Biosynthesis of lemongrass essential oil and the underlying mechanism for its insecticidal activity

Lemongrass (Cymbopogon flexuosus) is an aromatic perennial grass grown extensively for its essential oil. Lemongrass oil is chiefly a mixture of various cyclic and acyclic bioactive monoterpenes. We reviewed lemongrass oil and its biosynthesis in the present chapter along with its biochemical composition. Furthermore, we attempted to explore both the possible routes for essential oil biosynthesis in lemongrass, i.e. mevalonate and non-mevalonate pathways and how these pathways interwind with each other. Lemongrass oil has high commercial potential in medicinal, cosmetic, food and energy industries. Regarding the pharmacological properties, a wide array of biological activities has been observed in lemongrass oil such as antimicrobial, insecticidal, analgesic and anti-cancer properties as well as its efficacy as insect-repellent. The later sections were dedicated for the analysis of insecticidal property of the lemongrass oil and the mechanism working behind this phenomenon where it was observed that in addition to synergistic effects, various components of lemongrass oil can also induce specific neurotoxic and cytotoxic responses in the insects.


Introduction
Lemongrass (Cymbopogon flexuosus) is a perennial crop from grass family Poaceae with numerous industrial applications. The term lemongrass is coined after its 'lemony' essence. Lemongrass is endogenous to India, Sri Lanka, Brazil and Myanmar. India is a major exporter of lemongrass among these with an exportation capacity of about 1000 tons worldwide every year, predominantly to America, England, Australia (Ganjewala and Gupta, 2013).
Lemongrass is chiefly grown for its essential oil (EO) that has multiple medicinal (anticancer, analgesic and antimicrobial) and cosmetic usage (Chandrashekar and Prasanna, 2010;Ganjewala, 2009). In addition to this, lemongrass is a rich source of Vitamins A, B-complex, C, Folate and mineral nutrients including magnesium phosphorous, manganese, copper, potassium, calcium, zinc and iron. It is also used in Thai cuisine as well as in the form of herbal tea (Figure 1). Recent developments have also explored lemongrass as biofuels enhancing its worth even more (Dhinesh et al., 2016;Alagumalai, 2015). In addition to these, lemongrass essential oil has extensive utilisation in the synthesis of eco-friendly pesticides because of its insect-repellent nature (Zheljazkov et al., 2011;Ganjewala, 2009).
Given the high amount of plant volatiles, lemongrass oil has also been used in the making of high-grade deodorant or air fresheners (Srivastava et al., 2013).

Lemongrass Essential Oil
In principle, plants synthesise essential oils (EOs) for defence and communication (Hassoun and Çoban, 2017;Pavela and Benelli, 2016). They provide defence against multiple phenomena including but not limited to bacteria, virus, fungi and insects (Swamy et al., 2016;Vergis et al., 2015). Due to these, EOs find high use in pharmaceutical, cosmetic and food industries. As of now, almost 10-15% of total about 3000 different EOs are being used on a commercial scale for various purposes (Hassoun and Çoban, 2017). The EOs have a strong flavour or essence and thus are called as 'essential' oils. This property of EOs is highly exploited by perfumery and flavour industries (Pavela and Benelli, 2016).
Lemongrass EO is one from about 400-500 commercially produced EOs (Tisserand and Young, 2013). Lemongrass EO has rich spectra of compounds enriched with rigorous medicinal properties, e.g. antibacterial, antiviral, antifungal, antifungal, anticancer and antitumor (Sharma et al., 2009). It also has limonene and borneol that are attributed for analgesic and anaesthetic properties (Sharma et al., 2009). However, other closely related species to lemongrass do not have the identical chemical composition of their EOs.

Essential Oil Biosynthesis in Lemongrass
Biosynthesis of monoterpenes has been a point of interest since long (Banthrope et al., 1980(Banthrope et al., , 1972Croteau, 1988Croteau, , 1987. Various approaches were adopted with the advent of technology, including the use of radioactive compounds (Ganjewala and Luthra, 2007a). Though these attempts provided significant insights, essential oil biosynthesis in lemongrass could not be entirely comprehended and still has research gaps.
The essential oil in lemongrass is primarily a mixture of cyclic and acyclic monoterpenes which are chiefly derived from geranyl diphosphate (GPP) which acts as the precursor for monoterpenes biosynthesis (Ganjewala and Gupta, 2013). The GPP is formed by the condensation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) units.
This IPP can either be synthesised in the plastid (MEP-pathway) or in the cytoplasm (MVApathway). The IPP units generated by MVA-pathway could be transferred to plastids for synthesising monoterpenes along with other IPP units generated through MEP-pathway. Therefore, essential oil biosynthesis in lemongrass leaves occurs either through cytoplasmic-MVA pathway or through the plastidic-MEP pathway (Ganjewala and Gupta, 2013).

Mevalonate-independent Pathway (MEP Pathway)
The MVA pathway was considered to be the only pathway for IPP biosynthesis in plants until an alternate non-mevalonate pathway was discovered (Rohmer, 1999). This mevalonateindependent pathway, also known as the MEP pathway starts with the conversion of glyceraldehyde 3-phosphate and pyruvate to 1-deoxy-xylulose-5-phosphate (DXP) (Richard et al., 2001). This conversion is catalysed by DXP synthase (E.C

Essential Oil Storage
Lemongrass plant stores EO in its leaf parenchymal cells which are also known as 'oil cells' (Ganjewala and Gupta 2013). Expanding lemongrass leaves synthesise EO more rapidly during the early days of their development. Acetylation potential is also on the peek at this stage. This renders higher content of geranyl acetate and citronellyl acetate than geraniol and citronellol, respectively. The cytosolic-MVA pathway is more active in younger leaves that explains higher EO content in young lemongrass leaves. In older leaves, geranyl acetate and citronellyl acetate content start to diminish as a result of negligible acetylation capacity (Ganjewala and Luthra 2007a). Moreover, the photosynthetic rate lowered down in older leaves that might further account for decreased yield (i.e. EO content) in the older lemongrass leaves (Maffei et al., 1988).

Insecticidal Property of Lemongrass Essential Oil
Our heavy dependence on chemical pesticides till date has posed serious health concerns over the decade. These pesticides are a serious threat to non-target hosts, including humans that   by the fact that EOs might ultimately have the potential to hijack various receptors associated with GABA pathway and neurotransmission such as octopamine and acetylcholine esterase receptors in insects .
Furthermore, EOs can also induce cytotoxicity in mitochondria and damage its membrane by reducing membrane potential through depolarisation (Feroz, 2020). This also alters the permeability of the mitochondrial membrane (Doll-Boscardin et al., 2012). Mitochondrial dehydrogenase activity and in turn, the overall mitochondrial metabolic rate could thus be reduced under EOs exposure (Feroz, 2020).
A major constituent in lemongrass EO, citral, can regulate cell proliferation by interacting with intracellular oxygen radicals and oxidative stress (Sanches et al., 2017;Kapur et al., 2016).
Additionally, citral along with other EO components might act on neuroreceptors, hinder signal transduction, cause hormonal imbalance, membrane damage as well as cytotoxicity in the host (Feroz, 2020;Rattan, 2010). In addition to citral, different other components might target different sites in insect, enabling essential oils to attack multiple target sites simultaneously (Suwannavod et al., 2019). Components of EOs could exhibit a synergistic effect in insects, which further intensifies their insecticidal properties.
Interestingly, insects can minimize the toxic effects of various major components through their defence and detoxification system (Scalerandi et al., 2018). However, the underrated minor components are the ones that could impart graver consequences in insects by evading their detoxification (Scalerandi et al., 2018). Such constituents can induce cytotoxicity in various insect cell lines, including ovarian cell line Brari and Thakur, 2015).
Essential oils might also target specific vital metabolic enzymes. This can be correlated to various acidic phenolic components of EOs including geranial and neral, given their elicited reactivity with most enzyme active sites which can even lead to inactivation of the active sites (Ngongang et al., 2019). Phenoloxidase is one such enzyme that is considered as an important marker for insect defence system (Feroz, 2020;Huang et al., 2016). It provides resistance against various insecticides, and any change in its activity can be used to determine the toxicity level of any chemical (Bali and Kaur, 2013). Essential oils might interfere with the activation cascade of phenoloxidase and thus hampering its activity (Kalita and Devi, 2016). Such downregulation in phenoloxidase activity by EOs weakens insect immune system and exhibit serious damage to the insects in the long run. Similarly, lactate dehydrogenase activity was also found to be enhanced with EOs application (Qari et al., 2017). Lactate dehydrogenase activity is a potential marker for the evaluation of cellular damage and the enhancement in its concentration is directly proportional to the damage caused in the organism (Brown et al., 2012). It is thought that EOs might modulate the expression of genes associated with the biosynthesis of this enzyme (Qari et al., 2017).
Essential oils from different Cymbopogon spp. were also traced to hamper haemocyte viability in two different insect larvae populations (Feroz, 2020). Haemocytes are considered to have a significant part in maintaining the cellular and humoral immune system in insects (Kalita and Devi, 2016). Others (Kalita et al., 2017;Kalita and Devi, 2016) suggested that haemocytes viability can be modulated either by targeting constituents of insect immune systems or by regulating immune responses. Such alterations negatively influence the hematopoietic system along with the aberration in endocrine gland activity (Feroz, 2020). This retards overall insect growth and development.
Components of EOs can be effective against various insects to a different degree. Moreover, they can target different insect systems with varying intensity. These variations could be . Essential oils, which is a mixture of such components, thus can target the nervous system, respiratory system, reproduction system as well as the defence system in insects (Devi et al., 2019). Various recent reports have suggested that EOs can utilise neurological mechanism to impart substantial damage in insects (Moore, 2014;Kain et al., 2013;Hamid et al., 2011;Isman, 2006).

Conclusion and Future Perspective
Lemongrass plant is mainly cultivated for its essential oil that has numerous pharmacological and industrial applications. Apart from its heavy use in cosmetics and food industries, lemongrass oil has substantial potential in the synthesis of environment-friendly insecticides.
Essential oil constituents of lemongrass have a wide spectrum of biological activities. These components could also exhibit cytotoxic as well as neurotoxic response in different insects of