Micropropagation is an efficient method of propagating large numbers of genetically uniform plants (Honda & Kobayashi, 2004), although serious problems concerning specific steps including explant sterilization, media manipulation, and acclimatization phase, can often invalidate its success, making the plantlets production a cost-intensive process.

*In vitro* bacterization of potato plantlets has been shown to enhance their transplant stress tolerance thereby eliminating the need of an expensive greenhouse hardening step, which even now is commonly used by pre-elite seed potato producers. Plants bacterized *in vitro* with *Pseudomonas fluorescens* strains CHA0 and IP10 were found to have a significantly higher fresh shoot weight compared to non-bacterized plants in the same system. Arbuscular mycorrhizal (AM) fungi have also been shown to reduce drought stress and increase disease resistance.

We have investigated the possibility of using the PGPR *Azospirillum brasilense* Sp245 to improve the micropropagation of cherry plum (*Prunus cerasifera*) trees (Russo et al., 2008). We have examined the ability of *A. brasilense* to promote rooting of explants during *in vitro* culture in growth-chamber tests and to promote plant growth and plant health during *ex vitro* acclimatization in greenhouse. In the presence of indolebutyric acid (IBA), both rooting and growth of *P. cerasifera* cuttings were significantly improved by bacteria inoculation. During the acclimatation phase, the main positive effect of inoculation was an increased biomass production, as compared with uninoculated control, suggesting that during acclimatation the rhizobacterium produced phytohormones, increased the nutrient uptake of the roots, and caused an improvement of overall plant performance. An ability to protect plants against pathogen attack was also observed, with a plant survival rate of nearly 100% in inoculated plants as compared to 0% in the negative control. This protective effect was demonstrated both *in vitro* and *in vivo* against the pathogenic fungus *Rhizoctonia* spp.

The effectiveness of *A. brasilense* cells application to micropropagated plantlets at the time of transplanting from *in vitro* culture to acclimatization conditions, has been furthermore assessed on three different fruit tree rootstocks: Mr.S2/5 (*Prunus cerasifera* X *P. spinosa*), GF 677 (*Prunus persica* X *P. amygdalus*), MM 106 (Northen spy X M1). This is a critical phase, in which plantlets are subjected to numerous environmental stresses that may lead to significant plant loss. Plant growth and plant health at the end of *post-vitrum* acclimatization, both in growth-chamber and greenhouse trials, respectively, were evaluated.

After 60 days, growth parameters were positively affected by Sp245 inoculum. In the case of Mr.S 2/5, an increase in rootstock stem length and node number by 37% and 42%, respectively, compared to the control was noted. In the case of GF 677, the bacterial inoculum increased stem length and node number by up to the 75% and 65%, respectively, compared to the control. The inoculum did not exert on MM 106 for both parameters suggesting that the effects of Sp245 could depend on a specific clone-microbe association. In all the cases, however, a higher vigor, consistent with a wider leaf area, was present in the inoculated plantlets demonstrating that the use of *Azospirillum* can significantly contribute to optimize plant performance during the phase of adaptation of plants to *post-vitrum* conditions (Vettori et al., 2010).

Considering that the main obstacles to an intensive and widespread use of beneficial microorganisms, at the commercial level, have been so far the so called "inconsistent" results in field application, mainly related to biotic and abiotic adverse environmental factors, the *in vitro* and *post vitro* inoculation with bacteria may be a way of overcoming a part of these difficulties.

#### **3.2 Co-inoculation strategies**

In recent years, a number of studies on co-inoculation of two or more beneficial microorganisms for better crop productivity have been reported. Positive effects, such as increase in biomass parameters, nitrogen-content and yield have been found in legumes inoculated with *Rhizobium* and *Azospirillum*. These positive effects may be attributed to early and increased nodulation, enhanced N2 fixation rates, and a general improvement of root development. Stimulation of nodulation following the inoculation with *Azospirillum* may be derived from an increase in production of lateral roots, root hair density and branching, but

We have investigated the possibility of using the PGPR *Azospirillum brasilense* Sp245 to improve the micropropagation of cherry plum (*Prunus cerasifera*) trees (Russo et al., 2008). We have examined the ability of *A. brasilense* to promote rooting of explants during *in vitro* culture in growth-chamber tests and to promote plant growth and plant health during *ex vitro* acclimatization in greenhouse. In the presence of indolebutyric acid (IBA), both rooting and growth of *P. cerasifera* cuttings were significantly improved by bacteria inoculation. During the acclimatation phase, the main positive effect of inoculation was an increased biomass production, as compared with uninoculated control, suggesting that during acclimatation the rhizobacterium produced phytohormones, increased the nutrient uptake of the roots, and caused an improvement of overall plant performance. An ability to protect plants against pathogen attack was also observed, with a plant survival rate of nearly 100% in inoculated plants as compared to 0% in the negative control. This protective effect was

demonstrated both *in vitro* and *in vivo* against the pathogenic fungus *Rhizoctonia* spp.

growth-chamber and greenhouse trials, respectively, were evaluated.

conditions (Vettori et al., 2010).

**3.2 Co-inoculation strategies**

part of these difficulties.

The effectiveness of *A. brasilense* cells application to micropropagated plantlets at the time of transplanting from *in vitro* culture to acclimatization conditions, has been furthermore assessed on three different fruit tree rootstocks: Mr.S2/5 (*Prunus cerasifera* X *P. spinosa*), GF 677 (*Prunus persica* X *P. amygdalus*), MM 106 (Northen spy X M1). This is a critical phase, in which plantlets are subjected to numerous environmental stresses that may lead to significant plant loss. Plant growth and plant health at the end of *post-vitrum* acclimatization, both in

After 60 days, growth parameters were positively affected by Sp245 inoculum. In the case of Mr.S 2/5, an increase in rootstock stem length and node number by 37% and 42%, respectively, compared to the control was noted. In the case of GF 677, the bacterial inoculum increased stem length and node number by up to the 75% and 65%, respectively, compared to the control. The inoculum did not exert on MM 106 for both parameters suggesting that the effects of Sp245 could depend on a specific clone-microbe association. In all the cases, however, a higher vigor, consistent with a wider leaf area, was present in the inoculated plantlets demonstrating that the use of *Azospirillum* can significantly contribute to optimize plant performance during the phase of adaptation of plants to *post-vitrum*

Considering that the main obstacles to an intensive and widespread use of beneficial microorganisms, at the commercial level, have been so far the so called "inconsistent" results in field application, mainly related to biotic and abiotic adverse environmental factors, the *in vitro* and *post vitro* inoculation with bacteria may be a way of overcoming a

In recent years, a number of studies on co-inoculation of two or more beneficial microorganisms for better crop productivity have been reported. Positive effects, such as increase in biomass parameters, nitrogen-content and yield have been found in legumes inoculated with *Rhizobium* and *Azospirillum*. These positive effects may be attributed to early and increased nodulation, enhanced N2 fixation rates, and a general improvement of root development. Stimulation of nodulation following the inoculation with *Azospirillum* may be derived from an increase in production of lateral roots, root hair density and branching, but also from the differentiation of a greater number of epidermal cells into root hairs susceptible for infection by rhizobia. Nodulation by rhizobia co-inoculated with *Azospirillum* may also be enhanced by an increased secretion of root flavonoid substances that are involved in the activation of the nodulation genes in *Rhizobium* (Dobbelaere et al., 2001). Considerable results have also been obtained on grain yield, N, P, K content in wheat coinoculated with *Azospirillum brasilense* and *Rhizobium meliloti* (Askary et al., 2009).

The effects of co-inoculation of bacteria and fungi has also been reported. *Azospirillum*–AM fungus combination seems suitable for sustainable agriculture practices, since both types of microorganisms are compatible with each other. The stimulatory effect of the *Azospirillum* inocula on root growth did not significantly influence the mycorrhization, regardless of the AM fungus involved, either in wheat or in maize plants, grown in the greenhouse and/or under field conditions. The effect of *Azospirillum brasilense* Sp245 was assessed at greenhouse level in three different cultivars of durum wheat, in the presence of indigenous AM fungi, and in maize plants artificially inoculated with *Glomus mosseae* and *Glomus macrocarpum*. At field level, the establishment of natural AM fungal symbiosis was evaluated with the commercial strain *Azospirillum lipoferum* CRT1 in maize plants (Russo et al., 2005). Positive effects of *Azospirillum brasilense* and arbuscular mycorrhizal colonization on rice growth and drought resistance have also been attested (Ruíz-Sánchez et al., 2011).

On the other hand, the combination of two rhizobacteria had no synergistic or comparable effects on plant biomass, with respect to their single applications. Indeed, individual inoculation of *B. subtilis* and *A. brasilense* Sp245 positively affected the growth in dry weight of both shoots and roots of tomato plants, but the combination of the two rhizobacteria had no synergistic or comparable effects on plant biomass. *In vitro* tests and cellular analysis of root tips revealed a growth inhibition of the primary root, which is not related to a reduced persistence in the rhizosphere of one or both bacteria (Felici et al., 2008). Moreover coinoculation with mycorrhiza and rhizobia of different bean genotypes resulted in the reduction in the trehalose content and the authors concluded supporting the idea of using rhizobial or mycorrhizal inoculation separately (Ballesteros-Almanza et al., 2010).

These results suggest that mixing different microorganisms in the same inocula/treatment can cause interferences and consequent bad or lower than expected performances. Interactions and antagonist phenomena in contaminant species against *Bradyrhizobium japonicum*, obtained from the same soybean inocula preparation, had already been evidenced in the '80s.

#### **3.3 Bioremediation and phytoremediation**

Phytoextraction, actuated by hyperaccumulating or non-hyperaccumulating species, could be improved by using a plant-microbe system (Zhuang et al. 2007), thus contributing to novel promising methods for the cleaning-up of soils contaminated by heavy metals. Rhizobacteria of the genus *Azospirillum* have been extensively used for crop phytostimulation as above stated, thanks to the positive interaction between bacteria and plants at root level (Dobbeleare et al., 2001; Dobbeleare et al., 2003; Russo et al., 2005; Russo et al., 2008).

The implementation of lead phytoextraction in contaminated industrial soils by applying *A. brasilense* Sp245 to plants of indigenous species belonging to Mediterranean forestry was investigated. The possible phytoextraction ability was evaluated in *Myrtus communis*  L*.* and *Laurus nobilis* L., previously selected among other plant species that were found able to grow in the contaminated areas, on the basis of the Pb content (Emission Spectrophotometer Atomic Plasma, ICP-AES), the growing speed and the vegetative *habitus*. By trials carried out in greenhouse, it was shown that *A. brasilense* Sp245 can enhance the plant growth in Pb contaminated soil and affect the plant total lead content. Greenhouse trials were performed for 2 and 9 months, and plants were grown in pot in the presence of two level of Pb (312 and 4345 ppm).

The presence of Sp245 positively affected the total amount of Pb that was removed by plants, either as total biomass produced (Figure 1) or as specific Pb concentration, as a consequence of the incremented root growth, attesting the synergic effect of plants and microorganisms in a bioremediation system, and as higher specific Pb concentration (Table 1 and Table 2). Moreover the bioconcentration factor (Pb in plant tissues/Pb in soil) and translocation factor (Pb in leaves and shoots/Pb in roots) were significantly affected by the presence of *A*. *brasilense* Sp245, attesting the synergetic effect of plants and microorganisms in a rhizoremediation system.

Fig. 1. Effect of *Azospirillum brasilense* Sp245 inoculation on root system in *Laurus nobilis* and *Myrtus communis* after nine months of pot cultivation with Pb polluted soil.

Table 1. Effect of *A. brasilense* Sp245 inoculation on Pb uptake (mg/Kg d.m.) by each plant of *Laurus nobilis* and *Myrtus communis* after two and nine months of pot cultivation with soils at different Pb concentration (Pb- : 312 mg/Kg and Pb+ : 4345 mg/Kg). Within each shrub species and each period means with the same letter were not significantly different according to the SNK'test (P ≤0.05).

Table 2. Effect of *A. brasilense* Sp245 inoculation on biomass produced (g/plant d.m.) by *Laurus nobilis* and *Myrtus communis* after two and nine months of pot cultivation with Pb polluted soil. Within shrub species means with the same letter were not significantly different according to the SNK'test (P ≤0.05).

#### **3.4 Naturalistic engineering and endotherapy**

The increased root density and branching, the improving in rooting of cuttings and the better adaptation to biotic and abiotic stresses derived from beneficial microorganisms, may represent an advantage in soil bioengineering and landscape construction. The role of vegetation in slope stability and restoration of steep rock faces with shrubs and trees is difficult due to extreme microclimatic and edaphic conditions (Beikircher et al., 2010). Inoculation with *A. brasilense* Sp245 of plant material used for restoration of drought-prone sites during preconditioning, can increase the drought tolerance and can play a synergetic and pivotal role in that phase. Work in progress with some angiosperm species, known for their vulnerability, gives us good expectation.

Another promising field could be the use of selected beneficial microorganisms in endotherapy, or trunk injection. This is an alternative method of treatment of urban woodland plant, with many advantages compared to traditional air treatments, including the absence of spraying of chemicals, and hence the complete harmlessness for the health of citizens, birds and other animals (Sánchez-Zamora & Fernández-Escobar, 2004; Hubbard & Potter, 2006).

#### **4. Conclusions**

66 Innovations in Biotechnology

Spectrophotometer Atomic Plasma, ICP-AES), the growing speed and the vegetative *habitus*. By trials carried out in greenhouse, it was shown that *A. brasilense* Sp245 can enhance the plant growth in Pb contaminated soil and affect the plant total lead content. Greenhouse trials were performed for 2 and 9 months, and plants were grown in pot in

The presence of Sp245 positively affected the total amount of Pb that was removed by plants, either as total biomass produced (Figure 1) or as specific Pb concentration, as a consequence of the incremented root growth, attesting the synergic effect of plants and microorganisms in a bioremediation system, and as higher specific Pb concentration (Table 1 and Table 2). Moreover the bioconcentration factor (Pb in plant tissues/Pb in soil) and translocation factor (Pb in leaves and shoots/Pb in roots) were significantly affected by the presence of *A*. *brasilense* Sp245, attesting the synergetic effect of plants and microorganisms

Fig. 1.  Effect of *A. brasilense* Sp245 inoculation on Pb uptake (mg/Kg d.m.) by each plant of *Laurus nobilis* and *Myrtus communis* after two and nine months of pot cultivation with soils at different Pb concentration (Pb- : 312 mg/Kg and Pb+ : 4345 mg/Kg). Within each shrub species and each period means with the same letter were not significantly different

*Myrtus communis* after nine months of pot cultivation with Pb polluted soil.

Shrub species Pb uptake

the presence of two level of Pb (312 and 4345 ppm).

in a rhizoremediation system.

according to the SNK'test (P ≤0.05).

Agriculture is the oldest economic sector in the world, and is more dependent on fertile soils and a stable climate than any other trade. At the same time, it has a huge influence on the ecological balance, water and soil quality, and on the preservation of biological diversity. Since the middle of the last century, agricultural techniques and economic framework conditions worldwide have undergone such a radical transformation that agriculture has became a major source of environmental pollution.

The investigation about ecologically compatible techniques in agriculture and environmental sciences can take essential advantage from the use of beneficial microorganisms as plantmicrobe interactions fulfil important ecosystem functions.

Plant diseases are a major cause of yield losses and ecosystem instability worldwide. Use of agrochemicals to protect crop against plant pathogens has been increasing along with the intensification of agricultural production over the last few decades.

New biotechnological methods for crop protection are based on the use of beneficial microorganisms applied as biofertilizers and/or biocontrol agents; this approach represents an important tool for plant disease control, and could lead to a substantial reduction of chemical fertilizer use, which is an important source of environmental pollution. Nevertheless, despite dedicated efforts to study beneficial microorganisms, relatively few products have been registered for agricultural use and they count for a very small fraction of the potential market. This is a consequence of several limitations affecting their commercial expansion, which are mainly related to the survival capability of microorganisms under various environmental conditions.

Moreover, nowadays, microbial inoculants, some of which have a historical record for safe use since 1896 (the well-known rhizobia, for the inoculation of legumes) or since the 1930s (e.g. *Bacillus thuringiensis*, for the biological control of invertebrate pests) are being widely applied in modern agriculture as biofertilizers and biocontrol agents. Other interesting applications include micropropagation, bioremediation and phytoremediation, phosphate solubilization, soil aggregation, sewage treatment, bioleaching, oil recovery, coal scrubbing and biogas production, and represent incoming fields of application.

In short, from the examples and references cited above, it is manifest that useful microorganisms of agricultural importance represent an alternative and ecological strategy for disease management, in order to reduce the use of chemicals in agriculture and to improve cultivar performance. At the same time, their application is a highly efficient way to resolve environmental problems, for example through bioremediation and bioengineering. However, although beneficial microorganisms hold a great promise for dealing with different environmental problems, it is important to aknowledge that much of this promise has yet to be realized. Indeed, much needs to be learned about how microorganisms interact with each other and with the environments. For the future development of biotechnology in this field, the contribution of a combination of scientific disciplines is of primary importance to promote sustainable practices in plant production system, as well as in conservation and ecosystem restoration.

#### **5. References**

New biotechnological methods for crop protection are based on the use of beneficial microorganisms applied as biofertilizers and/or biocontrol agents; this approach represents an important tool for plant disease control, and could lead to a substantial reduction of chemical fertilizer use, which is an important source of environmental pollution. Nevertheless, despite dedicated efforts to study beneficial microorganisms, relatively few products have been registered for agricultural use and they count for a very small fraction of the potential market. This is a consequence of several limitations affecting their commercial expansion, which are mainly related to the survival capability of microorganisms under

Moreover, nowadays, microbial inoculants, some of which have a historical record for safe use since 1896 (the well-known rhizobia, for the inoculation of legumes) or since the 1930s (e.g. *Bacillus thuringiensis*, for the biological control of invertebrate pests) are being widely applied in modern agriculture as biofertilizers and biocontrol agents. Other interesting applications include micropropagation, bioremediation and phytoremediation, phosphate solubilization, soil aggregation, sewage treatment, bioleaching, oil recovery, coal scrubbing

In short, from the examples and references cited above, it is manifest that useful microorganisms of agricultural importance represent an alternative and ecological strategy for disease management, in order to reduce the use of chemicals in agriculture and to improve cultivar performance. At the same time, their application is a highly efficient way to resolve environmental problems, for example through bioremediation and bioengineering. However, although beneficial microorganisms hold a great promise for dealing with different environmental problems, it is important to aknowledge that much of this promise has yet to be realized. Indeed, much needs to be learned about how microorganisms interact with each other and with the environments. For the future development of biotechnology in this field, the contribution of a combination of scientific disciplines is of primary importance to promote sustainable practices in plant production

Aroca, R. & Ruíz-Lozano, J.M. (2009). Induction of plant tolerance to semi-arid

Arzanesh, M.; Alikhani, H.; Khavazi, K.; Rahimian, H. & Miransari, M. (2011). Wheat

Barea, J.M.; Pozo, M.J.; Azcón, R. & Azcón-Aguilar, C. (2005). Microbial cooperation in the

Ballesteros-Almanza, L.; Altamirano-Hernandez, J.; Peña-Cabriales, J.J.; Santoyo, G.;

stress. *World Journal of Microbiology and Biotechnology*, Vol.27, pp.197-205 Askary, M.; Mostajeran, A.; Amooaghaei, R.; Mostajeran, M. (2009). Influence of the co-

Reviews 2. (Lichtfouse, E., Ed.) Springer, Netherlands pp.121-135

rhizosphere. *Journal of experimental botany*, Vol.56, pp.1761-1778

environments by beneficial soil microorganisms (a review) En: *Climate Change, Intercropping, Pest Control and Beneficial Microorganisms, sustainable Agriculture*

(*Triticum aestivum* L.) growth enhancement by *Azospirillum* sp. under drought

inoculation *Azospirillum brasilense* and *Rhizobium meliloti* plus 2,4-D on grain yield and N, P, K content of *Triticum aestivum* (cv. Baccros and Mahdavi). American-Eurasian Journal of Agricultural and Environmental *Science*, Vol.5, No.3 pp.296-307

Sanchez-Yañez, J.M.; Valencia-Cantero, E.; Macias-Rodriguez, L.; Lopez-Bucio, J.;

and biogas production, and represent incoming fields of application.

system, as well as in conservation and ecosystem restoration.

various environmental conditions.

**5. References**

Cardenas-Navarro, R. & Farias-Rodriguez, R. (2010). Effect of co-inoculation with mycorrhiza and rhizobia on the nodule trehalose content of different bean genotypes. *Open Microbiol J*., Vol.17, No.4, pp.83-92

with *Rhizobium* on *Phaseolus vulgaris* flavonoids and Nod factor production under salt stress. *Soil Biology & Biochemistry,* Vol.40, pp. 2713-2721

Dimkpa, C.; Weinand, T. & Asch, F. (2009). Plant-rhizobacteria interactions alleviate abiotic

Dobbelaere, S.; Croonenborghs, A.; Thys, A.; Vande Broek, A. & Vanderleyden, J. (1999).

Dobbelaere, S.; Vanderleyden, J. & Okon, Y. (2003). Plant growth-promoting effects diazotrophs in the rhizosphere. *Crit Rev Plant Sci,* Vol.22, pp.107–149 Felici, C.; Vettori, L.; Toffanin, A. & Nuti, M. (2008). Development of a strain-specific

Glick, B.R.; Patten, C.L.; Holguin, G. & Penrose, D.M. (1999). Biochemical and genetic

Hamdia, A.B.E.; Shaddad, M.A.K. & Doaa M.M. (2004). Mechanisms of salt tolerance and

Honda, H. & Kobayashi, T. (2004). Large scale micropropagation system of plant cells. *Adv.*

Hubbard, J.L. & Potter A.D. (2006). Managing Calico Scale (Hemiptera: Coccidae) Infestations on Landscape Trees. *Arboriculture & Urban Forestry*, Vol.32, No.4, pp138-147 Jousset A, Rochat L, Lanoue A, Bonkowski M, Keel C, Scheu S. (2011). Plants respond to

Kokalis-Bourelle, N.; Vavrina, E.N.: Rosskopf, E.N. & Shelby, R.A. (2002). Field evaluation of

Kokalis-Burelle, N.; Kloepper, J.W. & Reddy, M.S. (2006). Plant growth-promoting

rhizosphere microorganisms. *Applied Soil Ecology*, Vol. 31, No.1-2, pp.91-100 Long, H.H.; Schmidt, D.D. & Baldwin, I.T. (2008). Native Bacterial Endophytes Promote

Mano, H. & Morisaki, H. (2008). Endophytic Bacteria in the Rice Plant. *Microbes and*

Result in Common Growth Responses. PLoS One, Vol. 3, p.2702

under salt stress conditions. *Plant Growth Regulation,* Vol.44, pp.165-174 Hardoim, P.R.; Van Overbeek, L.S. & Elsas, J.D.V. (2008). Properties of bacterial endophytes and their proposed role in plant growth. *Trends in Microbiology,* Vol.16, pp. 463-471 Hermosa, R.; Botella, L.; Alonso-Ramírez, A.; Arbona, V.; Gómez-Cadenas, A.; Monte, E. &

rhizosphere of tomato. *FEMS Microbiol Ecol*., Vol.65, No.2, pp.289-98 Glick, B.R. (1995). The enhancement of plant growth by free-living bacteria. *Can. J. Microbiol.*

altered in IAA production on wheat. *Plant and Soil,* Vol*.*212, pp. 153-162 Dobbelaere, S.; Croonenborghs, A.; Thys A.; Ptacek, D.; Vanderleyden, J.; Dutto, P.;

salt stress. *Soil Biology & Biochemistry,* Vol.40, pp. 2713-2721

*of Plant Physiology,* Vol.28, pp.871-879

*Biochem. Eng. Biotechnol*., Vol.91, pp.105-34

*Environments*, Vol.23, pp. 109-117

bacteria. *Mol Plant Microbe Interact.*, Vol.24, pp.352-358

Vol.41, pp. 109-117

London, UK

pp.257-266

stress conditions. *Plant Cell and Environment*, Vol.32, pp. 1682-1694

with *Rhizobium* on *Phaseolus vulgaris* flavonoids and Nod factor production under

Phytostimulatory effect of *Azospirillum brasilense* wild type and mutant strains

Labandera-Gonzalez, C.; Caballero-Mellado, J.; Aguirre, J.F.; Kapulnik, Y.; Brener, S.; Burdman, S.; Kadouri, D.; Sarig, S. & Okon, Y. (2001). Responses of agronomically important crops to inoculation with *Azospirillum*. *Australian Journal*

genomic marker for monitoring a *Bacillus subtilis* biocontrol strain in the

mechanisms used by plant growth-promoting bacteria. Imperial College Press,

interactive effects of *Azospirillum brasilense* inoculation on maize cultivars grown

Nicolás, C. (2011). Biotechnological applications of the gene transfer from the beneficial fungus *Trichoderma harzianum* spp. to plants. *Plant Signal Behav*., Vol.6, No.8

pathogen infection by enhancing the antifungal gene expression of root-associated

plant growth-promoting rhizobacteria amended transplant mixes and soil solarization for tomato and pepper production in florida. *Plant Soil*, Vol.238,

rhizobacteria as transplant amendments and their effects on indigenous

Host Growth in a Species-Specific Manner; Phytohormone Manipulations Do Not


**Author details**

Danica Dolničar\* and Bojana Boh Podgornik Faculty of Natural Sciences and Engineering, University of Ljubljana, Ljubljana, Slovenia

\*Address all correspondence to<EMAIL_ADDRESS>
© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


**3. Results**

In this section, the results are presented and discussed. The transformer oil testing samples based on mineral oil were abstracted from a South African transformer manufacturer after laboratory analysis. The BCSVM training phase was ingested with 70% of the oil testing samples and tested using 30% of the transformer oil samples. This investigation was conducted by employing the classification learner app in the MATLAB\_R2018a software platform. Once the oil samples dataset was trained, the veracity of the distinctive SVMs was corroborated. The configuration matrix of a particular SVM that provides the highest degree of accuracy was selected and imported to predict the new testing data sample. The fault class prediction for the new data sample is carried out by utilizing Equation (7).

$$yfit = fitecocc(Tbl, \text{ }ResponseVarName) \tag{7}$$

Here,

*Tbl*¯Multiclass predictor variables in table *ResponseVarName*¯Response variables


*2.2. Body Weight and Tissue Weights of Con- and FO-Fed Mice*

The mean energy intake was similar between the Con- and the FO-fed mice during the 10-day administration period (Con, 17.6 ± 0.8 kcal/day; FO, 17.7 ± 0.6 kcal/day, *p* = 0.97). Although final body weight (BW) was not different between the Con- and FO-fed mice, the BW gain in the FO-fed mice was significantly lower than that in the Con-fed mice during the 10-day period (Con, 10.3 ± 1.4%; FO, 6.6 ± 1.0%, *p* < 0.05). The weights of subcutaneous WAT (subWAT) in the FO-fed mice were not different from those in the

FO-fed mice (*p* = 0.07, Table 1). However, the weights of epididymal WAT and mesenteric WAT in the FO-fed mice were lower than those in the Con-fed mice. The weight of BAT was not affected by FO supplementation.

**Table 1.** BW and weights of tissues in Con- and FO-fed mice.

Values are mean ± SEM (n = 7). \* *p* < 0.05 vs. Con-fed mice. Significant differences between two groups were tested by Student *t*-test. BW: body weight; Con: control; FO: fish oil; BAT: brown adipose tissue; WAT: white adipose tissue.

#### *2.3. Serum Chemicals of Con- and FO-Fed Mice*

Because the weights of the epididymal WAT and mesenteric WAT in the FO-fed mice were lower than those in the Con-fed mice, we analyzed serum concentrations of glucose, non-esterified fatty acid (NEFA), triglyceride (TG) and total cholesterol (TC). The concentrations of serum glucose in the Con- and the FO-fed mice were the same (Table 2). However, the serum concentrations of NEFA, TG and TC in the FO-fed mice were significantly lower than those in the Con-fed mice (Table 2).

**Table 2.** Serum chemicals of Con- and FO-fed mice.

Values are mean ± SEM (n = 7). \*\* *p* < 0.01, \*\*\* *p* < 0.001 vs. Con-fed mice. Significant differences between two groups were tested by Student *t*-test. Con: control; FO: fish oil; NEFA: non-esterified fatty acid; TG: triglyceride; TC: total cholesterol.

#### *2.4. Effects of FO Supplementation on BAT*

To confirm the mechanism of increase of DIT in the FO-fed mice, we examined expression profiling of the *Ucp1* gene and UCP1 protein. FO supplementation resulted in a 1.5-fold increase in *Ucp1* mRNA in BAT (Figure 3a). UCP1 protein expression was also analyzed (n = 7 in each group), and representative data (n = 2 in each group) indicating a 1.2-fold increase in expression are shown in Figure 3b. The mRNA expression of *Pparα*, which is one of the nuclear transcription factors whose activation leads to increased fatty acid β-oxidation [39], was significantly increased by FO supplementation (Figure 3a). However, FO supplementation did not affect the mRNA expressions of target genes carnitine palmitoyltransferase I (*Cpt I*), acyl-CoA oxidase (*Aco*) and medium-chain acyl-CoA dehydrogenase (*Mcad*) (Figure 3a). Fibroblast growth factor 21 (*Fgf21*) expression was also not increased by FO administration (Figure 3a). The expression of the mitochondria biogenesis marker peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) and that of crucial thermogenesis biomarker type 2 iodothyronine deiodinase (Dio2) in the mice was not different between the two groups (Figure 3a). No difference in β3-adrenergic receptor (β3-AR) mRNA was observed in BAT (Con, 100.0 ± 7.0%; FO, 93.0 ± 11.4%).

**Figure 3.** Effect of fish oil (FO) supplementation on gene expression and UCP1 protein levels in brown adipose tissue. mRNA levels of *Ucp1* and *Pparα* and its target genes (**a**) and UCP1 protein (**b**) were assessed by quantitative RT-PCR or western blotting. β-actin was used as the normalization control. The percent of mRNA and protein levels relative to those of Con-fed mice are indicated. White and gray columns represent data from the Con- and FO-fed mice, respectively. Values are mean ± SEM (n = 7). \* *p* < 0.05, \*\*\* *p* < 0.001 vs. Con-fed mice. Significant differences between two groups were tested by Student *t*-test.


**2. Can vegetable oils and nuts be the natural solution for CVD?**

Oxidative stress is common in many clinically important cardiac disorders, including ischae‐ mia/reperfusion (I/R) injury, diabetes and hypertensive heart disease [42-46]. Several animal models suggest that when endogenous anti-oxidant systems are compromised, as is the case under oxidative stress conditions, exogenous antioxidant supplementation can be used for preventive and/or therapeutic intervention of CVD [42, 43, 47-49].

the prevention of coronary heart disease (CHD) stems from the observed beneficial effects of the Mediterranean diet [57], which includes high consumption of olive oil. MUFAs are less susceptible to oxidation when compared to PUFAs. This in turn leads to increased availability of antioxidants in the active form and better stability of olive oil [58-61]. Olive oil also contains some antioxidant micronutrients, namely polyphenols and squalene [58, 62-64]. The main MUFA in the human diet is oleic acid (C18:1n-9), which has one double bond. MUFA intake has been associated with a slight cardioprotective effect [65]. MUFAs are known to have a beneficial effect on the serum lipid profile and thus decrease the risk of CVD [66-68]. Further‐ more, these fatty acids are stable in oxidative stress conditions and are less likely to react with reactive oxygen species (ROS) when compared with PUFA [58-59]. However, studies reporting associations between dietary intake of MUFAs and CHD risk have been inconclusive [69-71].

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PUFAs are naturally occurring endogenous substances, present in almost all tissues and are essential components of all mammalian cells. They are essential for survival and cannot be synthesized in the body. Hence, they have to be obtained in our diet and are therefore essential [54, 72]. There are two types of naturally occurring PUFAs in the body, the (n-6) PUFAs derived from linoleic acid (LA, C18:2) and the (n-3) PUFAs derived from α-linolenic acid (ALA, C18:3). They are categorized depending on the location of their first double bond: (n-3) PUFAs have their first double bond located at the third carbon molecule and (n-6) PUFAs at the sixth. Both of these two forms of PUFAs are metabolized by the same set of enzymes as their respective long-chain metabolites [73]. The differences between (n-3) and (n-6) PUFAs are shown in Table

Vegetable oils are the predominant sources of alpha linolenic acid (ALA). ALA is found in legumes, flax seeds, walnuts, pinto beans, soybeans and spinach [74]. Dietary intake of ALA among Western adults is typically in the range of 0.5–2g/d [75]. The (n-6) PUFA is the main PUFA in most Western diets and is typically consumed in greater amounts than ALA [75, 76]. The evidence for a beneficial role of dietary (n-6) PUFAs is less convincing and for the purpose of this chapter we will focus on the (n-3) PUFA. The three main forms of (n-3) PUFAs are ALA, eicosapentaenoic acid (EPA, C20:5 n-3) and docosahexaenoic acid (DHA, C22:6 n-3) [77], with ALA being the simplest form. The (n-3) PUFAs are a family of biologically active fatty acids. The simplest member of this family, ALA, can be converted to the more biologically active and very long-chain (n-3) PUFAs; EPA and DHA. This process, as shown in Figure 1, occurs by a series of desaturation and elongation reactions, with stearidonic acid being an intermediate in

Research has shown that long-chain (n-3) PUFAs protect against CVD [77, 79-82]. The cardi‐ oprotective effects of (n-3) PUFAs have long been recognized. Epidemiologic data suggest that (n-3) PUFAs derived from fish oil reduce CVD. Fish oil is a rich source of EPA (C20:5 n-3) and DHA (C22:6 n-3) (Table 1) [67, 83, 84]. The cardioprotective roles of these two forms of (n-3) PUFA are extensively reviewed by Bester and co-workers [48]. Fish oil may also reduce mortality after a cardiovascular incident, as it plays a role in reducing potentially fatal arrhythmias ([85-87]. There are several prospective studies relating the use of fish or the intake

*2.1.4.  Composition and health benefits of vegetable oils**

### *2.1.1. Dietary fats*

Fats are the most concentrated form of energy for the body. They also aid in the absorption of fat-soluble vitamins (A, D, E and K) and other fat-soluble biologically active components [50]. Chemically, most of the fats in foods are triglycerides, made up of a unit of glycerol combined with free fatty acids, each of which may be the same or different. Other dietary fats include phospholipids, phytosterols and lipoproteins associated with cholesterol [50-52]. A balanced diet, including oils and fats that supply energy and essential fatty acids is needed for good health.

The different types of fatty acids are the most important characteristics of dietary fats. According to the degree of unsaturation (double bonds and hydrogen content), fatty acids are largely classified into three major types: saturated fatty acids, monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA). A fourth form, the trans fatty acids, are mainly produced by partial hydrogenation of polyunsaturated oils in food processing but also occur naturally in animal foods in small amounts [53].

Fatty acids consist of a hydrocarbon chain with a hydrophobic methyl group at one end and a hydrophilic carboxyl group at the other end. Greek letters (α, β, γ, ω) have been used to identify the location of the double bonds in fatty acids. The "alpha" carbon is the carbon closest to the carboxyl group. The methyl group of the molecule is also referred to as the omega end and the terminal carboxyl group is located at the delta end. Current chemical numerical terms number the carbon chain form one to "n", with n being the last carbon at the methyl end. The terms "n" and "omega" are synonymous [54].

#### *2.1.2. Saturated fat*

Saturated fatty acids contain no double bond; they are fully saturated with hydrogen. The main saturated fatty acids are lauric acid (C12:0), myristic acid (C14:0), palmitic acid (C16:0) and stearic acid (C18:0). Saturated fats are found in animal-based products, such as milk, cream, butter and cheese, meat from most land animals, palm oil and coconut oil, as well as manu‐ factured products made from these, such as pies, biscuits, cakes and pastries [55].

#### *2.1.3. Monounsaturated fat (MUFA)*

MUFAs are predominant in vegetable oils, such as olive oil, canola oil and peanut oil and are also found in high proportions in animal fats [56]. Much of the interest in the role of MUFA in the prevention of coronary heart disease (CHD) stems from the observed beneficial effects of the Mediterranean diet [57], which includes high consumption of olive oil. MUFAs are less susceptible to oxidation when compared to PUFAs. This in turn leads to increased availability of antioxidants in the active form and better stability of olive oil [58-61]. Olive oil also contains some antioxidant micronutrients, namely polyphenols and squalene [58, 62-64]. The main MUFA in the human diet is oleic acid (C18:1n-9), which has one double bond. MUFA intake has been associated with a slight cardioprotective effect [65]. MUFAs are known to have a beneficial effect on the serum lipid profile and thus decrease the risk of CVD [66-68]. Further‐ more, these fatty acids are stable in oxidative stress conditions and are less likely to react with reactive oxygen species (ROS) when compared with PUFA [58-59]. However, studies reporting associations between dietary intake of MUFAs and CHD risk have been inconclusive [69-71].

### *2.1.4.  Can vegetable oils and nuts be the natural solution for CVD?**

preventive and/or therapeutic intervention of CVD [42, 43, 47-49].

**2.1. Composition and health benefits of vegetable oils**

212 Antioxidant-Antidiabetic Agents and Human Health

occur naturally in animal foods in small amounts [53].

terms "n" and "omega" are synonymous [54].

*2.1.3.  Dietary fats*

*2.1.2. Saturated fat*

health.

Oxidative stress is common in many clinically important cardiac disorders, including ischae‐ mia/reperfusion (I/R) injury, diabetes and hypertensive heart disease [42-46]. Several animal models suggest that when endogenous anti-oxidant systems are compromised, as is the case under oxidative stress conditions, exogenous antioxidant supplementation can be used for

Fats are the most concentrated form of energy for the body. They also aid in the absorption of fat-soluble vitamins (A, D, E and K) and other fat-soluble biologically active components [50]. Chemically, most of the fats in foods are triglycerides, made up of a unit of glycerol combined with free fatty acids, each of which may be the same or different. Other dietary fats include phospholipids, phytosterols and lipoproteins associated with cholesterol [50-52]. A balanced diet, including oils and fats that supply energy and essential fatty acids is needed for good

The different types of fatty acids are the most important characteristics of dietary fats. According to the degree of unsaturation (double bonds and hydrogen content), fatty acids are largely classified into three major types: saturated fatty acids, monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA). A fourth form, the trans fatty acids, are mainly produced by partial hydrogenation of polyunsaturated oils in food processing but also

Fatty acids consist of a hydrocarbon chain with a hydrophobic methyl group at one end and a hydrophilic carboxyl group at the other end. Greek letters (α, β, γ, ω) have been used to identify the location of the double bonds in fatty acids. The "alpha" carbon is the carbon closest to the carboxyl group. The methyl group of the molecule is also referred to as the omega end and the terminal carboxyl group is located at the delta end. Current chemical numerical terms number the carbon chain form one to "n", with n being the last carbon at the methyl end. The

Saturated fatty acids contain no double bond; they are fully saturated with hydrogen. The main saturated fatty acids are lauric acid (C12:0), myristic acid (C14:0), palmitic acid (C16:0) and stearic acid (C18:0). Saturated fats are found in animal-based products, such as milk, cream, butter and cheese, meat from most land animals, palm oil and coconut oil, as well as manu‐

MUFAs are predominant in vegetable oils, such as olive oil, canola oil and peanut oil and are also found in high proportions in animal fats [56]. Much of the interest in the role of MUFA in

factured products made from these, such as pies, biscuits, cakes and pastries [55].

PUFAs are naturally occurring endogenous substances, present in almost all tissues and are essential components of all mammalian cells. They are essential for survival and cannot be synthesized in the body. Hence, they have to be obtained in our diet and are therefore essential [54, 72]. There are two types of naturally occurring PUFAs in the body, the (n-6) PUFAs derived from linoleic acid (LA, C18:2) and the (n-3) PUFAs derived from α-linolenic acid (ALA, C18:3). They are categorized depending on the location of their first double bond: (n-3) PUFAs have their first double bond located at the third carbon molecule and (n-6) PUFAs at the sixth. Both of these two forms of PUFAs are metabolized by the same set of enzymes as their respective long-chain metabolites [73]. The differences between (n-3) and (n-6) PUFAs are shown in Table 1 below.

Vegetable oils are the predominant sources of alpha linolenic acid (ALA). ALA is found in legumes, flax seeds, walnuts, pinto beans, soybeans and spinach [74]. Dietary intake of ALA among Western adults is typically in the range of 0.5–2g/d [75]. The (n-6) PUFA is the main PUFA in most Western diets and is typically consumed in greater amounts than ALA [75, 76]. The evidence for a beneficial role of dietary (n-6) PUFAs is less convincing and for the purpose of this chapter we will focus on the (n-3) PUFA. The three main forms of (n-3) PUFAs are ALA, eicosapentaenoic acid (EPA, C20:5 n-3) and docosahexaenoic acid (DHA, C22:6 n-3) [77], with ALA being the simplest form. The (n-3) PUFAs are a family of biologically active fatty acids. The simplest member of this family, ALA, can be converted to the more biologically active and very long-chain (n-3) PUFAs; EPA and DHA. This process, as shown in Figure 1, occurs by a series of desaturation and elongation reactions, with stearidonic acid being an intermediate in the pathway [54, 75, 78].

Research has shown that long-chain (n-3) PUFAs protect against CVD [77, 79-82]. The cardi‐ oprotective effects of (n-3) PUFAs have long been recognized. Epidemiologic data suggest that (n-3) PUFAs derived from fish oil reduce CVD. Fish oil is a rich source of EPA (C20:5 n-3) and DHA (C22:6 n-3) (Table 1) [67, 83, 84]. The cardioprotective roles of these two forms of (n-3) PUFA are extensively reviewed by Bester and co-workers [48]. Fish oil may also reduce mortality after a cardiovascular incident, as it plays a role in reducing potentially fatal arrhythmias ([85-87]. There are several prospective studies relating the use of fish or the intake of long-chain (n-3) PUFAs to lower risk of CVD [88, 89]. Long chain (n-3) PUFAs have several beneficial cardiovascular properties, including antiatherothrombotic, antiarrhythmic, antiinflammatory, antihypertensive and triglyceride lowering [81, 90, 91]. In summary, studies investigating the dietary roles of fatty acids demonstrate that dietary supplementation with (n-3) PUFAs decreases cardiac deaths, nonfatal cardiovascular events and all-cause mortality. These benefits are most apparent in high-risk patients. (n-3) PUFA supplementation appears to confer additional benefits in patients eating a Mediterranean diet.

The original observation is from almost 57 years ago, when Hugh M. Sinclair [92] published his observations on the negative effects of essential fatty acid deficiency on CVD. He strength‐ ened his hypothesis by noting the low mortality rate from CHD (coronary heart disease) in Greenland Eskimos, a population consuming a high fat diet, but rich in (n-3) PUFAs [92]. Clinical studies suggest that (n-3) PUFAs reduce mortality from coronary heart disease and the rate of sudden cardiac death [92-95]. Significant antiarrhythmic effects of (n-3) PUFAs were observed in some but not all human studies on atrial fibrillation [96, 97]. In addition, animal studies show strong antiarrhythmic effects of (n-3) PUFAs [98-102].

**Table 1.** Molecular structure, types and food sources of (n-3) and (n-6) PUFAs.

Long-chain (n-3) PUFAs are important constituents of all cell membranes and confer on membranes properties of fluidity and thus, determine and influence the behaviour of mem‐ brane-bound enzymes and receptors [103-107]. These PUFAs are found in abundance in the myocardium, retina, brain and spermatozoa, and are essential for the proper functioning of these tissues and growth, being important modulators of many physiological processes. The fact that these tissues have developed the cellular machinery to preferentially incorporate these minor dietary components into their membranes suggests that these PUFAs play a role in the proper function of the cell [108-110].

myocardial membrane phospholipids are rich in (n-3) PUFAs after fish oil consumption [111, 112]. Diet-induced changes in the PUFA composition of a cell membrane have an impact on the cell's function, partly because these fatty acids represent a reservoir of molecules that perform important signalling roles within and between cells. In particular, dietary (n-3) PUFAs compete with dietary (n-6) PUFAs for incorporation into all cell membranes [113,114]. (n-3) PUFAs modulate the expression of adhesion proteins such as selectins [115] and exert an effect by modulating the intracellular signalling pathways associated with the control of transcrip‐ tion factors (e.g., nuclear factor-κB) and gene transcription [116,117]. Research has shown that enrichment of monocyte membranes with (n-3) PUFAs results in the synthesis and secretion

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**Figure 1.** The biosynthesis of (n-3) PUFA.

The fatty acid composition of myocardial membrane phospholipids, in particular, is sensitive to the type of fatty acid consumed in the diet. Studies show that indeed the myocardium and

**Figure 1.** The biosynthesis of (n-3) PUFA.

of long-chain (n-3) PUFAs to lower risk of CVD [88, 89]. Long chain (n-3) PUFAs have several beneficial cardiovascular properties, including antiatherothrombotic, antiarrhythmic, antiinflammatory, antihypertensive and triglyceride lowering [81, 90, 91]. In summary, studies investigating the dietary roles of fatty acids demonstrate that dietary supplementation with (n-3) PUFAs decreases cardiac deaths, nonfatal cardiovascular events and all-cause mortality. These benefits are most apparent in high-risk patients. (n-3) PUFA supplementation appears

The original observation is from almost 57 years ago, when Hugh M. Sinclair [92] published his observations on the negative effects of essential fatty acid deficiency on CVD. He strength‐ ened his hypothesis by noting the low mortality rate from CHD (coronary heart disease) in Greenland Eskimos, a population consuming a high fat diet, but rich in (n-3) PUFAs [92]. Clinical studies suggest that (n-3) PUFAs reduce mortality from coronary heart disease and the rate of sudden cardiac death [92-95]. Significant antiarrhythmic effects of (n-3) PUFAs were observed in some but not all human studies on atrial fibrillation [96, 97]. In addition, animal

First double-bond on the sixth carbon counting from the

methyl end (the "nth" carbon)

Linoleic acids (LA) [C18:2] Arachidonic acid (AA) [C20:4]

Corn oil(LA) Soybean oil (LA) Sunflower oil (LA) Poultry (AA) Meats (AA)

Long-chain (n-3) PUFAs are important constituents of all cell membranes and confer on membranes properties of fluidity and thus, determine and influence the behaviour of mem‐ brane-bound enzymes and receptors [103-107]. These PUFAs are found in abundance in the myocardium, retina, brain and spermatozoa, and are essential for the proper functioning of these tissues and growth, being important modulators of many physiological processes. The fact that these tissues have developed the cellular machinery to preferentially incorporate these minor dietary components into their membranes suggests that these PUFAs play a role in the

The fatty acid composition of myocardial membrane phospholipids, in particular, is sensitive to the type of fatty acid consumed in the diet. Studies show that indeed the myocardium and

to confer additional benefits in patients eating a Mediterranean diet.

studies show strong antiarrhythmic effects of (n-3) PUFAs [98-102].

**(n-3) PUFA (n-6) PUFA**

First double-bond on the third carbon counting from the methyl end (the

Eicosapentaenoic acid (EPA) [C20:5] Docosahexaenoic acid (DHA) [C22:6]

**Table 1.** Molecular structure, types and food sources of (n-3) and (n-6) PUFAs.

"nth" carbon)

214 Antioxidant-Antidiabetic Agents and Human Health

**Types** α-Linolenic acid (ALA) [C18:3]

Canola oil (ALA) Soybean oil (ALA) Oily fish (EPA/DHA) Fish oil capsules (EPA/DHA)

proper function of the cell [108-110].

**Food sources** Flaxseed oil (ALA)

**Molecular structure**

> myocardial membrane phospholipids are rich in (n-3) PUFAs after fish oil consumption [111, 112]. Diet-induced changes in the PUFA composition of a cell membrane have an impact on the cell's function, partly because these fatty acids represent a reservoir of molecules that perform important signalling roles within and between cells. In particular, dietary (n-3) PUFAs compete with dietary (n-6) PUFAs for incorporation into all cell membranes [113,114]. (n-3) PUFAs modulate the expression of adhesion proteins such as selectins [115] and exert an effect by modulating the intracellular signalling pathways associated with the control of transcrip‐ tion factors (e.g., nuclear factor-κB) and gene transcription [116,117]. Research has shown that enrichment of monocyte membranes with (n-3) PUFAs results in the synthesis and secretion

of reduced quantities of cytokines (e.g., tumour necrosis factor-α, interleukin-1β) that are involved in the amplification of the inflammatory response [117,118]. Therefore, at a cellular level, (n-3) PUFAs from fish oils can directly or indirectly modulate a number of cellular activities associated with inflammation.

Of all the vegetable oils, RPO has the highest content of tocotrienols with γ-tocotrienol the most abundant. This form of vitamin E has been demonstrated to reduce cholesterol produc‐ tion and platelet aggregation [147-151]. RPO may also exert a neutral or positive effect on the serum lipid profile through the effects of its fatty acid composition and tocotrienols [152-155]. Investigations into vitamin E showed that tocotrienols are more potent than tocopherols as antioxidants. The tocotrienols present in palm oil have been shown to offer protection from myocardial I/R injury in an isolated perfused rat heart model [156, 157]. Animal studies with tocopherols and tocotrienols that investigate these compounds' potential against chronic diseases are extensively reviewed by Aggarwal and co-workers [158]. These authors argue that the evidence overwhelmingly suggests that tocotrienols may be superior in their biological properties than tocopherols and that their anti-inflammatory and antioxidant activities could

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Carotenoids are nature's most widespread pigments, well known for their orange-red to yellow colours, which they impart to many fruits and vegetables. These fat-soluble phyto‐ chemicals have also received substantial attention because of their provitamin A and antioxi‐ dant roles [159]. Carotenoids are polyenoic terpenoids with conjugated trans double bonds. They include carotenes (*β*-carotene and lycopene), which are polyene hydrocarbons and xanthophylls (lutein, zeaxanthin, capsanthin, canthaxanthin, astaxanthin and violaxanthin) that have oxygen in the form of hydroxy, oxo, or epoxy groups [160]. The majority of the 600 carotenoids found in nature are 40 carbons in length and may be pure hydrocarbons, called carotenes, or possess oxygenated functional groups, in which case they are called xanthophylls [161]. The long-chain conjugated polyene structure accounts for the ability of these compounds to absorb visible light, but also makes them quite susceptible to oxidation. This latter property

The properties and therefore functions of a carotenoid molecule are primarily dependent upon its structure and hence its chemistry [163]. In particular, the conjugated C = C double bond system is associated with energy transfer reactions, such as those found in photosynthesis [164]. In human plasma and tissues, several carotenoids have been well characterized includ‐ ing cyclic (such as β-carotene and α-carotene) and acyclic carotenes (such as lycopene and phytoene), together with a number of xanthophylls (such as zeaxanthin, lutein and betacryptoxanthin), all of which can be directly derived from dietary sources [165]. Carotenoids have generated considerable interest as several studies have suggested an inverse association between the dietary intake of carotenoids and the risk for CVD [166, 167]. Conversely proox‐

As mentioned earlier, RPO supplementation does offer protection against myocardial I/R injury via several suggested mechanisms. Amongst the proposed mechanisms are the NO– cyclic GMP pathway, phosphorylation of mitogen-activated protein kinases and scavenging

prevent CVD among other chronic diseases.

is closely related to their ability to act as antioxidants [162].

idant roles of these phytochemicals have also been reported [168-170].

of deleterious reactive oxygen species by RPO [42, 43, 47, 48].

*2.1.8. Possible mechanism(s) of action*

*2.1.7. Carotenoids*

#### *2.1.5. Polyphenols*

Polyphenols constitute one of the most numerous and ubiquitously distributed groups of plant secondary metabolites, with more than 8000 phenolic structures currently known. Natural polyphenols can range from simple molecules (phenolic acids, phenylpropanoids, and flavonoids) to highly polymerised compounds (lignins, melanins, tannins), with flavonoids representing the most common and widely distributed sub-group [119]. These secondary plant metabolites are known to have potential antioxidant activity and radical scavenging capacity [120-124]. Polyphenols are gaining increased importance due to their beneficial effects on health. Flavonoids are the most abundant polyphenols in our diets. They can be divided into several classes according to the degree of oxidation of the oxygen heterocycle: flavones, flavonols, isoflavones, anthocyanins, flavanols, proanthocyanidins and flavanones [125]. A complication of the epidemiological observations regarding members of the flavonoid family is that subtle differences in their chemical structures can translate into marked differences in their absorption, metabolism and bioactivities [126]. South African herbal teas, rooibos (*Aspalathus linearis*) and honeybush (*Cyclopia ssp*.) are currently gaining popularity worldwide [127, 128], owing to their anti-oxidant, anti-cancer and anti-mutagenic properties [129-131]. Rooibos is a herbal tea made from the leaves and stems of the indigenous South African plant, *Aspalathus linearis* (*Brum.f*) *Dahlg.* (*family Fabaceae; tribe Crotalarieae*) [132,133]. Research has demonstrated that this herbal tea is rich in flavonoids [127, 134]. Animal studies that have investigated the cardioprotective effects of natural or synthetic flavonoids have focused mainly on the acute pharmacological activity of these compounds. For example, *in vivo* studies using animal models have reported acute cardioprotection obtained from intravenous injections of natural or synthetic flavonoids [135,136].

#### *2.1.6. Vitamin E*

Natural vitamin E is composed of eight chemical compounds: α-, β-, γ- and δ-tocopherols and their corresponding tocotrienols. α-Tocopherol is the most active form of vitamin E *in vitro*. The tocopherols are saturated forms of vitamin E, whereas the tocotrienols are unsaturated and have an isoprenoid side chain. Tocopherols possess a chromanol ring and a 15-carbon tail. The presence of three *trans* double bonds in the tail distinguishes tocopherols from tocotrienols [137-139]. This may account for the differences in their efficacy and potency *in vitro* and *in vivo* [140,141].

Red palm oil (RPO) is a rich source of vitamin E. It contains 560–1000 parts per million of vitamin E, of which approximately 18–22% are tocopherols and 78–82% tocotrienols [142-144]. RPO has been shown to offer protection against I/R injury [42, 43, 47, 48] leading to a reduction in oxidative stress [145]. It has also been suggested that palm oil may have some anti-arrhyth‐ mogenic effects, which may reduce sudden death after ischaemic incidents [146].

Of all the vegetable oils, RPO has the highest content of tocotrienols with γ-tocotrienol the most abundant. This form of vitamin E has been demonstrated to reduce cholesterol produc‐ tion and platelet aggregation [147-151]. RPO may also exert a neutral or positive effect on the serum lipid profile through the effects of its fatty acid composition and tocotrienols [152-155]. Investigations into vitamin E showed that tocotrienols are more potent than tocopherols as antioxidants. The tocotrienols present in palm oil have been shown to offer protection from myocardial I/R injury in an isolated perfused rat heart model [156, 157]. Animal studies with tocopherols and tocotrienols that investigate these compounds' potential against chronic diseases are extensively reviewed by Aggarwal and co-workers [158]. These authors argue that the evidence overwhelmingly suggests that tocotrienols may be superior in their biological properties than tocopherols and that their anti-inflammatory and antioxidant activities could prevent CVD among other chronic diseases.