*2.7.6 Antiaging activity*

An important factor in aging or age-associated degenerative diseases, the free radicals and oxidative stress have been believed as an antioxidant systems are declined during aging. Antiaging activities is explained by different mechanisms and revealed by antioxidant phytochemicals [34–37].

#### *2.7.7 Protective action on Alzheimer's disease*

It is particularly susceptible due to high concentration of free radicals without appropriate levels of anti-oxidation. In elderly people, the pathogenesis of dementia or AD shows oxidative stress. The study on walnuts shows that polyphenolic compounds helps to release the oxidant and decrease the inflammatory signs on the brain cells. It also repairs, the increased neurogenesis, inter-neuronal signaling, upgrade isolation of insoluble toxic protein accumulates, that play a role in preventing AD.  It is revealed that flavonoids are of special attraction and bioactive compounds in plant providing defensing effects. A study shows that in mice, it provides protection against cancer of skin which are caused through ultraviolet radiation or chemical carcinogens by consumption of tea and its polyphenolic constituents [39–43].

### *2.7.9 Miscellaneous*

Plant PC provide a means for preventing the side effects that fungal toxins (mycotoxins) and also serving in detoxification [41]. Many of the volatile PCs, such as the main PC of cloves i.e. eugenol (a hydroxyphenyl propene), or a typical component of oregano i.e. carvacrol (phenolic terpene), curcuminoids or Curcumin (diferuloylmethane) are which found only in the rhizomes of *Curcuma longa* [turmeric] are achieved. Curcumin as we all known plays an important role of various illnesses from cancer to autoimmune, neurological, cardiovascular, and diabetic etc. in the form of preventing and treating diseases [33, 42–45].


**5. Innovation project: use of ozone in the harvesting and processing of nopal**

#### **5.1 Introduction**

The nopal vegetable (*Opuntia ficus-indica*) grows in semi-arid zones around the world, it has a high dietary fiber content, and is usually consumed Mexico [40, 41]. Physical and morphological characteristics of nopal allow a quick adaptation to low precipitation and excessive heat (**Figure 3**). The nopal consist of two main parts: the edible part (84–90% water), and prickly pear (45–67% of reducing sugars) also called "tuna" [42]. Relative humidity is a crucial variable in nopal for development of pests and diseases. The most commonly microorganisms present in nopal are: cactus weevil *Cactophagus spinulae* (*Gyllenhal*), [43]; boll weevil (*Cylindrocopturus biradiatus Champs*), [40]; white worm (*Lanifera cyclades Druce*), zebra worn (*Olycella nephelepsa Dyar*), [44]; and cochineal (*Dactylopius coccus costa*), [45]. The most important include the bacterial stain (*Bacterium sp*.), rotting of the epidermis, anthracnose, prickly pear gold [46], the thickening of cladodes and the presence of bold among others [47]. The objective was to give added value to nopal-vegetable (raw and processed) using ozonated foliar spraying and ozonated water irrigation.

**77**

**Figure 4.**

*Innovation in Food Products Using Ozone Technology: Impact on Quality Assurance*

Due to the nature of nopal, good manufacture practices in soil and water are required to control outbreaks of diseases. The experimental plantation was located in Matehuala, San Luis Potosi; México. The producers gave light watering to nopal seedlings every 8 or 15 days during the dry months as shown in **Figure 4**, and the irrigation program was monitored periodically to establish the ozone generator requirements. Salinity, hardness, pH and sedimented solids were registered for 15 days before installing the irrigation system. The ozone solubility control included the measurement of pressure and temperature, concentration of carbonates, nitrates and metals [6]. Once the nopal "pencas" (thick and fleshy central nerve that has leaves) were cut, they were placed in recyclable plastic boxes preserving the

Ozonated water favored the organoleptic integrity of the nopal, reducing oxidation reactions and retaining green color in sliced raw nopal. Other goals achieved were the reduction in microbial load and the increase in shelf life (data not shown). The final product was packaged and sealed in plastic bags with 500 g and 1000 g. An investment proposal to install a rural cooperative company with capacity of

Technical specifications and cost of a nopal sider, an immersion washing machine, a centrifugal dryer, and a vibrator dryer with cold were included in the

*DOI: http://dx.doi.org/10.5772/intechopen.96681*

thorns to avoid the oxidation reactions [48].

investment project for \$1,500,000.00 pesos.

*Sustainable management of the nopal irrigation system.*

processing 500 kg per day was presented to the cooperative.

**5.3 Results and discussion**

**5.2 Methodology**

*Manual harvesting of nopal in México.*

**Figure 3.**

*Innovation in Food Products Using Ozone Technology: Impact on Quality Assurance DOI: http://dx.doi.org/10.5772/intechopen.96681*

**Figure 3.** *Manual harvesting of nopal in México.*

#### **5.2 Methodology**

*Innovation in the Food Sector Through the Valorization of Food and Agro-Food By-Products*

The research team of Pérez-Nafarrate [36] designed processes using ozone gas and ozone water for disinfection and packaging of mango in an industrial plant in Guerrero, Mexico. The accomplishment of concise food safety regulations was according to studies realized by Tran et al. [37]. In order to preserve the physicochemical characteristics of mango fruit, the next premises were followed:

Ozone gas used in a regular basis (one dose every third day) should eliminate

Gas monitoring was performed using an ozone analyzer (Model IN-2000,

Ozone concentrations (1.5, 2.5, 3.5, 5 ppm) applied (96 h, 30 ± 3 °C) in the storage container should reduce the ethylene content generated as part of the fruit

The most effective ozone dose (3.5 ppm) preserved the organoleptic characteristics of the fruit and delayed the ripening for 15 days. According to producers, optimal shelf life of mango is 10 days [37]. Soluble solids, color, flavor and empiric firmness were improved (data not published) and a sensory evaluation of the fruit was developed by the operators. More and Rao [38, 39] used a combination of UV-C irradiation (210–280 nm), guar gum (2.5%), and ozone water (400 mg-h-1) to preserve organoleptic characteristics of mangoes at 32 ± 3 °C after 19 days.

Compliance with good manufacturing practices diminish the fruit deterioration, and the economic profit increased. Harvesting of climacteric fruits, including mangoes, papaya, and banana; requires storage temperature and relative humidity similar to that observed in subtropical environments. According to Pérez-Nafarrate [36], key factors to conduct an ozonated water experiment consist on reliability and accuracy of the measurement devices, type and growth stage of the microorganism,

**5. Innovation project: use of ozone in the harvesting and processing of**

The nopal vegetable (*Opuntia ficus-indica*) grows in semi-arid zones around the world, it has a high dietary fiber content, and is usually consumed Mexico [40, 41]. Physical and morphological characteristics of nopal allow a quick adaptation to low precipitation and excessive heat (**Figure 3**). The nopal consist of two main parts: the edible part (84–90% water), and prickly pear (45–67% of reducing sugars) also called "tuna" [42]. Relative humidity is a crucial variable in nopal for development of pests and diseases. The most commonly microorganisms present in nopal are: cactus weevil *Cactophagus spinulae* (*Gyllenhal*), [43]; boll weevil (*Cylindrocopturus biradiatus Champs*), [40]; white worm (*Lanifera cyclades Druce*), zebra worn (*Olycella nephelepsa Dyar*), [44]; and cochineal (*Dactylopius coccus costa*), [45]. The most important include the bacterial stain (*Bacterium sp*.), rotting of the epidermis, anthracnose, prickly pear gold [46], the thickening of cladodes and the presence of bold among others [47]. The objective was to give added value to nopal-vegetable (raw and processed) using ozonated foliar spraying and ozon-

ozone concentration and time of contact with the product.

**4.2 Methodology**

ripening process.

**nopal**

**5.1 Introduction**

gradually the presence of pests.

L2-LC, in USA Incorporated).

**4.3 Results and discussion**

**76**

ated water irrigation.

Due to the nature of nopal, good manufacture practices in soil and water are required to control outbreaks of diseases. The experimental plantation was located in Matehuala, San Luis Potosi; México. The producers gave light watering to nopal seedlings every 8 or 15 days during the dry months as shown in **Figure 4**, and the irrigation program was monitored periodically to establish the ozone generator requirements. Salinity, hardness, pH and sedimented solids were registered for 15 days before installing the irrigation system. The ozone solubility control included the measurement of pressure and temperature, concentration of carbonates, nitrates and metals [6]. Once the nopal "pencas" (thick and fleshy central nerve that has leaves) were cut, they were placed in recyclable plastic boxes preserving the thorns to avoid the oxidation reactions [48].

#### **5.3 Results and discussion**

Ozonated water favored the organoleptic integrity of the nopal, reducing oxidation reactions and retaining green color in sliced raw nopal. Other goals achieved were the reduction in microbial load and the increase in shelf life (data not shown). The final product was packaged and sealed in plastic bags with 500 g and 1000 g. An investment proposal to install a rural cooperative company with capacity of processing 500 kg per day was presented to the cooperative.

Technical specifications and cost of a nopal sider, an immersion washing machine, a centrifugal dryer, and a vibrator dryer with cold were included in the investment project for \$1,500,000.00 pesos.

**Figure 4.** *Sustainable management of the nopal irrigation system.*

#### *Innovation in the Food Sector Through the Valorization of Food and Agro-Food By-Products*

Good manufacturing practices are essential to accomplish nopal processing with ozone. Water quality and soil requirements monitoring were key factors to improve the postharvest life of nopal opuntia. An innovative option for rural population centers was presented to detonate the local economy. Almost 50% of inhabitants in Mexico are distributed in rural areas, therefore ozone technology should be affordable to most of the population.


**3.5. Piracy**

164 Risk Management – Current Issues and Challenges

countries [40].

the authorities.

illegal product is discovered.

the freight routes and port activities in the same way as legal supply chain does. Among the receiver countries, Europe and the US are favorites, just as Africa is the favorite for transit activities. Confiscated products that have not been produced in Africa, like jewelry and CDs, show this, because the African market does not have the ability, in general, to buy that type of product. Countries in Central and South America act like magnets for counterfeited products. Purchases of counterfeited goods to launder money occur in larger numbers there than anywhere else in the world [40]. Large stocks of illegitimate products are easily shipped from parts of South America to Central America, where they are big consumers of that type of product. Organized crime also uses Central America as the base for shipments of illegitimate goods to North America. The situation in Europe makes it the most lucrative market for counterfeited products. The types of confiscated goods at the external borders of the EU are different from other places in the world. This indicates that the dealers of counterfeited products adjust products to each market's special condition. They look at the fashion, culture, and buying habits of individual

Both types of Illegal supply chains use the international flow of containers to transport their products all over the world, regardless if the product is counterfeited or an illegal drug. Criminals try to delude customs' watchfulness by "breaking" their way through from the area of production to the area of supply, and avoiding direct paths that are well known to

A problem linked to smuggling is the manufacturing of products without intellectual rights, or the production of counterfeited goods. Everything that has been produced can be reproduced by someone else. In order to bring counterfeited products from the production site to the end user, they may have to cross several national boundaries as well as intellectual property legislation. The counterfeiting business evolves constantly within current trends and technologies. The production and distribution of illegal products is performed under the risk for detection and this diversity leads to a different design of the supply chain [41]. The location of the production facilities is subject to the risk of discovery. Normally, illegal production units are placed where the risk for detection is low, in line with the normal legal business problem of where to produce according to different types of costs and quality aspects. Then, the illegal products are distributed by trade routes and port activities in the same way as legal products [42]. The pollution of illegal products in the legal transport network is a serious problem. The most common countermeasure against smuggling is the inspection of cargo carriers when they cross a national border. The mere existence of these inspections creates disturbances in the transport network, even if no

The counterfeiting business evolves constantly within current trends and technologies. The illegal products are then distributed by trade routes and port activities in the same way as legal products [42]. According [43], the discourse on the gray market is filled with the idea of a criminal underworld in order to separate it from the legal/normal upper-world. In reality is it very difficult to establish the underworld/upper-world image. The legal The modern types of pirates do not act officially of any specific courtiers' order but research has indicated relationship between piracy and weakness of central governances [44]. In essence is piracy an international crime against all states and the perpetrators can be brought to justice in all everywhere [45]. In recent years has the threat from piracy against sea shipment (direct threat) and also against the different supply chains utilizing sea shipment (indirect threat) received increasing attention [44, 46, 47, 48]. The real increasing threat from pirates, primary at the horn of Africa [45, 47], has resulted into both a changes in shipping routes (Sullivan, 2010) and also a naval response from several countries [46] that are depended on a smooth passage of cargo carrying ships. According [49], was there 489 attacks last year (2010) and over a twelve year period was there about 347 attacks/year globally [43]. Piracy is an increasing problem, especially near Somalia [48]. The pirates are changing their tactics and targets to use more sophisticated weaponry and apply more advanced techniques, all in order to improve their own success ratio. Table 2 presents the current trends and patterns in piracy.

**Table 2.** Trends and numbers in piracy 2006-2010 [48]

As stated in table 2, piracy is an increasing threat and especially alarming is the increasing use of violent and kidnapping of crew members. According to [48], piracy cost the maritime industry between 7 - 12 billion dollars a year. In addition to this shall also the cost for rerouting to avoid pirate infested waters, ransom payments and support from various organizations be added.


**1. Introduction**

Sesquiterpenoids are an extremely wide group of secondary metabolites found in the plant kingdom [1,2]. Their main carbon backbone consists of 15 atoms and several synthases action leads to a great variety of chemical structures. In particular, bicyclic sesquiterpenoids have raised attention in the bio-pharmacological field, even as essential oils. Among them, β-caryophyllene is one of the major component of essential oils extracted from spice and food plants [3–7], such as black pepper (*Piper nigrum* L.), rosemary (*Rosmarinus o*ffi*cinalis*), cinnamon (*Cinnamomum* spp.) oregano (*Origanum vulgare* L.) basil (*Ocimum* spp.), thyme (*Thymus vulgaris*), sage (*Salvia o*ffi*cinalis*), mint *(Mentha piperita***)**, ginger (*Zingiber o*ffi*cinale*), chinotto (*Citrus Myrtfolia* Raf) [8], and cloves (*Syzygium aromaticum*) [9,10]. It was also found in citronella, (*Cymbopogon*), pine tree (*Pinus*), *Chenopodium ambrosioides*, *Cannabis sativa*, in plants of the genus *Copaifera*, *Artemisia*, *Murraya*, *Cordia*, *Spiranthes*, *Ocimum*, *Croton* [9,10], and in the leaves of *Annona Cherimola* [11]. This plant compound was approved by Food and Drug Administration (FDA) and European Food Safety Authority (EFSA) and it is used as flavour enhancer [9] and in cosmetics [12]. In nature, BCP mainly occurs as *trans*-caryophyllene ((E)-BCP) (**1**) mixed with small amount of the isomers (Z)-β-caryophyllene ((Z)-BCP) (**2**) and α-humulene (α-caryophyllene) (**3**) [12], and its oxidation derivative, β-caryophyllene oxide (BCPO) (**4**) (Figure 1).

**Figure 1.** Structures of sesquiterpenes (**1**–**4**).

BCP belongs to cannabinoid family, which are ligands of the cannabinoid receptors present in the organism. Cannabinoid receptors CB1-R and CB2-R are metabotropic receptors are G protein (protein binding GTP)-coupled receptors, involved in the regulation of neurotransmitters responsible for maintaining an energetic balance, in the metabolism, and in the immune response. The aforementioned receptors are bound and activated by endogenous cannabinoids, derivatives of arachidonic acid, including 2-arachidonoylglycerol and *N*-arachidonoylethanolamine, better known as anandamide. Both receptors are bound by a lot of proteins in various pathways, acting as mediators of cellular responses to biological molecules.

Unlike the main traditional cannabinoids, such as Δ8-tetrahydrocannabinol (**5**), Δ9- tetrahydrocannabinol (**6**) and cannabinol (**7**) (Figure 2), able to activate both receptors, BCP has a very different chemical structure and it is a selective agonist of CB2-R. Furthermore, BCP has no side effects, not activating CB1-Rs (mainly expressed in the central nervous system, but also in the liver, lungs, heart, blood vessels and digestive tract). CB2-Rs are mainly found in peripheral tissues and in immune system cells (B and NK lymphocytes, macrophages, mast cells) and, to a lesser extent, in the central nervous system (brain, neurons, microglia) [13]. The expression of CB2-Rs in the central nervous system is increased in neurodegenerative pathologies, such as Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS) and in some tumours like gliomas [14].

The binding of BCP to CB2-Rs is responsible for many cellular events:

**Figure 2.** Structures of cannabinoids (**5**–**7**).

In addition to the involvement in physiological functions, CB2-Rs are implied in numerous pathological processes, so they can represent an interesting target in order to obtain agonist molecules for the treatment of many pathological conditions, including neuropathic pain, inflammation, neuroinflammatory and neurodegenerative pathologies (Parkinson's disease, Alzheimer's disease, multiple sclerosis, and amyotrophic lateral sclerosis), spinal and brain injuries, stroke, ischemia, anxiety disorder, depression, colitis, fibrosis and liver ischemia, atherosclerosis, osteoporosis, osteoarthritis, diabetes, obesity, and some types of cancer [9,12–14,16–21].

Molecular docking studies showed that BPC interacts with CB2-R on the binding site of CP55,940 (an agonist), precisely in a hydrophobic pocket, engaging with hydrophobic amino acid residues (valine-113, phenylalanine-117, isoleucine-198, tryptophan-258 and methionine-265) [12]. The double bond with conformation E of BCP is fundamental for receptor binding. BCPO lacks a double bond in position C4-C5 and it is not able to bind CB2-R [10].

### **2. Biological properties of** β**-caryophyllene**

In the last few years, BCP has represented an important subject of study [9–15,22–65]. In particular, a lot of researches about the its effects in vitro and in vivo on animals have been conducted, thus experimental data about its biological properties have been verified. However, further studies are needed in order to translate the findings in animal models into promising pre-clinical and clinical trials on humans. Pre-clinical studies have revealed that BCP is a modulator of nervous system and exerts beneficial effects on numerous neurodegenerative and inflammatory pathologies. Moreover, it is able to act on the liver and bones, and has antibiotic properties.

Table 1 shows the principal studies on β-caryophyllene.

### **Table 1.** Preclinical studies on β-caryophyllene.

**Table 1.** *Cont.*

**Table 1.** *Cont.*

### *2.1.* β*-Caryophyllene and Nervous System*

Many studies report the beneficial effects of BCP on central nervous system [22–44], in particular against neuroinflammatory and neurodegenerative pathologies.

Neuroinflammation is a process leading to nervous system degeneration, characterised by the activation of monocytes, macrophages, mast cells, lymphocytes, and the production of inflammation mediators, such as nitric oxide (NO), various cytokines (IL-1β, IL-6 and TNF-α), the protein NF-κB (nuclear factor kappa B) and the prostaglandins.

In detail, BCP, administered intraperitoneally in Wistar rats, at the dose of 50 mg/kg, has reduced the activity of inducible nitric oxide synthase (iNOS) and, consequently, nitric oxide production, thus decreasing brain oxidative stress and leading to the inhibition of lipid peroxidation and the depletion of glutathione stores [22], causing striatal and cortical neurotoxicity [9]. Moreover, nitric oxide is involved in the activation of cyclooxygenase (COX), the enzyme synthetizing prostaglandin H2 (PGH2), which is the precursor of the other prostaglandins, in particular Prostaglandin D2 (PGD2) and Prostaglandin E2 (PGE2), responsible for inflammation and pain. In this contest a frontiers of research for BCP and BCPO will be their influence on microRNA, small molecule able to regulate gene expression, considering that they are involved and proposed as biomarkers, in neuroinflammation and pain [66,67].

The essential oil of *Erymanthos erythropappus*, rich in BCP, has been shown to have anti-inflammatory in Wistar rats [23].

It has also been demonstrated that BCP, at the dose of 10 mg/kg, inhibits the transcription of iNOS, IL-1β, IL-6, and COX-2 in C6 microglia cells [9,24].

Furthermore, BCP, tested on the mouse BV2 cell line at the concentrations of 10, 25 and 50 μM, has inhibited NF-κB activation and reduced the production of nitric oxide and PGE2, thus suppressing hypoxia-induced neuroinflammatory response [25,26].

In the case of central nervous system pathologies, Iba-1, analogous protein of Aif-1, present only in macrophages and microglia, is over expressed (for example, in ischemia) [27], whereas GFAP (glial fibrillary acidic protein), which forms intermediate filaments, is expressed in a lot of central nervous system cells (including astrocytes and ependymal cells). In addition, it is involved in cell communication, in the interaction neuron-astrocytes, in the functioning of the blood–brain barrier (BBB), particularly during mitosis, in which it modulates the filament network, and in the repair following brain injuries [9,28]. It has been demonstrated that BCP administered intraperitoneally at the dosage of 50 mg/kg, reduces the activation of astrocytes and microglia, by decreasing Iba-1 and GFAP expression, thus avoiding the death of dopaminergic neurons [22].

Amyloid plaques, formed by β-amyloid peptides, composed in turn by 36–43 amino acids and derived from amyloid precursor protein (APP), characterize Alzheimer's disease. These peptides are responsible for direct toxicity (death of neurons) and indirect toxicity (production of molecules stimulating inflammation). A considerable decrease of the β-amyloid peptide-induced overexpression of TLR4 (Toll-like receptor 4), which determines the activation of monocytes and microglia, has been noted in BV2 microglia cells treated with BCP, and, consequently, the sesquiterpene leads to a clear reduction of the biosynthesis of IL-6, IL-1β, PGE2, TNF-α, NF-κB and nitric oxide. In addition, also a reduced expression of COX-2 and iNOS has been observed [26].

Another common condition affecting central nervous system is Parkinson's disease, a neurodegenerative pathology characterized by neuroinflammation, oxidative stress, mitochondrial dysfunction and cell death, particularly in dopaminergic neurons. An in vitro study on SH-SY5Y cells showed that the treatment with BCP inhibits reactive oxygen species (ROS) production, restoring mitochondrial functionality and the levels of the antioxidant glutathione. BCP prevents apoptosis, by inhibiting the expression of Bax and caspase-3 and increasing Bcl-2 one. It reduces the phosphorylation of JNK (c-Jun N-terminal Kinase), which determines the increase of HO-1 (heme oxygenase-1) expression in this pathological condition. All these effects are related to CB2/Nrf2 pathway [29]. Research on a mouse model of Parkinson's disease conducted by Viveros-Paredes revealed that the treatment with BCP, at the dose of 10 mg/kg, enhances motor coordination in mice and protects the dopaminergic neurons from degeneration, reducing the production of inflammatory cytokines, in particular IL-1β, IL-6, and TNF-α [30].

BCP is also cytoprotective towards the central nervous system due to its modulation of the redox state and inflammation, useful during chemotherapy. The main mechanism of action regarding this aspect is the stimulation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) following the activation of cannabinoid receptors CB2. Nrf2 is a transcription factor, whose activity is stimulated by oxidative stress (in particular, reactive oxygen species or ROS) and oncogenes, such as KRAS, BRAF and MYC. Nrf2 increases the expression of the genes involved in cell survival and in the reduction of the inflammatory process and the oxidative stress in SNC. Moreover, nerve growth factor (NGF) is stimulated through Tropomyosin receptor kinase A (Trk A) pathway, which stimulates in turn PI3K-Akt pathway [68].

BCP, through Nrf2 activation, increases the expression of antiapoptotic genes (B-cell leukemia/lymphoma-2 (BCL-2), Mouse double minute 2 (mdm-2), COX-2, c-Myb) and, at the same time, reduces the one of proapoptotic genes (Bax, Bak-1, caspase-8, caspase-9) [31].

All these cellular events determine cell survival and proliferation and angiogenesis [9].

BCP also diminishes the expression of matrix metallopeptidase (MMP-9) and increases the one of occludin, claudin-5, Tight Junction Protein ZO-1 and Growth Associated Protein 43 (GAP-43) [32].

Furthermore, BCP, at the concentrations of 0.5 and 1 μM, exerts cytoprotective effect on C6 glioma cells, increasing the antioxidant activity, through CB2-R-dependent Nrf2 pathway [33]. In addition, administered orally in Wistar rats, at the dosages of 34, 102 and 306 mg/kg, it reduces oxidative stress and neuronal apoptosis, due to the increased expression of Nrf2 and OH-1.

BCP also inhibits the production of nitric oxide, hydrogen peroxide, TNF-α, interferon gamma (IFN-γ), interleukin-17 (IL-17) reducing the extent of macrophage infiltration [9].

BCP can induce neurogenesis with a mechanism independent from the activation of CB2-R, Nrf2 and NGF, stimulating Tropomyosin receptor kinase A (TrkA) in SH-SY5Y (Cell lines Homo sapiens, human, bone marrow) and PC12 (Cell line derived from a pheochromocytoma of the rat adrenal medulla) neuroblastoma cells [34].

There are numerous studies regarding the analgesic effects of the sesquiterpene, isolated or in mixture in essential oils. In fact, the essential oil of *Erymanthos erythropappus*, containing BCP as one of the main components, administered orally at the doses of 200 and 400 mg/kg, induces analgesia in Wistar rats [23]. Further, the essential oil of *Senecio rufinervis*, containing about the 6% of BCP, is responsible for the analgesic effect in mice, when administered orally at the doses of 50 and 75 mg/kg [35].

Other studies have reported the same results in mice at the doses of 1 and 5 mg/kg intraperitoneally and at the dosages of 2.6 mg/kg/die per *os*for 2 weeks, alone or in combination with the docosahexaenoic acid (DHA) [9]. The essential oil of *Vitex agnus-castus*(BCP accounts for about 7% of the oil), has exhibited analgesic effect in Wistar rats which were subjected to an immersion test.

BCP, at the dose of 25 mg/kg, decreases the extent of peripheral neuropathyin mice, by the activation of cannabinoid receptors CB2-R and the inhibition of the MAPK p38, resulting in a reduced transcriptional activity of NF-κB, involved in phlogosis [36].

It has been demonstrated in mice that BCP actually exerts its antinociceptive action by activating CB2-Rs, in particular towards primary sensory neurons. In fact, the analgesic effect manifests itself in wild-type mice, but not in the CB2-R knockout mice. The activation of cannabinoid system indirectly leads to the modulation of opioid system. In more detail, it stimulates β-endorphin release, activating opioid receptors μ on primary afferent neurons. BCP has been shown to boost the painkilling effect of morphine, used in the treatment of severe pain, making possible the reduction of the dose of the drug and, consequently, decreasing its side effects [37].

Instead, there was no indication of synergy between BCP and the other components of the essential oils [13].

The derivative BCPO has not elicited any interest as an analgesic [13], since it does not bind to cannabinoid receptors due to the different molecular structure.

BCP could result potentially useful in multiple sclerosis management (pathology characterized by axon demyelination and neuroinflammation), as demonstrated by previous study which investigated the therapeutic potential of BCP on experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS).

The research has shown that the treatment with BCP reduces interferon-γ (IFN-γ) production, the main activator of macrophages, and increases the levels of interleukin-10 (IL-10).

Moreover, it inhibits cell T migration. The oral treatment with 50 mg/kg of BCP twice a day has decreased hyperalgesia induced by the EAE and protected from brain damage, inhibiting cytokine biosynthesis and restoring the activity of catalase, superoxide dismutase and glutathione peroxidase, all enzymes involved in the detoxification of oxidant substances [38].

Two essential oils containing BCP, basil one, administered intraperitoneally (at the doses of 100, 150 and 200 mg/kg), and *Baccharis uncinella* one, administered per *os* (at the doses of 50 or 100 mg/kg), exhibit significant sedative effect [39,40].

These early results on the animal model could be attributed to BCP, being a valid alternative to the most common sedatives and anaesthetics.

The latter drugs have contraindications in patients with cardiovascular, neuromuscular and cerebrovascular diseases, and could cause many side effects, such as convulsions, insomnia, agitation, dependence and, in the most extreme cases, lead to death.

Basil essential oil has also exerted anxiolytic effect [39]. The same results have been obtained in mice, when BCP has been orally administered at the dose of 50 mg/kg [41].

Therefore, all these data suggest the possible use of the sesquiterpene as a substitute of the most common anxiolytics, like benzodiazepines and the selective serotonin reuptake inhibitors (SSRI), which cause many adverse effects, such as movement problems, sedation, dependence, muscle relaxation, anterograde amnesia, teratogenic risk, reduction of bone mineral density.

BCP also exhibits antidepressant properties, as revealed by a research conducted on albino Swiss mice receiving 50 mg/kg of the BCP per Os (oral somministration). The mechanism of this e ffect is due to the activation of CB2-R [41].

Various studies have shown muscle relaxant e ffect of BCP in animals. In this regard, the compound, alone or as a constituent of the essential oil from *Pterodon polygalaeflorus*, exerts antispasmodic activity on rat isolated ileum at variable concentrations (1 to 1000 μg/mL) [42].

BCP, orally administered at the dosages of 10, 30, and 100 mg/kg in C57BL/6 mice, exerted a dose-dependent anticonvulsant e ffect [9,43]. Furher research on mice has revealed that the sesquiterpene inhibits tonic-clonic seizures in MES (maximal electro shock seizure) test and reduces kainate-induced neurotoxicity, suggesting a possible use of the sesquiterpene for the treatment of epilepsy [44].


**1. Introduction**

324 Chromatography – The Most Versatile Method of Chemical Analysis

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[17] Surewicz WK, Szabo A G, Mantsch H H. Conformational properties of azurin in solution as determined from resolution-enhanced Fourier-transform infrared spectra.

carcinoma cells. Journal of Cancer Science and Therapeutics 2011;3(5) 104-111. [15] Fukuo T, Kubota N, Kataoka K, Nakai M, Suzuki S, Arakawa R. Matrix-assisted laser desorption/ionization and electro spray ionization mass spectrometry analysis of blue copper proteins azurin and Mavicyanin. Rapid Communication in Mass Spectrometry

elements in cytotoxicity. Biochemistry (Mosc) 2003;68 958-962.

*Pseudomonas aeruginosa*. Biochemical Journal 1976;157 423-430.

European Journal of Biochemistry 1987;167 519-523.

The apple tree belongs to the *Plantae* kingdom, *Magnoliophyta* class, *Rosales* order, *Rosaceae* family, *Pomoidae* sub-family and *Malus* genus. Originating in the mountains of Asia, the apple (*Malus domestica* Borkh) is a temperate fruit with an ancient history [1].

The center of the origin of the *Malus* cultivars is understood to be in Asia Minor, in the Caucasus, located between the Caspian and the Black Seas, the Indian Himalayas, Pakistan and Western China[2], all areas that had access to the Silk Road where marketable products were transported from the east to Europe since antiquity. This is a series of trade routes that crossed Eurasia by land and by stretches of sea or river for over 2,000 years until 1,500 BC, and which enabled the movement of important materials that promoted the exchange of ideas and innovations between different cultural groups. Over the centuries, gunpowder, the compass, the printing press, silk, mathematics and ceramics all migrated along this road, as well as stringed instruments. Resources, information and news were all spread in this fashion among many cultures for such a long period that is often difficult to identify the source of many traditions.

Whatever may have been the mechanism of transport of varieties of apples it is certain that animals, birds, cattle and bats, among others, played an important role [2]. The caravans, with their humans and camels, ate the fruit and left the seeds in fertile areas along the highway since the Neolithic period and many cultivars were perfectly defined in the Middle East around 4000 BC. These regions are characterized by a temperate climate, with low winter temperatures - important for the winter hibernation of apple trees. Commercial apples today are descendants of species from Asia, including the Gala, Golden Delicious and Fuji.

© 2012 Wosiacki et al., licensee InTech. This is an open access chapter 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. © 2012 Wosiacki et al., licensee InTech. This is a paper 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.


obtained by Ray X diffraction

Table 3. Molecular electrostatic potentials for all members of the series calculated at B3LYP/6-31G\* level of theory

For the N-(2'-furyl)-imidazole a region can be observed where the delocalization of charge decrease (blue region between two rings) due to the breaking-off planarity of the both rings, while the remain molecules show π-electron delocalizated surfaces that evidence stables aromatic systems, being smaller for the N-(2'-furyl)-imidazole compound.

These results can be related with the energies of the HOMO and LUMO for all molecules. Table 4 shows the comparison of the HOMO and LUMO orbitals energies values as well as the GAP energies values of them.

The corresponding values for the N-(2'-furyl)-imidazole show that this compound is the most stable of the series, as expected, because it presents the longer GAP values. On the

Structural and Vibrational Properties and NMR Characterization of (2'-furyl)-Imidazole Compounds 175

HOMO -0.21 -0.22 -0.19 -0.19 -0.18 -0.18 -0.19 -0.19 LUMO -0.01 -0.01 -0.01 -0.01 0.00 0.00 -0-01 -0-01 GAP 0.20 0.20 0.17 0.17 0.18 0.18 0.17 0.17

(kJ/mol) 535.0 542.9 464.2 464.2 495.4 495.4 466.8 466.8

Table 4. HOMO and LUMO orbitals Energíes (u.a.) and gap of energy (u.a.) for all members

All nuclear magnetic resonance spectra were recorded for diluted solutions in DMSD-d6 and the calculated chemical shifts of the 1H NMR and 13C NMR for the two conformers of each compound were obtained by GIAO method24 using the B3LYP/6-311++G\*\* theory level, as it is usually used for chemical shift NMR calculations on reasonably large molecules25,26. The calculations have been performed using the geometries optimized for this theory level and by using TMS as reference. A comparison of the results is present in Table 5 and it shows that for 2-(2'-furyl)-imidazole the calculated 13C chemical shifts from CSGT method are in accordance with the experimental values, while the closest values were obtained with the GIAO method and the 6-311++G\*\* basis set. Furthermore, the calculated shifts with both methods for the C(2), C(7), and C(1) atoms are higher than the experimental values. One important observation is that the results obtained from the conformational average have better agreement with data experimental, as expected, due to the presence of the two conformers in the solution. On the other hand, a comparison is present in Table 6 and it shows the calculated shifts with the two methods used where, for the H atom show significant differences with the experimental results. The addition of the polarization and diffuses functions at the basis set improves the results; however, the CSGT method predicts shifts practically different than the GIAO method for this atom. This disparity would be attributed to the fact that the GIAO method uses basis functions which depend on the field while the CSGT method achieves gauge invariance by performing a continuous set of gauge transformations, for each point, obtaining an

The peak belonging to H atom of the N–H bond appears at 11.37 ppm. A small shift of these peaks towards lower fields implies the existence of some intermolecular interaction between

Table 7 shows the experimental and calculated chemical shifts for 4(5)-(2'-furyl)-imidazole. These chemical shift values indicate that in the H atom is weakly held by two imidazole rings. At an intermediate rate of exchange, the H atom is partially decoupled, and a broad

*syn anti syn anti syn anti syn anti*

4-(2'-furyl)- Imidazole

5-(2'-furyl)- Imidazole

2-(2'-furyl)- Imidazole

Orbital N-(2'-furyl)-

GAP

of the series

Imidazole

**2.2 Nuclear magnetic resonance characterization**

accurately description of the current density17,18.

nonbonding electrons.

24 Ditchfield, 1974. 25 Cheeseman et al., 1996. 26 Keith Bader, 1993.

other hand, the calculated energy HOMO orbital value for 4-(2'-furyl)-imidazole is higher than the other ones due to the fact that it is the most reactive compound of this series.

Fig. 3. Electrostatic Surface Potential calculated on the molecular surface of the conformers for all compounds of the series. Color, in u.a.: from red -0.103 to blue 0.103.

Table 4. HOMO and LUMO orbitals Energíes (u.a.) and gap of energy (u.a.) for all members of the series

#### **2.2 Nuclear magnetic resonance characterization**

All nuclear magnetic resonance spectra were recorded for diluted solutions in DMSD-d6 and the calculated chemical shifts of the 1H NMR and 13C NMR for the two conformers of each compound were obtained by GIAO method24 using the B3LYP/6-311++G\*\* theory level, as it is usually used for chemical shift NMR calculations on reasonably large molecules25,26. The calculations have been performed using the geometries optimized for this theory level and by using TMS as reference. A comparison of the results is present in Table 5 and it shows that for 2-(2'-furyl)-imidazole the calculated 13C chemical shifts from CSGT method are in accordance with the experimental values, while the closest values were obtained with the GIAO method and the 6-311++G\*\* basis set. Furthermore, the calculated shifts with both methods for the C(2), C(7), and C(1) atoms are higher than the experimental values. One important observation is that the results obtained from the conformational average have better agreement with data experimental, as expected, due to the presence of the two conformers in the solution. On the other hand, a comparison is present in Table 6 and it shows the calculated shifts with the two methods used where, for the H atom show significant differences with the experimental results. The addition of the polarization and diffuses functions at the basis set improves the results; however, the CSGT method predicts shifts practically different than the GIAO method for this atom. This disparity would be attributed to the fact that the GIAO method uses basis functions which depend on the field while the CSGT method achieves gauge invariance by performing a continuous set of gauge transformations, for each point, obtaining an accurately description of the current density17,18.

The peak belonging to H atom of the N–H bond appears at 11.37 ppm. A small shift of these peaks towards lower fields implies the existence of some intermolecular interaction between nonbonding electrons.

Table 7 shows the experimental and calculated chemical shifts for 4(5)-(2'-furyl)-imidazole. These chemical shift values indicate that in the H atom is weakly held by two imidazole rings. At an intermediate rate of exchange, the H atom is partially decoupled, and a broad

174 Magnetic Resonance Spectroscopy

other hand, the calculated energy HOMO orbital value for 4-(2'-furyl)-imidazole is higher than the other ones due to the fact that it is the most reactive compound of this series.

Fig. 3. Electrostatic Surface Potential calculated on the molecular surface of the conformers

for all compounds of the series. Color, in u.a.: from red -0.103 to blue 0.103.

24 Ditchfield, 1974.

25 Cheeseman et al., 1996.

26 Keith Bader, 1993.

Structural and Vibrational Properties and NMR Characterization of (2'-furyl)-Imidazole Compounds 177

 Exp.b Theorc Exp.b Theorc 10 104.3 103.55 6.54 (d, J=3.3 Hz, 1H) 6.29 11 111.3 108.82 6.43 (dd, J=1.8, J=3.3 Hz, 1H) 6.17 5 114.7 111.03 7.33 (d, J=0.9 Hz, 1H) 6.98

12 135.6 138.1 7.71 (d, J=0.9 Hz, 1H) 7.26 2 141.2 141.35 7.39 (dd, J=0.9, J=1.5 Hz, 1H) 7.16

N-H 11.37 (bs,1H) 7.86

Table 7. Experimental and calculated Chemical shifts (in ppm) for 4(5)-(2'-furyl)-imidazole

A comparison between the experimental and calculated chemical shifts for the C and H atoms for N-(2'-furyl)-imidazole are given in Tables 8 and 9, respectively. The calculation results show that the GIAO method reproduces quite well the 13C and 1H experimental chemical shifts values as shown by the calculated root mean square deviations (RMSD) values for each conformer. Furthermore, the shifts of the H atoms for both structures are similar in both conformers and practically equal to the average values. As expected, the latter values have a better agreement with the experimental data, probably because both

Chem. Shifts C10 C11 C5 C4 C12 C2 C6 RMSD

*anti* b 96.5 112.7 117.7 133.2 135.4 140.4 149.5 *0.9 syn* b 93.6 112.7 116.1 132.9 134.7 139.1 149.7 *0.9*  **AVERAGEB** 95.0 112.7 116.9 133.1 135.0 139.7 149.6 *0.9*

Table 8. 13C Experimental and calculated Chemical shifts (in ppm) for N-(2'-furyl)-imidazole

Chem. Shifts H14 H15 H8 H9 H16 H7 RMSD

*anti* b 5.87 6.29 7.05 7.05 7.16 7.30 *0.10 syn* b 5.73 6.28 6.81 7.01 7.08 7.62 *0.10*  **AVERAGE**B 5.80 6.29 6.93 7.03 7.12 7.46 *0.09*

Table 9. 1H Experimental and calculated Chemical shifts (in ppm) for N-(2'-furyl)-imidazole

Exp.a **6.14 6.44 7.13 7.22 7.26 7.81**

Exp.a 96.2 111.6 117.5 130.0 135.4 138.9 144.5

Position C H, multiplicidad in ppm

4 131.6 132.40

8 149.0 153.20

conformers are present in the solution.

a Related to DCCl3; b GIAO, B3LYP/6-311++G\*

a Related to DCCl3; b GIAO, B3LYP/6-311++G\*

a B3LYP/6-311++G\*\*, average values; b In CDCl3; c Related to TMS

N–H peak results. The most intense peak of this spectrum belongs to the C atom of the reference, while the following four peaks belong to the C atoms of the furyl group. The calculated chemical shifts are in agreement with the experimental ones, with RMSD values of 2.53 and 0.32 ppm for the 13C and 1H atoms, respectively. The agreement with our experimental data in these solvents is good, except for the H atom of the H–N bond in chloroform. This is because the calculations are for the gas phase, while the experimental values are for the CDCl3 solution, where the molecular interactions are important. It is important to mention that the registered NMR spectra at room temperature and low temperature were not sufficient to identify the tautomeric mixture of 1, because the speed of exchange of protons is higher than the response time of NMR.


**Author details**

Stella Despoudi1,2

1 University of Western Macedonia, Grevena, Greece

2 Aston University, Birmingham, UK

\*Address all correspondence to<EMAIL_ADDRESS>
© 2021 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.


**Author details**

Tanya Kitova1 \*, Borislav Kitov2 , Denis Milkov4 and Aida Masmoudi3

\*Address all correspondence to<EMAIL_ADDRESS>
1 Department of Anatomy, Histology and Embryology, Medical University of Plovdiv, Plovdiv, Bulgaria

2 Department of Neurosurgery, Medical University of Plovdiv, Plovdiv, Bulgaria

3 University of Tunis—El Manar, Faculty of Medicine of Tunis, Center of Maternity and Neonatology of Tunis, Tunis, Tunisia

4 Medical Faculty, Medical University of Plovdiv, Plovdiv, Bulgaria