**6. Selected examples**

There are several plant secondary metabolites including among others alkaloids, terpenes, flavonoids, and glycosides, which can be produced by plant tissue culture techniques using different strategies [3, 13, 14]. Two examples were selected to be described in this chapter: the production of important anticancer compounds and the production of metabolites from *Lavandula* spp.

#### **6.1. Anticancer compounds**

As mentioned before in this chapter, over 60% of anticancer drugs are directly or indirectly derived from plants [7]. The search for anticancer compounds from plants started in the 1950s when the alkaloids vinblastine and vincristine from *Catharanthus roseus* (L.) G. Don and podophyllotoxin from *Podophyllum* spp. were discovered. The United States National Cancer Institute initiated an extensive program in 1960 that led to the discovery of many novel chemotypes with cytotoxic activities [55], taxanes and camptothecins being some of the examples [7]. Camptothecin, podophyllotoxin, taxol, vinblastine, or vincristine are the most important plant-derived anticancer compounds [19, 56]. Most compounds with anticancer properties are alkaloids, and some of them have a complex structure, with multiple rings and chiral centers, and therefore the chemical synthesis is prohibitively expensive [17]. Plant cell and tissue culture techniques appear as environmentally friendly alternative methods for the production of these secondary metabolites [17, 19].

Taxanes from *Taxus* spp., terpenoid indole alkaloids from *C. roseus*, camptothecin from *Camptotheca acuminata* Decne among other species, and podophyllotoxin from *Podophyllum* and *Linum* spp. are the main compounds produced by using biotechnological approaches (**Table 1**). For the production of taxanes, cell suspension cultures are definitively the most adequate culture system. However, some studies demonstrated that differentiated tissues are more adequate than undifferentiated cells to produce other anticancer compounds. For instance, intact plants of *C. acuminata* contain around 0.2–5 mg/g dry weight (DW) of camptothecin while callus and suspension cultures produced only 0.002–0.004 mg/g DW or lesser [57]. Hairy root cultures have also proven to be a good option for *in vitro* production of secondary metabolites as indole alkaloids due to their higher level of cellular differentiation and improved genetic or biochemical stability. The hairy roots of *Ophiorrhiza pumila* Champ. ex Benth showed a high capacity to produce camptothecin (0.1% DW), although the callus culture failed to produce this compound [58].

cultures, it has been possible to enhance production yields. Several factors have been optimized, such as nutrients, carbon source, plant growth regulators, or culture environmental conditions, and several biotic and abiotic elicitors have been tested. Studies have also been focused on the elucidation and regulation of biosynthetic pathways and on aiming the increase of production yields of anticancer compounds as taxanes [41] and indole alkaloids [59] by using elicitors to activate genes involved in metabolic pathways. In spite of all the advantages of producing anticancer compounds by using plant cell and tissue culture techniques and the significant advancements in the last years, the examples of the production of plant anticancer compounds on an industrial level are scarce. As previously mentioned in this chapter, the best success example is the production of taxanes by the Germany company Phyton Biotech [51]. Plant cell and tissue culture techniques have also been applied for the propagation of several anticancer plants. *In vitro* propagation allows the rapid mass multiplication of true-to-type plants within a short span of time which is particularly important in the case of endangered species. Some recently selected examples comprise plants producing the important anticancer

**Table 1.** Some examples of studies reporting the production of plant anticancer compounds using biotechnological

**Compound(s) Group Source (plant species) Culture** 

Camptothecin Monoterpene indole

alkaloid

**system**

http://dx.doi.org/10.5772/intechopen.76414

CC [77]

CSC [78]

HRC [58]

CSC [84]

HRC [86, 87]

*Camptotheca acuminata* Decne HRC [75]

Production of Plant Secondary Metabolites by Using Biotechnological Tools

*Camptotheca acuminata* Decne CSC [76]

*Ophiorrhiza alata* Craib HRC [79] *Ophiorrhiza mungos* Linn. CSC [80] *Ophiorrhiza prostata* D. Don ARC [81]

*Corylus avellana* L. CSC [85]

*Nothapodytes foetida* (Wight)

*Nothapodytes nimmoniana*

*Ophiorrhiza pumila* Champ.

*Linum album* Kotschy ex

Sleumer

(J. Grah.)

ex Benth.

Boiss.

G. Don

Podophyllotoxin Aryltetralin lignan *Linum* spp. HRC [82, 83]

Taxanes (taxol) Diterpene alkaloids *Taxus* spp. CSC [27, 28]

ARC: adventitious root culture; HRC: hairy root culture; CSC: cell suspension cultures; CC: callus culture.

Vinblastine and vincristine Terpene indole alkaloids *Catharanthus roseus* (L.)

approaches.

**Reference(s)**

89

compounds camptothecin [60, 61] and podophyllotoxin [62, 63].

Several researchers have focused on studies aiming for the optimization of biomass growth conditions and on the application of biotechnological strategies to increase production yields of anticancer compounds. By manipulating empirical factors related to plant cell and organ


for hairy and adventitious root cultures [14]. The interested reader can find more important details about the scale-up process in the works by Murphy et al. [14], Yue et al. [13],

There are several plant secondary metabolites including among others alkaloids, terpenes, flavonoids, and glycosides, which can be produced by plant tissue culture techniques using different strategies [3, 13, 14]. Two examples were selected to be described in this chapter: the production of important anticancer compounds and the production of metabolites from

As mentioned before in this chapter, over 60% of anticancer drugs are directly or indirectly derived from plants [7]. The search for anticancer compounds from plants started in the 1950s when the alkaloids vinblastine and vincristine from *Catharanthus roseus* (L.) G. Don and podophyllotoxin from *Podophyllum* spp. were discovered. The United States National Cancer Institute initiated an extensive program in 1960 that led to the discovery of many novel chemotypes with cytotoxic activities [55], taxanes and camptothecins being some of the examples [7]. Camptothecin, podophyllotoxin, taxol, vinblastine, or vincristine are the most important plant-derived anticancer compounds [19, 56]. Most compounds with anticancer properties are alkaloids, and some of them have a complex structure, with multiple rings and chiral centers, and therefore the chemical synthesis is prohibitively expensive [17]. Plant cell and tissue culture techniques appear as environmentally friendly alternative methods for the produc-

Taxanes from *Taxus* spp., terpenoid indole alkaloids from *C. roseus*, camptothecin from *Camptotheca acuminata* Decne among other species, and podophyllotoxin from *Podophyllum* and *Linum* spp. are the main compounds produced by using biotechnological approaches (**Table 1**). For the production of taxanes, cell suspension cultures are definitively the most adequate culture system. However, some studies demonstrated that differentiated tissues are more adequate than undifferentiated cells to produce other anticancer compounds. For instance, intact plants of *C. acuminata* contain around 0.2–5 mg/g dry weight (DW) of camptothecin while callus and suspension cultures produced only 0.002–0.004 mg/g DW or lesser [57]. Hairy root cultures have also proven to be a good option for *in vitro* production of secondary metabolites as indole alkaloids due to their higher level of cellular differentiation and improved genetic or biochemical stability. The hairy roots of *Ophiorrhiza pumila* Champ. ex Benth showed a high capacity to produce camptothecin (0.1% DW), although the callus cul-

Several researchers have focused on studies aiming for the optimization of biomass growth conditions and on the application of biotechnological strategies to increase production yields of anticancer compounds. By manipulating empirical factors related to plant cell and organ

and Isah et al. [3].

*Lavandula* spp.

**6. Selected examples**

88 Secondary Metabolites - Sources and Applications

**6.1. Anticancer compounds**

tion of these secondary metabolites [17, 19].

ture failed to produce this compound [58].

**Table 1.** Some examples of studies reporting the production of plant anticancer compounds using biotechnological approaches.

cultures, it has been possible to enhance production yields. Several factors have been optimized, such as nutrients, carbon source, plant growth regulators, or culture environmental conditions, and several biotic and abiotic elicitors have been tested. Studies have also been focused on the elucidation and regulation of biosynthetic pathways and on aiming the increase of production yields of anticancer compounds as taxanes [41] and indole alkaloids [59] by using elicitors to activate genes involved in metabolic pathways. In spite of all the advantages of producing anticancer compounds by using plant cell and tissue culture techniques and the significant advancements in the last years, the examples of the production of plant anticancer compounds on an industrial level are scarce. As previously mentioned in this chapter, the best success example is the production of taxanes by the Germany company Phyton Biotech [51].

Plant cell and tissue culture techniques have also been applied for the propagation of several anticancer plants. *In vitro* propagation allows the rapid mass multiplication of true-to-type plants within a short span of time which is particularly important in the case of endangered species. Some recently selected examples comprise plants producing the important anticancer compounds camptothecin [60, 61] and podophyllotoxin [62, 63].

#### **6.2.** *Lamiaceae* **spp. metabolites**

The mint family (*Lamiaceae*) contains about 236 genera and more than 7000 species with cosmopolitan distribution [64]. Some of the most important genera are *Hyptis*, *Lavandula*, *Nepeta*, *Salvia*, *Scutellaria*, *Thymus*, and *Teucrium.* Species from the family inhabit different natural ecosystems, and many are already cultivated. Most of the species belonging to this family are aromatic (possess essential oils) and are widely used in traditional medicine to cure various disorders. They also have great economic value due to their use in culinary or as ornamentals, and for cosmetic, flavoring, fragrance, perfumery, pesticide, and pharmaceutical applications [65]. Many *Lamiaceae* contain high levels of phenolics, which are probably the most relevant group of secondary metabolites synthesized by plants due to their health promotion effects [64]. Among phenolic compounds, rosmarinic acid is present in the tissues of many of these species being used as a chemical marker of the family [64, 66, 67]. In some species, this compound is accumulated as the main phenolic compound at a concentration above 0.5% dry weight [64]. Several species in the *Lamiaceae* family can also accumulate high levels of other phenolic acids, flavonoids, or phenolic terpenes [64]. There are some phenolic compounds as carnosic and clerodendranoic acids that are exclusive from this family [68, 69]. The interested reader can find an excellent overview on the phytochemical characterization and biological effects of *Lamiaceae* species in Trivellini et al. [64].

biotechnological production of this compound but its large-scale production still requires further optimization. The molecular understanding of its biosynthesis and the application of metabolic engineering tools are crucial to improve the biotechnological production.

**Table 2.** Selected examples of elicitation treatments applied in cultures of *Lamiaceae* species to increase the production

**Plant species Tissue Elicitor Reference** *Coleus blumei* Benth. HRC Methyl jasmonate [88] *Coleus forshohlii* (Willd.) Briq. HRC Methyl jasmonate [73] *Lavandula officinalis* Chaix CSC Jasmonic acid [72] *Lavandula vera* MM CSC Vanadyl sulphate [89] *Lavandula vera* MM CSC Methyl jasmonate [90] *Mentha* × *piperita* CSC Methyl jasmonate [91] *Rosmarinus officinalis* L. L UV-B [74]

Production of Plant Secondary Metabolites by Using Biotechnological Tools

http://dx.doi.org/10.5772/intechopen.76414

91

Plant cell and tissue culture techniques are an attractive system for the cultivation of a broad range of secondary metabolites, including important alkaloids with anticancer properties and bioactive phenolics. This alternative provides a continuous, sustainable, economical, and viable production of secondary metabolites, independent of geographic and climatic conditions, which is particularly useful for the production of species at risk. Despite the great progresses in this area in the last decades, in some cases, production occurs at very low yields, and there are many difficulties in scaling up the production, and limited commercial success is achieved. Incomplete knowledge about the biosynthetic pathways of bioactive molecules limited the improvement of the production yields. Exploiting modern molecular biology techniques emerged as an alternative that needs to be harnessed to improve production efficiency by engineering biosynthetic pathway(s) of the molecules in plant cells. Also promising are new elicitors and permeabilizing agents such as coronatin or cyclodextrins. The production of bioactive molecules in endophytes also appears as an attractive alternative, although till date,

This work is supported by National Funds—FCT (Fundação para a Ciência e a Tecnologia, I.P.), through the project UID/BIA/4325/2013. S. Gonçalves acknowledges a grant from the FCT (SFRH/BPD/84112/2012) financed by POPH-QREN and subsidized by the European

**7. Conclusions and prospects**

of rosmarinic acid.

HRC: hairy root culture; CSC: cell suspension cultures; L: leaves.

there is no reported commercial exploitation.

**Acknowledgements**

Science Foundation.

Phenolic compounds are generally produced as a defense mechanism or as a response to stressful environment conditions [9]. The activation of these protective mechanisms by applying stress stimulus can be used as a strategy to increase the production of phenolic compounds in plant cell and organ cultures [70]. Recently, several attempts were made regarding the production of secondary metabolites by several *Lamiaceae* species (mainly phenolics) using plant tissue cultures particularly applying elicitation as a strategy to achieve higher production yields [64]. These studies involve mainly the use of chemical elicitors like jasmonic acid (or methyl jasmonate), or physical elicitors as UV-B and ozone (O3 ), to increase the production of many compounds as essential oil constituents, phenylpropanoids, flavonoids, and phenolic acids. Overall, the results demonstrated that these elicitors had an immediate effect on enhancing the production of phenolics [64].

The revised study showed that a high number of studies reported an increase in the production of rosmarinic acid after elicitation of cultures of several *Lamiaceae*, such as *Coleus*, *Lavandula,* and *Salvia* genera [64, 66, 71]. Several studies reported the increase in rosmarinic acid production through the application of elicitors (**Table 2**). Elicitation with jasmonic acid induces a 4.6-fold increase of rosmarinic acid production in *L. officinalis* L. cell suspension cultures [72], and elicitation with methyl jasmonate induces a 3.4-fold increase in *C. forskohlii* (Willd.) Briq. hairy root cultures [73]. The production of this compound also increased (2.3 fold) in leaves of *Rosmarinus officinalis* L. after 14 days of UV-B exposure [74]. Recently, rosmarinic acid attracted the attention of the scientists due to its broad range of biological activities, such as anti-inflammatory, antioxidant, cognitive-enhancing, cancer chemoprotection effects, among others [71]. In the last years, there are many progresses in the


**Table 2.** Selected examples of elicitation treatments applied in cultures of *Lamiaceae* species to increase the production of rosmarinic acid.

biotechnological production of this compound but its large-scale production still requires further optimization. The molecular understanding of its biosynthesis and the application of metabolic engineering tools are crucial to improve the biotechnological production.
