidales_S24-7_group, Lactobacillaceae and Prevotellaceae [198].Essential oil emulsions of Satureja hortensis, Petroselinum crispum and Rosmarinus officinalis were also administered to humanized mice harbouring gut microbiota derived from patients with ischemic heart disease and Type 2 diabetes mellitus.Overall, the essential oil emulsions in mice supplemented with Lcarnitine showed prebiotic effects on beneficial commensal bacteria, mainly the Lactobacillus genus [199].Furthermore, a review carried out on the modulation of gut microbiota by essential oils and inorganic nanoparticles showed that essential oils are widely studied for their gastrointestinal interference, being potent modulators for gut inflammation, metabolic reactions and microbiota diversity [200].

Discussion and Future Perspectives
Studies on monoterpenes' effect on ageing hallmarks have been carried out with many compounds presenting in their chemical structure a hydroxyl group or a phenol group that might justify the observed effects.However, an accurate chemical structure-activity relation is difficult to establish due to the multitude of study models used, concentrations tested and observed effects that hamper direct comparisons.Figure 1 shows the chemical structures of the compounds summarized in Tables 1-6.Importantly, some monoterpenes like carvacrol, 1,8-cineole, limonene, perillyl alcohol and thymol have been largely assessed, showing relevant effects on different ageing hallmarks.Overall, inflammation is the hallmark mostly addressed with more than 1500 studies identified in a PubMed bibliographic search combining "monoterpenes" and "inflammation".On the other hand, some hallmarks remain elusive, lacking studies on the effect of these compounds, namely epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, stem cell exhaustion and altered intercellular communication (Figure 2).longevity and decrease the incidence and development of ageing-related diseases are in the spotlight.In this context, monoterpenes and/or essential oils emerge as promising compounds with beneficial effects on the drivers of ageing-related diseases.These compounds can also be considered for prevention strategies, thus meeting the new developments in geriatric medicine that is gradually focusing on pre-intervention rather than post-disease treatment.As shown by the review herein presented, monoterpenes seem to be very promising anti-ageing compounds, but it is important to highlight that the majority of the studies assess the hallmarks individually and not in an ageing context.Indeed, several compounds are studied in a cancer setting or are performed to justify the mechanism of action associated with other properties of these compounds, namely antimicrobial or insecticidal.In these situations, monoterpenes also exerted a beneficial effect but through an enhancement of the studied hallmark, as cancer cell or microbe eradication is aimed.In addition, regarding senescence studies, the majority of the studies do not address, as recommended, at least three markers.Additional studies resorting to ageing models are, therefore, needed and could include monoterpenes with anti-inflammatory properties, as these will most likely exert a senomorphic effect by modulating components of the SASP.Moreover, essential oils have also shown very promising effects, and one cannot neglect possible synergistic effects between several compounds, including monoterpenes, present in the mixture.Therefore, further studies are still required to elucidate the real potential of monoterpenes to boost natural defences during ageing and extend healthy lifespan in the elderly.The majority of the studies include in vitro and in vivo approaches, and, in line with this, clinical trials have also been carried out, although in much lesser extent.Interestingly, carvacrol and geraniol are the monoterpenes mostly included in clinical trials.Once again, several features of ageing hallmarks are assessed but not in an age-related context.Indeed, many of the trials are conducted in patients with lung disorders [201][202][203] or intestinal diseases like irritable bowel disease [204,205].Furthermore, clinical translation of these compounds remains a challenge mainly due to their volatility and low water solubility and stability.Moreover, pharmacokinetic studies are sparse, and more information on the absorption, metabolism, distribution and elimination of volatile monoterpenes and essential oils is required for safe and effective use in humans.To overcome these issues, encapsulation and targeting strategies have been considered to improve bioavailability and efficacy, as reviewed elsewhere [206].Furthermore, more large-scale and well-controlled human clinical trials taking into account the heterogeneity of ageing need to be conducted to validate the potential of these compounds and develop innovative anti-ageing strategies.
Overall, as ageing is not a disease per se, strategies that promote healthy ageing and longevity and decrease the incidence and development of ageing-related diseases are in the spotlight.In this context, monoterpenes and/or essential oils emerge as promising compounds with beneficial effects on the drivers of ageing-related diseases.These compounds can also be considered for prevention strategies, thus meeting the new developments in geriatric medicine that is gradually focusing on pre-intervention rather than post-disease treatment.

Figure 1 .
Figure 1.Chemical structures of monoterpenes with effects on ageing hallmarks.Figure 1.Chemical structures of monoterpenes with effects on ageing hallmarks.

Figure 1 .
Figure 1.Chemical structures of monoterpenes with effects on ageing hallmarks.Figure 1.Chemical structures of monoterpenes with effects on ageing hallmarks.

Figure 2 .
Figure 2. Hallmarks of ageing.Hallmarks with studies on the effect of monoterpenes are highlighted on the top part, whereas on the bottom part are shown the hallmarks lacking studies.Created with BioRender.com.

Figure 2 .
Figure 2. Hallmarks of ageing.Hallmarks with studies on the effect of monoterpenes are highlighted on the top part, whereas on the bottom part are shown the hallmarks lacking studies.Created with BioRender.com.

Table 1 .
Effect of monoterpenes on genomic instability.

Table 2 .
Effect of monoterpenes on mitochondrial dysfunction.

Table 3 .
Effect of monoterpenes on cellular senescence.

Table 4 .
Effect of monoterpenes on autophagy.

Table 5 .
Effect of monoterpenes on inflammation.
and 100 mg/Kg, i.p.; days 7 to 35 post FCA injection, on alternate days)