Keywords

1 Introduction

Interest in medicinal plants has increased in past few decades due to their low cost and safety. Herbal drug is a quick growing sector worldwide; it is estimated that the world trade in medicinal plants will reach US$ 5 trillion by 2050 (Anonymous 2000). Terpenoids are naturally occurring hydrocarbons of plant origin with general formula (C5H8)n. The C5H8 unit is called isoprene unit (2-methyl-1,3-butadiene). Terpenoids constitute about 30,000 identified compounds (Dzubak et al. 2006; Mufflera et al. 2011). It is reported that terpenoids are probably the largest group of phytochemicals. They include essential oils phytohormones (cytokinin and gibberellins), resins, steroids, carotenoids, and others (Lohr et al. 2012). Terpenoids or isoprenoids are a group of structurally diverse phytochemicals known for their wide range of pharmacological activities. The term terpene is used for compounds with C5 (isoprene) units. Terpenoids are classified on the basis of number of isoprene units (Table 8.1).

Table 8.1 Classification of terpenoids
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The isoprene units have a branched and an unbranched ends, earlier termed as head and the later as tail. Although head-tail arrangements of the isoprene units are the commonest type of linking, e.g., in mono-, sesqui-, di-, and sesterterpenoids, tail-tail arrangements are at center of tri- and tetraterpene molecules. Polyisoprenoids like rubber and gutta are two of the many other naturally occurring compounds in which non-terpenoid structural parts are linked to terpenoids. Structure and examples of terpenoids are depicted in Table 8.2.

Table 8.2 Few examples and structures of terpenoids with molecular weight and compound identity (CID) from PubChem (National Center for Biotechnology Information 2021a, b, c, d, e, f, g, h, i, j, k, l, m, n, o, p)
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Apart from this, lower plants like bryophytes are also reviewed for the presence of terpenoids. It is estimated that in the past 40 years, around 1600 terpenoids have been reported from this plant group (Asakawa 1982, 1995; Asakawa et al. 2013). Ludwiczuk and Asakawa (2019), in their review on bryophytes, have enlisted over 128 studied pharmacologically active terpenoids from liverworts and mosses. Many of them are species specific like dumortane type (Dumortiera hirsute), hodgsonoxanes (Lepidolaena hodgsoniae), bergamotanes, and clavigerins (L. clavigera) (Chen et al. 2018). A mini review by Pai and Joshi (2014) have enlisted about 56 plant species producing triterpenoid betulinic acid. Very recently, 7 undescribed terpenoids, alongside 26 known compounds with isolated from aerial parts of Elsholtzia rugulosa (Yang et al. 2021). In a separate study, Yu et al. (2020) reported six new terpenoids from Eclipta prostrata, thus indicating continuation of adding new terpenoids to the family. Below is the list of some plants, parts, and terpenoids reported (Table 8.3).

Table 8.3 Various terpenoids of plant origin
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Isoprenoids have reported to be having functionalities in plant cells. They serve as starting material in the formation of various plant hormones. Differential expression of pathway isozymes; metabolic regulation and network, alongside regulation to light, external stimuli; and metabolic exchanges across the subcellular compartments have been well documented by Tholl (2015). Terpenoids like sterols function in fluidity of plasma membrane; polyprenyl dolichol is and sugar carrier lipid used in protein glycosylation; phytol is a part of chlorophyll, tocopherols, phylloquinones, carotenoids thus functioning in photosynthesis, and polyprenyl plastoquinone in electron transport chain (Lohr et al. 2012).

A number of genes coding enzymes and regulators in biosynthesis of terpene were studied with respect to their location, genome, and expression (Chen et al. 2011). Though much is understood about the terpene metabolism, recent findings are revealing new substrates and enzymatic reactions in its biosynthesis (Zhou and Pichersky 2020). On other hand, alternative strategies like chemical synthesis are impractical due to the complex nature of these compounds (Misawa 2011; Oksman-Caldentey and Inzé 2004). With limited commercial success, biotechnological methods give an attractive alternative to obtain these drugs. Comprehensive reviews have already been published on microbial transformation and its production using tissue culture methods (Parra et al. 2009; Malinowska et al. 2013). This chapter concentrates on in vitro production of terpenoids.

2 Biosynthesis Pathway

It is one of the large groups of phytochemicals in plant kingdom. They include representations in large number of plant molecules such as vitamins and hormones. Isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) are the two precursors which significantly contribute into the biosynthesis of terpenoids. Classical studies by a number of workers have elucidated biosynthesis of terpenoids via acetyl-CoA and mevalonate (Qureshi and Porter 1981; Bloch 1992; Bach 1995; Bochar et al. 1999). Thus, mevalonate is the best studied pathway for terpenoid biosynthesis which includes mevalonic acid as an intermediate. Recent studies have revealed variations in biosynthesis of terpenoids production from the common 2-C-methyl-d-erythritol 4-phosphate (MEP) and the mevalonate (MVA) pathways. The earlier (MEP) being working in plastids and later one (MVA) in cytoplasm, endoplasmic reticulum and peroxisomes (Vranová et al. 2013).

As reported by Habtemariam (2019), acetyl-CoA a primary metabolite undergoes a series of reaction to produce a six carbon molecule mevalonic acid (Fig. 8.1). This further produces DMAPP and IPP an interchangeable 5 carbon (C) building blocks. Isopentenyl diphosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) condense to produce geranyl diphosphate (GPP), which is considered as the intermediate precursor for all monoterpenes. The GPP serves precursor to monoterpene production, and further addition of a 5 carbon unit (IPP/DMAPP) produces farnesyl pyrophosphate (FPP) a precursor of sesquiterpenes (15 C) and its dimer to give triterpenes (30 carbon). The addition of another 5 carbon unit to FPP gives geranylgeranyl diphosphate (GGPP) as an originator of diterpenes (20 C). Dimerization of GGPP gives tetraterpenes (40 C). Mostly, head-to-tail arrangements by addition of 5 carbon units are observed, exceptionally head to head in tri- and tetraterpenes. Though we consider 5 carbon unit (IPP and DMAPP) for biosynthesis terpenoids, monoterpenes (10 C) are considered to be the smallest unit.

Fig. 8.1
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An overview of terpenoid biosynthesis (source: Habtemariam 2019)

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Studies also reveal that enzymes such as Nudix hydrolases and isopentenyl phosphate kinase (IPK) affect biosynthesis of terpene in cytosol as well as plastids by regulating IPP and DMAPP ratios (Zhou and Pichersky 2020).

3 In Vitro Production Methodology

Plants are looked as inexhaustible sources of new drugs. In vitro methods as alternative strategies are applied in two ways for secondary metabolite production: (1) biomass and (2) synthesis. Biomass aggregation would rely on in vitro culturing of shoot, root, callus, etc. (Guerriero et al. 2018; Hussain et al. 2012). Terpenoids are no different, and they play vital role in the organism. Apart from pharmacological properties of terpenoids, their cosmeceuticals, antioxidant, and nutraceutical properties of terpenoids are studied vastly (Bonfill et al. 2013). In vitro culture techniques coupled with bioengineering has demonstrated great applications. Artemisinin a sesquiterpene lactone drug from Artemisia annua is known for its antimalarial property. Rationally, overexpression of farnesyl diphosphate synthase (FDS) enzyme which condenses IPP with DMAPP to form (FPP) farnesyl diphosphate yields higher artemisinin. The same was achieved by placing a 35S CaMV promoter with FPS gene using Agrobacterium mediated transformation. This study showed about fourfold increase in the content (Exposito et al. 2010). Farzaei et al. (2017) included 55 mono- and sesquiterpenoids alongside other compounds in their review on pharmacological and phytochemical properties of Mentha longifolia. They also tabulated plant part used and the extraction method. Few of the plants alongside their in vitro methods studied and terpenoids identified are enlisted below (Table 8.4).

Table 8.4 Terpenoids with method of in vitro production
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4 Scale-up Techniques and Bioreactors

Commercially important secondary metabolites (SMs) need scale-up using bioreactors, as the traditional way of production may not suffice the need. It takes help of modern methods like genomics, proteomics, metabolomics etc. to modify the metabolic routes in an organism for scaled-up production of SMs (DellaPenna 2001). Shikimate, polyketide, and terpenoids pathways are the main routes for production of secondary metabolites. Terpenoids comprise around one-third of the total secondary metabolites (Verpoorte 2000). Scaling-up may include metabolic engineering, up- and downregulating pathways, redirecting common precursors, and targeting metabolites to specific cell compartments (Chandran et al. 2020).

Overexpressing of FaNES1 (Fragaria ananassa nerolidol synthase 1) a protein identified as sensor for production of both mono and sesquiterpene precursors. High levels of monoterpene linalool was achieved by CaMV 35S-driven promoter and engineered with wild strawberry FvNES1 (Fragaria vesca nerolidol synthase 1) plastid targeting region (Aharoni et al. 2006). It was also reported that, compared to the monoterpenes, sesquiterpene engineering in plants is difficult as precursor pool required is less. In another study, Rodríguez et al. (2014) observed that downregulating production of terpene D-limonene is inversely correlated by lowering fungal infection in transgenic oranges. Redirecting common precursor, viz., prenyl pyrophosphate (C5), to provide an adequate flux of IPP and DMAPP can be achieved by manipulation of genes regulating MVA pathway or by inserting MEP pathway in organisms having only MVA pathway (Vavitsas et al. 2018; Yang et al. 2016). Escherichia coli and Saccharomyces cerevisiae are termed as industrial workhorses and are used to produce high-valued terpenoids in bioreactors. Zhang and Hong (2020) in their mini review have well documented a list of strategies for production of terpenoids by targeting it to specific cell compartments.

Gupta et al. (2021), in an overview on pharmaceutical properties and biotechnological advancements of Withania coagulans, have reported that the plant is on the verge of extinction. Withanolides are a group of steroidal lactone triterpenoids which can also be synthesized by terpenoids forming mevalonate pathway (Kreis and Muller-Uri 2010). Seven different methods have been highlighted for increased production of bioactive compounds from the plant; they include Agrobacterium-mediated transformation, metabolic engineering, plant tissue culture, germination by seeding, abiotic/biotic elicitors, gene transfer, and nanoparticle synthesis. To infer the work of Gupta et al. (2021), in vitro system is suggested to be the attractive option to achieve the goal.

Apart from the above, Zhang et al. (2019) used Agrobacterium-mediated transformation studies to functionally expedite terpenoids biosynthesis in Tripterygium wilfordii. In a separate study, pharmaceutically important metabolites were enhanced by adding silver nitrate (linalool, estragole) and yeast extract (rutin, isoquercetin) in Ocimum basilicum suspension cultures (Açıkgöz 2020). Polzin and Rorrer (2018) reported selective production of β-myrcene (monoterpene) from Octodes secundiramea on nutrient perfusion cultivation with bromide-free medium. High levels of amorphadiene a precursor of artemisinin were produced by using Bacillus subtilis as terpenoids cell factory (Pramastya et al. 2021). Finding of new genes and computational biology has boosted production of new terpenoids using microbial methods. Expression of plant enzymes in microorganisms has helped biosynthesis of diverse class of complex terpenoid derivatives (Belcher et al. 2020).

5 Extraction and Detection Techniques

A number of extraction and detection methods are employed for determining the contents of phytochemicals from plants (Fig. 8.2). Range of methods from conventional, simple maceration to recent ultra-sonication and microwave-assisted extractions are deployed for extracting terpenoids. In a study, phenolic and terpenoids were identified using reversed-phase high-performance liquid chromatography (RP-HPLC) and head space solid phase microextraction gas chromatography coupled with mass spectrometry (HS-SPME-GC/MS), respectively (Açıkgöz 2020; Muñoz-Redondo et al. 2020)

Fig. 8.2
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Various extraction methods reported

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More recently, Vaníčková et al. (2020) have identified species-specific terpenoid markers to chemo-taxonomically distinguish four species and one subspecies of Dracaena using solid-phase microextraction – coupled to a gas chromatography – ion trap tandem mass spectrometry (SPME-GC × GC-MS). Percolation and maceration extraction methods have been reported for extraction of terpenoids from stem and root material of Eurycoma longifolia. Supercritical fluid extraction and microwave-assisted extraction are some of the non-conventional methods utilized and reported for extraction in many plants including Andrographis paniculata for terpenoids andrographolide, deoxyandrographolide, and its variants (Aziz et al. 2021). Plants reported with different extraction and detection methods alongside terpenoids identified is tabulated in Table 8.5.

Table 8.5 Method of extraction and detection used in various plant terpenoids
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6 Biological Activities

The significance of terpenoids is because of their anticancer, anti-inflammatory, antiplatelet, antibacterial, hypocholesterolemic, immune adjuvant, anti-viral, antibacterial, fungicidal, and antileishmanial agents like pharmacological properties (Abdelrahman and Jogaiah 2020). Anticancer and antimicrobial properties are some of the largely studied activities of terpenoids. Apart from cancer treatment and life style disorders, withanolides as bioactive terpenoid derivative from Withania has proved antianxiety activity in albino mice model (Gupta et al. 2021). On other hand, a range of biological activities may be attributed to terpenoids from cucurbit family Cucurbitacin, and its derivatives from Cucurbitaceae have been reviewed against inflammation, cancer, and many other properties (Montesano et al. 2018). The terpenoids are not only studied from plant system but are reported from marine bacteria and fungi with similar properties. Altemicidin, marinocyanins A-F, azamerone, and napyradiomycins were some of the potent monoterpenoids identified from bacteria (Gozari et al. 2021). Chen et al. (2012) reviewed Sinularia soft coral as a source of terpenoids for potential bioactivities such as antimicrobial, anti-inflammatory, and cytotoxic activities. Terpenoids like andrographolide, glycyrrhetinic acid, ursolic acid, costunolide, β-elemene, glaucocalyxin A, and cucurbitacin B have been reviewed and reported to show anti-liver fibrosis by Ma et al. (2020).

Plant essential oils are a media of communication within and between the plants. These are chemical terpenes containing repeated units of isoprenes. Depending on the occurrence in plant parts, they may be for attracting pollinators or to repel predators. They are generally present on leaves in secretary glands (e.g., mint, sage, basil, pine), in flowers (e.g., orange, chamomile, clove), in wood (e.g., sandalwood, balsam, camphor), in fruits (e.g., star anise, fennel, apple), in rhizomes (ginger, turmeric), and in seeds (cardamom, nutmeg, pepper). Geraniol (geranium), linalool (mint), myrcene (thyme), and ß-ocimene (basil) are some of the important essential oils (and plant name) having terpenes (Böttger et al. 2018). They are termed biopesticides due to their repellent and insecticidal properties.

The sequence of outbreaks of viral infection particularly corona, starting from the year 2003, SARS-CoV; 2012, MERS-CoV; and 2019, SARS-CoV-2, raised global health concerns. Conventional drug limitations forced researchers to look into plant-based bioactive compounds. Bhattacharya et al. (2021), in their review, updated on antiviral drugs of plant origin which are potential players in counteracting these viruses. Rajan et al. (2021) in a recent commentary on promising antivirals from ayurvedic herbs against COVID-19 have mentioned 26 natural compounds along with terpenoids as a potential candidate based on in silico approach. Table 8.6 enlists some important bioactivities of terpenoids or terpenoid-derived compounds majorly reviewed by Martin-Smith and Sneader (1969).

Table 8.6 Important bioactivities of terpenoids
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7 Commercial Utilization and Prospects

Commercial utilization and prospects should include lucrative applications of terpenes in various industries. For example, rubber, a polyterpene with repeating units of isoprene, is extensively studied, known and one of the largest industries. Still, it is important to find and concentrate on such targets which are of real concern in the coming years. Industries like therapeutics, food, flavor, perfumery, or even fuel are highly commercially utilized, in relation to environment. However, without better environment and sustainable resources, all other industries will have poor prospects. Thus, environment and ecology being one of the three such points will be explored in this section.

7.1 Environment and Ecology

Terpenes are the most abundantly found and studied natural product. Out of the three main categories of natural products, it is estimated that terpenoid accounts for 55%, alkaloid 27%, and phenolic 18% (Croteau et al. 2000), thus overstating their importance in nature and for mankind. Cheng et al. (2007) have discussed the role of terpene in ecological point of view. Terpenes are assumed to have a role in earth’s atmosphere by interacting with solar radiations. The 500 teragrams of isoprene emission annually is being estimated which influence formation of ozone (Zwenger and Basu 2008). Beyond plant-insect interaction, terpenes also act as messengers in plant defense mechanism, which can be further utilized commercially for their insect repellent or even attractants.

7.2 Therapeutic, Perfumery, Food, Flavor

Advent of bioinformatics and progressions in molecular databases have contributed in understanding the synthesis and pathways much better. Commercially, they have contributed largely to therapeutics, perfumery, food, and flavor industries. Also their role in essential oils and cosmetics can’t be underestimated. Developing agronomic traits in medicinal plants is an important area of research explored in the past few decades. A combination of engineering to use “omics” technologies and computational and systems biology, along with synthetic chemistry, shows an imperative prospect in commercialization. Artemisinin, squalane, sandalwood, and patchouli oils are few of the examples discussed by Leavell et al. (2016) as potential terpenes in pharma and fine chemicals. The volatility of terpenoid supplies and prices has been attributed to smaller market size and the dependency on the plant source for its production. Thus, alternative strategies for stable production using fermentation are an attractive option. Amyris Inc. is a synthetic biotechnology company making its presence in all the four sectors for terpenoids, artemisinin (pharma), squalane, numerous fragrance oils (fine), isoprene, farnesene for lubricants, polymers (as commodity chemicals), and farnesene for diesel and jet fuel (fuels). Antimicrobial-resistant (AMR) bacteria or even superbug issues in present-day list of drugs can be updated by addition of phytocompounds chiefly terpenes (Mahizan et al. 2019; Zwenger and Basu 2008). Terpenes may reduce the rate of antibiotic resistance via livestock feed by substituting orthodox antibiotics.

7.3 Fuel

“Biofuel” a renewable source particularly from plant origin is another prospective application of terpenes in commercialization. Countries such as the USA, Brazil, and European Union are expressing interest and supporting research based on such alternative sources. Diesel obtained from Copaifera langsdorffii a natural biofuel largely contains terpenes. In a study, Mewalal et al. (2017) have summarized specific terpenes as appropriate standby or composite for present-day fuels. They have also made a comparison of such biofuels with much known fuels. It has been reported that terpenes could assist as standalone or blended up to 65% in diesel engines. There are more than a few challenges to leverage plant terpenes as a commercially viable source for replacing diesel or gasoline. A roadmap has been proposed by Mewalal et al. (2017) for commercial recovery of terpenes synthesized. Its tremendous potential as sustainable biofuel has been swotted and described.

The above-discussed points will raise concerns in the coming years. Thus, it was important to highlight the area of future research on terpenoids.

8 Conclusions

Terpenes are still being fully realized for their applications. Discovery of more and more number of terpenes will surely add to the ever-growing list of its utilization. Like most other bioactives, terpenoids from nature are supply limited. However, production at commercial level is driven by recent annotated technologies. Few important such terpenes are reviewed and discussed above. Differences in the challenges to produce terpenoids vary from one to another, thus making it non- comprehensive. It means that a method developed for one terpene cannot be applied to another. On the other hand, predictive engineering along with in vitro culture techniques can lead to selective production of terpenoids. With this, it is also projected that wide understanding of structure and activity liaisons of terpenes will open avenue for medicinally important phytocompounds. Further study on their activity as well as mechanism will be of great importance. Furthermore, work related to cost-effective and more contemporary way for production of biodiesels rather than the now conventional ethanol conversion from sugarcane or corn would be essential. Conclusively, owing to the ever-increasing number of terpenoids, a lot of study still can be undertaken for in vitro sustainable production, fundamental understanding of their role in biological processes, and commercial utilization.