**7. Biological activity of the antimalarial drug artemisinin**

The use of sesquiterpene lactone artemicin has been reported to have a variety of physiological effects on target cells, including disruption of mitochondrial function [18]. Artemisinin is a natural product synthesized by the Chinese plant *Artemesia annua* (sweet wormwood), which means that this molecule and its derivatives are now part of the front-line anti-malarial treatment. The effect of artemisinin on plant cells is unknown, but several studies have attempted to determine why this metabolism is toxic to the malaria parasite *Plasmodium falciparum* and other protozoa. Osteomycin is abnormal in possessing an endoperoxide moiety essential for cell function (**Figure 2**).

Artemisinin *Plasmodium falciparum* inhibits the absorption of oxygen, indicating that it may be the target respiratory chain [19]. In a new strategy, Li and colleagues explored the mechanism of action of artemisinin using a yeast sample and using yeast genetics to disrupt the mitochondrial membrane capacity of artemisinin [20]. Their work pointed out that the electron transport chain actually activates the mitochondrial depolarizing function of artemisinin. In contrast, Nagamun and colleagues found that artemisinin could not affect the mitochondrial membrane ability of another protozoan parasite *Toxoplasma gondii*, suggesting that mitochondria were not a primary target in the *T. gondii* [21]. In fact, there is now strong evidence that calcium affects homeostasis in the target species of artemisinin.

Eukaryotic cells use Ca2+ as a second messenger and generally maintain very low cytoplasmic concentrations of Ca2+ by dividing Ca2+ into segments, such as the endoplasmic reticulum. One of the key enzymes in this process is the sarcoplasmic/ endoplasmic reticulum Ca2+ -ATPase (SERCA). Heterologues host, *Xenopus lewis* using early work, demonstrated that artemisinin inhibits *P. falciparum* function [22] and recent experiments revealing the *T. gondii* in *S.cerevisiae* demonstrated that the *T. gondii* enzyme was inhibited by artemisinin. Physiological tests in many protozoa are consistent with the effects of artemisinin on calcium homeostasis, suggesting that it may account for a significant portion of the biological activity of this metabolite [23]. There are conflicting opinions on the biological functions of artemisinin with further studies to determine whether the malaria parasite *P. falciparum* and in fact plant organisms affect the mitochondria as the primary or secondary target of artemisinin. Artemisinin appears to cause specific nonspecific effects such as the production of free radicals and immune stimulation, and it is absolutely plausible that, like sorgoleone, artemisinin has more than one target or effect. The interactions between mitochondrial function, calcium signaling, and apoptosis should go unnoticed, and the effects that appear pleiotropic may actually become part of the same process.

### **8. Disruption of plasma membrane integrity**

#### **8.1 Importance of the fungal membrane as a target**

As previously highlighted, secondary metabolites plays an important role protection of plants against fungal pathogens. This is an important area of interest in modern agriculture and medicine with the fungal cell membrane for clinical medicine and agro-fungal drugs. The fungal membrane has unique features, especially sterol ergosterol other than cholesterol or stigmasterol, which is present in animal and plant membranes, respectively. Other differences include the presence of specific lipids on

the outer leaf of the membrane. Common antifungal compounds include amphotericin B, which binds to ergosterol, which leads to pore formation, azoles, and morpholine, which inhibit ergosterol biosynthesis. Evolution has failed to observe this effect on fungi and plants but develops different types of antifungal defense metabolites that target the membranes of phytopathogenic fungi. The well-understood of these are defensins and saponins.

#### **8.2 Plant defenses have specific binding sites in fungal membranes**

Defensins are the most basic, cysteine-rich peptides, typically 40–45 amino acids in length, produced by plants, insects and other animals as antimicrobial defensins molecules [24]. Molecular phylogenetic analysis while the evolutionary roots of these molecules were probably in plants, there was a significant functional difference in the family of cationic antimicrobial peptides (cAMPs) defensins by evolution [25]. A variety of defensins has been reported to have antiviral, antibacterial, and antifungal activity. The prevailing opinion is that the positive charge of peptides mediates specific non-binding with phospholipids, which leads to pore formation and loss of membrane integrity. Although this is a common feature of cAMPs, in recent years it has emerged that specific interactions play a role in the functioning of some cAMPs. For example, human α-defensins have been shown to inactivate adenovirus by binding directly to the viral protein, and endogenous targets for cAMPS have been identified in some bacteria. It is already known that plant defensins and some insect defensins have a specific binding target and mode of action. This discovery initially came from work using *S. cerevisiae* as a model to study the antifungal activity of plant defensins. Yeast strain DmAMP1, a mutation in a gene required for the synthesis of sphingolipids, altered sensitivity to plant defensins [26]. Sphingolipids are commonly found on the outer leaf of eukaryotic membranes and resemble phospholipids, except that the vertebrae are not made of diacylglyceride.

Many variants of Sphingolipids have some unique structures in different fungi in eukaryotic membranes. In a series of studies, some plant and insect defensins bind to different fungal sphingolipids or different nuclei in the same sphingolipids. Following binding, membrane infiltration occurs, but it is not yet known whether this is the result of the signal layer or the biophysical effect. However, it is clear that plant defensins do not particularly penetrate fungal membranes, creating pores and destroying membrane integrity. Interestingly, in *Candida albicans*, the anti-fungal activity of a plant called RsAFP2, which binds to glucosylceramide, was found to

**Figure 3.**

*The antifungal activity of plant defensins is specific and involves receptors and signal transduction pathways.*

#### *Biological Activity of Defence-Related Plant Secondary Metabolites DOI: http://dx.doi.org/10.5772/intechopen.101379*

involve the production of reactive oxygen species (ROS) suggesting that binding to the membrane ligand initiated a signal transduction cascade that culminated in the production of ROS and membrane infiltration (**Figure 3**).

Despite advances in the study of plant defensins, some serious questions and challenges remain to be resolved. First, most extensive work has been done with a limited number of specific defensins, and it remains to be determined whether this is the only procedure. Second, it is not known what signal transmission paths are activated in response to defensins. Third, it is not yet clear whether defensins are internalized after binding to or with sphingolipids. Working with human cAMPs is said to be at least absorbed by some bacteria and reported to be absorbed by the fungal cells of pea defensins [27].

#### **8.3 Lysis of fungal membranes by saponins**

Saponins are a structurally different class of secondary metabolites found in different plants. For example, a survey lists more than 200 plants that isolated saponins between 1998 and 2003. The basic structure of all saponins consists of the polar core and the polar glycosyl group or groups, which give the molecules ambiguous properties. Typically, saponins are classified as triterpenoid or steroidal, with a subset of steroidal alkaloids (steroidal glycoalkaloids) depending on the structure of the hydrophobic center. However, some authors consider steroidal glycolic colloids to be a unique natural product, and recently, a new saponin classification has been proposed into 11 different families depending on the structure of the spine. Saponins are present in significant concentrations in many traditional herbal medicines and a variety of beneficial functions, including common ingredients such as ginseng, are often attributed to the components of saponin.

Within plants, saponins are believed to provide protection against phytopathogenic fungi because they have powerful antifungal activity, are usually accommodated in the epidermal layers of plant tissues and have been shown to play a protective role in many pathogenic interactions. The amphibian nature of saponins represents a mechanism of action and it has been demonstrated that saponins penetrate fungal membranes. The proposed mechanism is that the hydrophobic core enters the outer membrane, forming a compound with ergosterol. Subsequent interaction between polar glycocytic sidechains leads to aggregation, pore formation, and loss of membrane integrity [28]. The ability to penetrate membranes has been demonstrated *in vitro* and *in vivo* in sample membranes of *S.cerevisiae* to explore the anti-fungal activity of the steroidal glycoalkaloid saponin. However, the study also showed that the alkaloid α-tomatine did not penetrate the membranes of the α-tomatine, which is more potent than the α-tomatine, in fact inhibiting ergosterol biosynthesis.

However, the study also showed that the aglycone of α-tomatine did not penetrate the membranes of the α-tomatine, was more potent than the α-tomatine, and actually inhibited ergosterol biosynthesis. Furthermore, several studies have proposed additional functions for α-tomatine and its derivatives. β2-tomatine (created by removing sugar from sugar α-tomatine) has been found to be capable of suppressing plant defense response, and α-tomatine has been reported to induce projected cell death in fungi called *Fusarium oxysporum*, lack of membrane penetration. Finally, studies with potato steroidal glycoalkaloids (saponins), α-chaconine and α-solanine, have identified various toxic effects on animal systems that differ from membrane penetrating activity. In conclusion, although membrane penetration activity contributes

to the antifungal activity of saponins, saponins may have other biological properties, including beneficial roles in human health [29].

#### **9. Anti-tumor activity of plant natural products**

Among the various properties associated with saponins, the ability of some saponin products to inhibit the growth of tumor cells *in vitro* attracts much attention. In fact, many saponins have been reported to have such activity, increasing the likelihood of developing novel saponin-based anti-cancer drugs [30]. Some commentators have questioned the relevance of these *in vitro* data and want to prove that their validity covers specific endogenous targets and is not related to membrane penetration. Significant progress in this direction is due to the synthesis of two different groups of legume triterpenoid saponins, avicins, *Acacia victoria* (**Figure 2**), and soyasaponin from soybean plants. This work was triggered by reports that Avicenna had apoptotic activity against human tumor cells. Several studies have established that it is mediated by mitochondrial dysfunction, indicating that the effects are twofold—disruption of the outer membrane energy and closure of the voltage-dependent ion channel (VDAC) in the mitochondrial membrane. The link between saponin-induced mitochondrial disfunction and apoptosis was further supported by a recent report showing that treatment of HeLa cells with soyasaponin products led to apoptosis via the mitochondrial pathway [31].

Other endogenous targets for specific avicin have also been reported; however, proapoptotic effects may involve multiple targets or indicate that different avicins have specific target processes. In the East model, evidence was obtained for the modulation or inhibition of RO-based signaling and CAMP/PKA signal transmission pathways. A more direct link to apoptosis or automation was obtained from studies with avicin D, where avicin D activates AMP-activated kinase (AMPK), thereby inhibiting mTORC1 and downstream targets. Although many studies of plant natural products have reported a "pro-apoptotic" function, it is worth noting here that there is not much difference between apoptosis and autoimmunity in the plant literature in general. In fact, although the end result is the same, the paths and processes involved are completely different and this is a topic that will require closer attention in the future. Autophagy is given more importance by discovering that the production of β-group soysasaponins reduces mTORC activity, this time apparently by activating another kinase, Akt. The general significance of apoptotic pathways as a target for plant natural metabolism is that other saponin non-metabolites, such as sesquiterpenoid helenalin, which inhibits telomerase, have proliferative effects on mammalian cells [32]. Some of the more than 5000 different flavonoids that occur naturally in plants have effects associated with apoptosis in the future. Nutmeg flavonoid, (−) catechins, for example, inhibit seed germination and cause cell death in sensitive species. This effect appears to involve the generation of reactive oxygen species and may also be linked to calcium signaling or homeostasis. Again, the relationship between ROS, calcium homeostasis, mitochondrial function, autoimmunity, and apoptosis [33] should be kept in mind.

#### **10. Conclusion**

Plant natural products, especially those involved in the protection against pathogens, can lead to biotechnological applications. However, beyond the phytochemical list and general studies, there is a need to go for experiments to identify specific screens and

*Biological Activity of Defence-Related Plant Secondary Metabolites DOI: http://dx.doi.org/10.5772/intechopen.101379*

functional patterns. It should take two forms, identifying the biological role of metabolism in the plant and determining the effects of metabolism on other organisms. The latter is a compelling argument for the use of unbiased genetic or proteomic methods and cell-based assessments to avoid confusion with specific nontargets. Finally, once the candidate goals have been identified, it is necessary to carry out detailed structural and functional studies of the interaction in the actual hosts. However, for preliminary screens and analyzes, plant natural product scientists must follow their biomedical counterparts in eukaryotes and *S. cerevisiae* using chemical genetics and molecular techniques.
