**3. "Nature-derived" anti-***H. pylori* **phytochemicals**

Phytochemicals have been used for centuries in the treatment of gastrointestinal disorders, such as dyspepsia, gastritis, and peptic ulcer disease [60]. Over the last two decades, phytotherapy has gained strength in the scientific community, prompted by the need of alternatives to the ineffectiveness of traditional antibiotics.

Plants synthesize a vast range of secondary metabolites with a significant portion consisting of phenolic and flavonoid compounds [61]. These secondary metabolites, other than providing plants with unique survival or adaptive strategies, are associated to a wide range of biological activities [62]. Phenolic compounds, namely wine polyphenols, from which resveratrol is the most studied, and olive oil polyphenols, mainly hydroxytyrosol, have been associated with anti-*H. pylori* activity [5]. Lipophilic compounds from the terpenes family can also be obtained from several plants. In the scope of anti-*H. pylori* strategies, these are described in more detail in the following section.

#### **3.1. Triterpenic acids**

Although *in vivo* studies using gastric-infected mice demonstrated that DHA was able to decrease only 50% of *H. pylori* gastric colonization, the DHA conjugation with antibiotic stan-

Another DHA feature is its ability to attenuate the host inflammatory response associated

DHA poor solubility in water, fast oxidation/degradation plus gastric settings drawbacks (namely low gastric residence time and low penetration through the gastric mucus layer) are challenging issues for its clinical translation [36, 50, 51]. To overcome these obstacles, cytocompatible lipid nanoparticles have been researched to encapsulate DHA [44, 52]. It was demonstrated that DHA lipid nanoparticles are able to destabilize *H. pylori* membranes, leading to disruption and leakage of cytoplasmic contents [32, 44]. Importantly, these lipid nanoparticles do not interfere with normal gut microbiota in opposite to dramatic changes described for the

LA, as fatty acids in general, is considered safe [53]. It is classified as an essential fatty acid, once it cannot be synthesized by the human body, being necessary to be supplied by the diet [54]. Its importance for biological processes is unquestionable. LA undergoes metabolic changes *in vivo* that ultimately lead to the formation of prostaglandins, thromboxanes, leukotrienes, and lipoxins [54]. Furthermore, the usefulness of LA as an antibacterial agent was also proved, being one of the most potent unsaturated fatty acids against *H. pylori* [7]. It also promotes the adhesion of *Lactobacillus casei* to mucosa surfaces, which indirectly hinders the growth of *H. pylori* [55]. Besides its bactericidal effect, LA is also important for the integrity of the gastric mucosa. It was already proposed that lower levels of essential fatty acids, such as LA, lead to decreased levels of prostaglandins and, consequently, to a higher susceptibility

Nanotechnology has been successfully used to load fatty acids, including LA [34]. As above mentioned, the oral administration of fatty acids is hindered by their poor solubility, especially at acidic pH, and their susceptibility to chemical degradation [57]. In fact, the carboxyl protonation under acidic pH at the stomach lumen decreases the efficacy of fatty acids after oral administration [53]. This was already shown *in vivo*, with no significant effect of plain LA in killing *H. pylori* on a mouse model [53]. Nevertheless, liposomes are promising bioengineering strategies to overcome these limitations. Due to the amphiphilic nature of fatty acids, they can be easily incorporated into the phospholipid bilayer of liposomes [57]. Hence, Obonyo et al. used liposomes of egg phosphatidylcholines, cholesterol, and LA to kill *H. pylori* [57]. They showed that LA-loaded liposomes were effective against *H. pylori* even in its coccoid form and regardless their resistance to antibiotics [57]. Interestingly, *H. pylori* developed resistance against free LA at subbactericidal concentrations, whereas it showed no resistance against LA when incorporated into the nanoparticles [57]. These results show the promising usefulness of nanotechnology not only to protect the fatty acid from its degradation, but also to improve its efficacy. The higher efficacy relies on their ability to fuse with the

dard treatment decreased the recurrence of *H. pylori* infection [42, 43].

114 Helicobacter Pylori - New Approaches of an Old Human Microorganism

with gastric infection [49, 50].

conventional antibiotic therapy [44].

of the gastric mucosa to ulcerogenic agents [56].

**2.2. Linolenic acid (LA)**

Terpenes are naturally occurring hydrocarbons, with the general formula (C5 H8 )n (▬(▬CH2 〓C(CH3 )▬CH〓CH2 )n), where n is the number of isoprene units. Depending on the number of isoprene building blocks, they are classified into several groups, such as monoterpenes, sesquiterpenes, diterpenes, triterpenes, and tetraterpenes (with 2, 3, 4, 6, and 8 isoprene units, respectively). These compounds can undergo chemical modifications by oxidation or rearrangement of the carbon skeleton, which leads to a vast group of compounds denominated terpenoids [63].

Pentacyclic triterpenoids are commonly isolated as active substances from different natural sources, mainly plant surfaces such as stem bark or leaf and fruit waxes [64]. Among them, pentacyclic triterpenes (C30H48) are being marketed as therapeutic agents or dietary supplements around the world due to their biological applications [65, 66]. Their antibacterial properties are also recognized. For instance, it was demonstrated that the acidic fraction of the total mastic extract without polymer (TMEWP) from the Chios Mastic Gum (resin of *Pistacia lentiscus* var. chia) is effective in killing *H. pylori* [67]. This antibacterial effect was attributed to their rich composition in oleanolic acid, isomasticadienolic acid, masticadienolic, and moronic acid [67]. Paraschos et al. demonstrated that the prophylactic treatment with the TMEWP was not able to prevent *H. pylori* infection in C57BL/6 mice infected with mouseadapted *H. pylori* SS1 strain [67]. Nevertheless, the number of *H. pylori* colonies significantly reduced (1.5 log colony forming units/g of tissue) when the animals were subjected to continuous administration of 0.75 mg of TMEWP for 3 months [67]. Shin et al. reported that betulinic acid and oleanolic acid, extracted from *Fosythia suspensa*, were able to inhibit the urease activity of *H. pylori* ATCC 43504 [68]. Furthermore, Parreira et al. reported that outer bark extracts of *Eucalyptus nitens* and *E. globulus*, rich in betulinic, betulonic, oleanolic, and ursolic acids (**Figure 4**), have anti-*H. pylori* activity against strains with distinct virulence degree [69]. Interestingly, the eucalyptus extracts had a lower minimal inhibitory concentration than the isolated pure triterpenic acids, which led to the conclusion that the final observed antibacterial effect was due to synergic effects [69].

**4. Translation to real-world scenario**

would require massive bacterial energy [32, 69].

**5. Conclusion**

Both fatty acids and triterpenic acids have been reported to exhibit similar performance against *H. pylori*. Nevertheless, their action mechanisms are fairly distinct: while fatty acids are reported to interact with the bacterial membrane, triterpenic acids are reported to be more involved in enzymatic inhibition, namely urease hindering [5]. Since both bioactives classes target crucial structures for *H. pylori* survival, emergence of resistance is not anticipated, as it

Nonantibiotic-Based Therapeutics Targeting *Helicobacter pylori*: From Nature to the Lab

http://dx.doi.org/10.5772/intechopen.81248

Despite the remarkable effects associated to fatty and triterpenic acids for gastric infection management, translation into real-world applications is still delayed. For that, it has contributed the fact that only in the last decade more attention has been paid to nature-derived molecules, counteracting the "chemical pharmacological" tendency that had been initiated in the beginning of the twentieth century. Also, there was a significant reduction of investment in the clinical development of antibiotics over the last years. In fact, only 1.6% of the drugs under clinical development by the world's largest drug companies are antibiotics [73]. This has boosted the search for other sources of antimicrobials. In addition, bioengineering emerged in the twenty-first century as a powerful tool to develop drug delivery systems and, consequently, to overcome the more generalized drawbacks associated with the lipophilic bioactive compounds discussed in this chapter [5]. Bioengineering approaches for fatty acids specific application against *H. pylori* are already on a "fast-track," while those for triterpenic acids are only now evolving, which explains the lack of solid studies coupling these bioactives with bioengineering strategies.

To the date and to the best of our knowledge, most of the herein described compounds are in *in vivo* studies phase, being expected that in the next few years some will cross the clinical trials barrier. There are several factors contributing to the anticipated success of these "naturebased" strategies. They are generally cost-effective, due to their abundance in nature, and they require low-cost extraction productions. Furthermore, the biotechnological improvements that include nanotechnological coupling to nature-derived molecules will hopefully contribute to reaching "real-life" applications. In addition, more "nature-based" molecules are reaching the market with FDA approval to treat infectious disease, such as antimalaria Artemisinin therapies, based on an herb employed in Chinese traditional medicine [74], which anticipates the future success of nature-inspired strategies for *H. pylori* eradication.

*H. pylori* infection is one of the most prevalent infections worldwide, which is also reflected onto the high prevalence of gastric cancer. Emerging antibiotic resistance leads to an urgent need of alternative treatments. Resourcing to widely available lipophilic natural bioactive compounds with anti-*H. pylori* activity, namely fatty or triterpenic acids, should be further considered as novel therapeutic options. In this context, nanotechnology emerges as a key player, as it allows overcoming the bioactive major drawbacks that have been holding back their "real-world" application.

Although not specifically designed toward *H. pylori* infection, different strategies to improve the oral bioavailability of triterpenic acids have been studied. For example, oleanolic acid bioavailability has been enhanced by using a phospholipids complex with hydroxyapatite [70]. Yang et al. have developed liposomes to increase ursolic acid bioavaliabitliy [71] and pharmacokinetic studies carried out by Ge et al. reported that the oral bioavailability of ursolic acid was 27.5-fold higher when it was incorporated in nanoparticles than when administered as a free compound [72].

The abovementioned advances in increasing the bioavailability of triterpenic acids using bioengineering strategies will enable, in the near future, to further pursue research of novel nonantibiotic and more effective "nature-inspired" therapies against *H. pylori*.

**Figure 4.** Chemical structures of triterpenic acids: betulinic (BA), betulonic (BOA), oleanolic (OA), and ursolic (UA) acids.
