9.1 Introduction
Various plant parts including secondary metabolites of medicinal plants are used as vernacular medicine in traditional treatment of diseases and ailments. Increment in the number of cases in opportunistic diseases related to side effect of synthetic drugs makes a pressure to increase the efforts to search for biological optional drugs with little side effect. Therefore, struggles are focused to elucidate plausible sources such as plants containing therapeutic agents (Patil et al. 2010). Newly and advanced techniques of extraction helped in better investigation of natural occurring compound of plant origin with more exactitude leading to separation of biomolecule from crude combinations of secondary metabolites (Wang and Weller 2006). Several ethnoplants have medicinal properties, and lemongrass is one of these plants. Lemongrass is recurrent grass broadly dispersed globally and most especially in countries with humid atmosphere (Francisco et al. 2011). The commercially significant grasses of lemongrass family are C4 tropical plants. Leaves of lemongrass contain major portion of all secondary metabolites of the plant that includes up to 1.5% (d.wt) aromatics and essential oils with high smell of lemon, yellow or amber in color (Adejuwon and Esther 2007). Most of the essential oils obtained from aromatic and medicinal plant species are useful in production of perfumes, soaps, toiletries, syrups, and sauces. Some of these oils are used in traditional and folk medicine for various medical purposes. Inquiries about the assessment of the biological actions of essential and aromatic oils of medicinal plants have discovered the therapeutic properties like antifungal, insecticidal, antiviral, and antibacterial. Essential oil mainly used in aromatherapy to treat serious skin diseases like superficial mycoses showed significant antimicrobial effect on skin pathogens (Tajidin 2012). Citral is the major component of lemongrass oil (LO) (Tajidin 2012), a natural combined form of geranial (a-citral) and neral (b-citral) (Pengelly 2004). Both geranial and neral are aldehydes and isomers to each other. The high citral content of lemongrass oil has made it significant for numerous therapeutic combinations. Citral, also known as 3,7-dimethyl-2,6-octadienal, is a monoterpene chemical that can be obtained from citrus fruits, herbs, plants, and grasses (Negrelle and Gomes 2007). Citral has been used as a natural preservative to foodstuff, maquillages, and drinks due to the presence of strong flavor and lemon aroma (Maswal and Dar 2013). The citral molecule can be move freely across the membranes due to its small size and hydrophobic nature. Citral has strong electrophile property due to the activity of α, β-unsaturated carbonyl and can be readily digested by mammal cells (Esterbauer et al. 1975; Diliberto et al. 1990).
Citral has antifungal, bactericidal, deodorizer, insecticidal, linctus, weak diuretics, stimulating, spasmolytic, and craving properties. Citral also has the mild effect on inflammation. Citral exhibited bacteriostatic effect and was found significant against Staphylococcus aureus with minimum inhibitory concentration (MIC) values at the range of 75–150 μg/ ml. Citral has no effect on cell wall but can degrade cell membrane by affecting its potential, obstructing efflux pump. Citral can reduce the load of staphylococcal infections in liver tissues and spleen according to dose-dependent manner which further decrease when combined dose of citral and norfloxacin is used. Citral has no effect on mortality or morbidity at the dose of 500 mg/kg body weight and can elongate effect of norfloxacin in the case of the post-antibiotic effect. Green and Berenbaum (1994) advised that being a volatile molecule, it can be worked as natural repellent and protect the plant from insects and other predators by repelling. They found citral as toxic agent for cabbage loopers (Trichoplusia ni), and they also observed that ultraviolet light can increase citral toxicity. Devi et al. (2011) reported calcium antagonist role of citral. The existing chapter recapitulates the work on citral content in various aspects carried out in the last decades in relation to their traditional and modern uses as culinary, medicinal, and cosmetic and also deals with quality issues and toxicity.
9.2 Source of Citral
Citral, chemically known as 3,7-dimethyl-2,6-octadienal, is an aromatic bio-active molecule present in the extracted essential oils obtained from lemongrass, citrus fruits, verbena (Verbena officinalis), and ginger. Citral is made up of monoterpenes (isomeric mixture of geranial and neral) and myrcene being found (Dudai et al. 2005). Zeng et al. (2015) proved that extract of ginger, obtained from steam distillation, has 30–40% citral.
9.3 Extraction and Characterization of Citral
Supercritical extraction of essential oil with the help of CO2 is most reliable technique in which dried powder of leaves is used. As compound with high molecular mass is started to extract at higher solvent density, yellowish semisolid mass extracted in place of yellow essential oil. At 90 bar and 50 °C, the optimum extraction citral yield is obtained from CO2 supercritical extraction. At above conditions, citral yield was 68% of the essential oil. The citral content was higher in hydrodistilled essential oil than that of supercritical extracted oil.
Schaneberg and Khan (2002) developed method to quantify the bio-active markers like neral, geraniol, geranial, citronellal, limonene, and β-myrcene, based on gas chromatography with flame ionization. They compared processes for the extraction of oils from C. citratus. These procedures were steam distillation, supercritical fluid extraction, and accelerated and simple solvent extraction.
Mei et al. (2010) determined the stability of citral in emulsions (oil-water) with octadecane in both liquid and solid phase at pH 3.0. The results of Schaneberg and Khan (2002) experiment showed faster degradation of citral in anionic sodium dodecyl sulfate stabilized in comparison with nonionic polyoxyethylene lauryl ether-stabilized emulsions.
Rapid degradation of citral was noted when octadecane crystallized in emulsions like nonionic polyoxyethylene lauryl ether and anionic sodium dodecyl sulfate. The solid and liquid phase of octadecane also affects the citral partitioning. In liquid phase of octadecane, 18–25% partitioning of the total citral was noted while 41–53% in solid phase. They suggested that increment in the rate of citral degradation is due to an increment in citral partitioning outside of emulsion (oil-water) droplets. These results emphasized the use of technology able to decrease citral partitioning and contact to acidic phases to enhance stability of citral in emulsions. Tian et al. (2018) prepared nanoparticles using solid lipid loaded with citral (citral SLNs) by a method of homogenization (high-pressure); the lipid known as glyceryl monostearate (GMS) is used and a mixture of 1:1 (weight ratio) of Span 80 and Tween 80 as the surfactant. The GC data indicated that citral stability increase and 67% of the total citral stayed in the suspensions of citral-SLN while only 12% in control. They concluded that covering of citral with solid lipid can increase citral stability in acidic phases.
Citral, a key molecule of lemongrass essential oil, can be isolated by using steam distillation (Rao et al. 2015). The analysis of results and conditions explained that the time of distillation and volume were 98.21 min and 0.0 53 μl, respectively. The citral yield was 85.1416% at optimum conditions. The 83.8% yield of citral was noted in revised and confirmation experiment. The data of refractive index, flash point, density, and specific gravity of isolated product were 1.488, 91 °C, 0.89031 g/cm3, and 0.8904 which were similar to data of above properties of standard citral.
9.4 Biological Properties of Citral
9.4.1 Anti-inflammatory Properties
There are several severe health issues in the world; inflammation is one of these issues.
The main causes of inflammation of tissue include physical stress and chemical inducers like lipopolysaccharides. The discharge of proinflammatory facilitators like prostaglandin E2 (PGE2) and nitric oxide (NO) by incubated lipopolysaccharides with macrophages can cause inflammation. The fluctuation in nuclear factor kappa-B cells (NF-κB), tumor necrosis factor-TNF-α, interleukins, reactive oxygen species (ROS), and cytokines are other factors that can induce inflammation. Several investigators reported that isolated citral has the strong property of anti-inflammation, while solvent extracts of lemongrass and polyphenol-rich extractants showed low to mild anti-inflammatory activities. The secondary metabolites of lemongrass including citral have anti-inflammatory effect on paw edema and peritonitis induced by carrageenan in model rat. Paw edema was reduced by using citral, and peritonitis was reduced due to mitigation of leukocyte conversion to peritoneal cavity. Generally, citral is dose reliant in decreasing protein expression, both alpha and gamma peroxisome proliferator-activated receptor, COX-2 mRNA in human macrophage (U937) induced by LPS (Katsukawa et al. 2010). Alpha and gamma peroxisome proliferator-activated receptor is cluster of nuclear receptor proteins that have important role to control the metabolism, differentiation, and cell development by acting as transcription factor (Kulinsky 2007). Citral also reduced the production IL-10, IL-6, and IL1-β resultant in the inhibition of cytokine in both LPS introduced peritoneal macrophage and animal as well as in control (Sforcin et al. 2009; Bachiega and Sforcin 2011). Treatment with citral oil in mice with lung injury induced by LPS inhibited IL-1β, TNF-α, and IL-6 levels both in vivo and in vitro, demonstrating that the citral can inhibit a possible inflammatory response (Shen et al. 2015).
It was also demonstrated that the alcoholic extract of lemongrass, which has, as major compound citral, reduced the generation of TNF-α in bronchoalveolar macrophages stimulated with LPS, enhancing the anti-inflammatory property of citral and indicating that modulation of the COX-2 and TNF-α genes can be one of the processes involved in such activity (Tiwari et al. 2010). Citral inhibited the phosphorylation interaction with inhibitory proteins kB (IkB), blocking translocation of the p50 and p65 subunits of NF-kB and leading to a low expression of inducing enzyme nitric oxide synthetase (iNOS) (Lee et al. 2008).
9.4.2 Antioxidant Properties
Free radicals, superoxide anion, and hydrogen peroxide are the major reactive oxygen species (ROSs) formed by the reaction of oxidation in tissue, cell, and organ systems of human (Heo et al. 2003). ROSs are very reactive and can damage various cell components and biomolecules such as DNA, structure and nature of proteins, cellular lipids, and cell membranes (Devasagayam et al. 2004). Furthermore, ROSs can induce health problems like muscle destruction, rheumatoid arthritis, and atherosclerosis. The body has antioxidants which are able to fight ROSs and can provide protection against oxidation effect of free radicals (Finkel 1998; Thannickal and Fanburg 2000). DPPH scavenging test showed the antioxidant potential of lemongrass oil. The data available in literature shows that extracts of both leaves and stalk have antioxidant potential which were dose reliant (Mirghani et al. 2012). Bouzenna et al. (2017) examined the antioxidant effect of citral and its possible protecting effects against toxicity induced by aspirin in in vitro condition. Ferric reducing antioxidant power (FRAP), β carotene/linoleic, and 1,1diphenyl-2-picrylhydrazyl (DPPH) are used generally to find out the antioxidant potential of various molecules including citral. Citral showed FRAP with effective concentration (EC 50) 125 ± 28.86 μg/ml and inhibits the oxidation of linoleic acid as well as moderates the DPPH. The combined dose of aspirin and citral reduced cell death induced by aspirin. Citral controlled the activities of superoxide dismutase (SOD) and glutathione. Citral also prevents the activation of MAPKs. The data obtained from above study suggested that citral can provide the protection to IEC-6 cells from oxidative stress, induced by aspirin. Oxidative stress is helpful to search new molecules from available natural substances with antioxidant potential. The antioxidant potential of pure citral is similar to the antioxidant potential of ascorbic acid. A high treatment dose of citral (50 mg/kg body weight) did not show any mutagenic effect on mice. High dose citral showed no harmful effect on direct ingestion (Rabbani et al. 2005). Citral has antioxidant potential, and it can serve as antioxidant defense to protect the plant from ROSs or free radicals.
9.4.3 Antibacterial Properties
Gupta et al. (2017) studied the combined action of norfloxacin and citral against Staphylococcus aureus (SA) and drug-resistant strains. Espina et al. (2017) studied the sterilizer power of carvacrol (500–2000 μl/l) or citral against mature biofilms of L. monocytogenes (EGD-e), S. aureus (SC-01), and E. coli (MG1655). Carvacrol at the rate of 1000 ppm reduced sessile cells (reduced 5 log cycles) making mature biofilms of all three studied species. The results of above study showed the potential of the citral capable to eradicate the biofilm of foodborne pathogens. In recent times, various plant materials were tested for antibacterial activity, and results are very promising which make positive approach to discover new biomolecules with antibacterial potential. The antibacterial potential of different extracts of lemongrass including essential oil has also been examined by various workers (Grace et al. 1984). α-citral and β-citral also known as geranial and neral, respectively, are among the main aromatic compound of lemongrass oil. Both α- and β-citral showed antibacterial activity against both gram-positive and gram-negative bacteria. Another component of lemongrass oil, myrcene, has no direct effect on bacteria individually but improves the activity when used in combination with other components (Grace et al. 1984). Use of the essential oil citral in the local therapy of infectious diseases triggered by S. aureus showed positive action. However, the existence of a harmful action or interference in the physiology and/or structures of bacterial cells of this natural product and its bioavailability when used in living beings are not well understood. The joint influence of the air pouch model, essential oil citral, and S. aureus, since the citral appears to be a potential therapeutic agent for treating local infections triggered by S. aureus. In conclusion, the treatment with essential oil citral in the infection triggered by S. aureus led to a reduction of some features of acute inflammation, including the number of monocytes. The TNF-α cytokine has proved to be a more sensitive biomarker, in ELISA and RT-qPCR array. By reducing TNF-α concentration, EOC promoted the reduction of transcription of genes related to proinflammatory cytokines. The action of the EOC seems to have a better response in a period of 4 h; thus, this suggests that the EOC can act as a modulator of the immune system by decreasing cellular migration and the production of proinflammatory cytokines following infection with S. aureus.
9.4.4 Anti-obesity and Antihypertensive Properties
Aqueous extract of citratus at a dose of 500 mg/kg/day reduced hypoglycemic index significantly in the presence of counter-regulators like glucan, cortisol, and catecholamine. It was noted that hypolipidemic effect reduced in blood stream with low lipid density level. The extracts of lemongrass including essential oil relaxed various tissues like rat mesentery, rat aortic rings, and rabbit ileum (Bastos et al. 2010; Devi et al. 2011, 2012). For example, citral produced a dose-reliant vasorelaxation in phenylephrine aortic rings (pre-constricted) of male SHRs or WKRs (Devi et al. 2012). Similarly, intravenous administration of citronellol (acyclic monoterpenoid) created a hypotensive response in Wistar rats. Factors like indomethacin, hexamethonium, and atropine have no effect on such type of hypotensive response (Bastos et al. 2010). Citronellol used endothelium-independent process to prompt relaxation to superior mesenteric artery of rat. The potassium channels dependent on tetraethylammonium has no relation with arteries without endothelium. Calcium channels operated by voltage inhibited Ca2+ influx to activate citronellol and regulate intracellular Ca2+ stores (caffeine gated) and IP3 (Bastos et al. 2010).
Citral was found to be a moderate inhibitor of mammalian alpha-amylase, with an IC50 of 120 μM and caused also a decrease of alpha-amylase levels in vivo (Najafian et al. 2011). Moderate lowering of postprandial glucose, alongside with normalization of blood lipid profile, was observed in diabetic rats upon treatment with the compound. Water intake and urine volume of diabetic rats are also showing a remarkable decrease with the use of 16 mg/kg of citral, which is in accordance with its effect on blood glucose, and interesting in terms of the therapeutic benefits that it could have on these discomforting consequences of diabetes in patients. Citral was also found to be able to promote weight loss and to decrease food intake. On the basis of above findings, Najafian et al. (2011) proposed citral as a possible antihyperlipidemic agent in diabetes and potential therapeutic in obesity.
9.4.5 Antinociceptive Properties
Lemongrass oil containing citral is used in experiments on three nociception models of mice to find out the antinociceptive properties. In hot plate test, intraperitoneal supply of essential oil increased the response to stimuli in mice, while induction by acetic acid exhibited that oral and intraperitoneal supply of essential oil inhibits the contraction in the abdomen in a dose-reliant manner. In another test with formalin, supply of essential oil through IP inhibited licking time in both (first and second) phases of experiment (Viana et al. 2000). They observed the role of opioid receptors in the action of antinociceptive as antagonist naloxone obstructed function of essential oil found in the extract. The investigators of the same group pronounced that differences in reports published previously might be due to chemotypes used in experiments.
Quintans-Junior et al. (2011) reported antinociceptive potential of citral isolated from lemongrass. They used acetic acid writhing and formalin-induced nociception to study the antinociceptive properties of citral. Conclusively, citral is able to exhibit antinociceptive property by inhibiting nociception and writhing.
9.4.6 Anti-fungi Properties
Citral showed antifungal activity by damaging cell wall and membrane of spore of Aspergillus flavus. Inside the cell, citral interacts with DNA and their mitochondrial processes and also aggregates protein-like molecule that leads further damage of the cell. All these events inside the cell lead disorder in metabolic reaction which diminished the germination ability of the spore (Luo et al. 2004). The three fungi known as F. subglutinans, C. gloeosporiodes, and C. musae, responsible for postharvest diseases of fruits, are affected by citral as it can alter the morphology of fungal hyphae (Garcia et al. 2008). The antifungal activity of citral is also reported for Penicillium digitatum, a postharvest pathogen of lemon fruit (Ben-Yehoshua et al. 1995).
Agar dilution method is used to determine the minimum lethal concentration (MLC) and minimum inhibitory concentration (MIC) of citral oil against different isolates of four dermatophytes (M. gypseum, E. floccosum, T. rubrum, and T. mentagrophytes). The data of MLC and MIC indicated that citral has mild effect on all isolates of dermatophytes than that of essential oil. M. gypseum was the most resistant which is followed by T. rubrum. The results of above study proved the antifungal activity of citral and lemongrass oil, and both can be used as fungicides. The hole diffusion assay was followed in vitro condition to study the effectiveness of cream with four different doses of oil of lemongrass. The cream containing 2.5% oil of lemongrass showed minimum concentration to control the fungal infection hence used to make antifungal cream for further clinical study (Wannissorn et al. 1996).
Desai and Parikh (2012) used a hydrotropic combined solution of sodium cumene sulfonate and sodium salicylate to extract the citral content from leaves of lemongrass (C. flexuosus). Plant material, temperature, solid loading, and hydrotrope concentration can affect directly the yield of citral. Taguchi method gave highest extraction in which both hydrotropes registered highest citral yield with conditions as 5% solid loading, temperature of 30 °C, and size of 0.25 mm of pieces of leaves. Lower performance of sodium cumene was noted for extraction than that of sodium salicylate. Extraction mechanism can be understood by microscopic analysis of leaves that provide insight of leaves. The efficiency of hydrotropes for extraction was checked from the kinetic study. The organic solvent can be avoided in the extraction of citral with help of hydrotropes under hydrotropic extraction. Hydrotropic extraction technique can be used to extract different biomolecules and oils from plants as this technique is very simple and environment friendly.
In another observation by OuYang et al. (2018), they noted that citral prevents the growth of P. digitatum by accumulation of ROS as a result of damage in cell membrane and oxidative phosphorylation.
9.4.7 Anticancer Properties
The anticancer property of citral was exposed when a report published to claim that caspase 3 activity induced by citral in the HL60 and U937 cell lines in 2005. The potential of citral to treat the cancer has not been completely explained, but citral is among the natural compound of plant origin that gave some promising results against several human cancer cells like HL60, ovarian cancer cells, U937, etc. (Liu et al. 2012). Another positive observation regarding citral is that it showed very little or negligible cytotoxic effect on normal epithelial cells but showed sufficient toxicity against breast cancer cell line and indicated cancer-specific effect of citral (Patel et al. 2015). In vitro condition, citral can induce the cell death in the cells of leukemia and breast cancer (Dudai et al. 2005; Xia et al. 2013). Maruoka et al. (2018) observed that citral alone or combined dose with chemotherapeutic agents can suppress proliferation of lung cancer cell by inhibiting Src/Stat3 activities. Naz et al. (2018) studied the potential of citral and mode of its action to inhibit the activity of microtubule affinity-regulating kinase 4 (MARK4).
Citral can bind the active site effectively and stabilize the complex with several interactions. The above observation is made by docking studies. They noted the strong stability in binding of citral with MARK4. The similar findings were obtained from fluorescence binding studies which also indicate that citral inhibits enzyme activity of MARK4 that measured through kinase inhibition assay. Citral-treated cells of MCF-7 showed inhibition in growth as these cells are arrest in cell cycle phase (G2/M phase) and citral-induced apoptosis. Citral treatment decreased synthesis of prostaglandin E2 within 48 h. The above study established the fact that citral can be used to treat the cancer by MARK4 inhibition (Chaouki et al. 2009). Dubey et al. (1997) noticed that citral has anticancer potential. Citral showed the cytotoxic effect on mouse leukemia cells (P388) at IC50 value (7.1 μg/mL). Micronucleus antimutagenic assay was used to study antimutagenic effect on mutagens like nickel metal (NiCl2), mitomycin C, and cyclophosphamide. High dose of citral was used to check the mutagenic potential, and the result showed that no significant change in micronucleus frequencies of erythrocytes that proved citral is nonmutagenic. Moreover, this study proposes that citral reduces nuclear injury prompted by the clastogens by utilizing antioxidant potential (Rabbani et al. 2005). White et al. (2017) examined effect of citral on immortalized rhabdomyosarcoma (RMS) cells and found significant death rate in cancer cells at and above the dose of 150 μM citral, and significant changes were noted in morphology of mitochondria of the cell incubated with 10 μM citral.
9.5 Conclusions and Future Prospects
Citral is one of secondary metabolites of lemongrass and has lemon-like aroma. Due to its aromatic nature, it involved to provide the fragrance to several formulations and take part in the formation of consumer products as flavor gradient. The citral molecule is unstable and lost its properties like flavor over time in watery solutions because of the oxidative reactions. The use of citral in food industry is a big challenge due to its unstable nature. Another challenge is to develop the delivery system of citral content for food industry. Colloidal systems are generally used to encapsulate and in delivery technique of citrate. There is need to focus the technical problems like stabilization of citral, use of cofactors, instability of citral under various environmental stresses, and the preparation of citral-based nanoparticle, etc. All above technical problems related with stability of citral and development of new techniques to use citral in the protection of various pathogenic diseases including cancer should be addressed as future prospects to develop particular formulation to treat particular disease without any side effect.