**2. Polyphenols in citrus fruits (classifications, structures and beneficial effects)**

Polyphenols are a large family of thousands of plant compounds that have been identified for their unique and characteristic structures which include a flavan ring system and many units of phenol [4, 5]. Polyphenols are classified majorly into flavonoids and non-flavonoids. Flavonoids are a class of polyphenols that have been well researched. They share a common flavan nucleus, which consists of a pair of aromatic rings A and B, aromatic and connected by a pyran ring (C). The location difference of the B-ring to C-ring linkage enables the classification among flavonoids, isoflavonoids and neoflavonoids (4-phenylbenzopyrans). Flavonoids, which are the most abundant may be also categorized into flavonols, dihydroflavonols, anthocyanidins, flavanols and flavonoids with no substitution at C3 (**Figure 2**). The detection of a double bond at the position of C2–C3 defines the difference between flavones and flavanones [14].

Flavanones could be considered the main substrate for the biosynthesis of flavonoids as they serve as the precursors of other flavonoid classes. Naringenin and hesperetin (with their aglycones and their glycosides) are flavanones that have sparked great curiosity and interest due to their abundant occurrence in foods. Hesperidin is an important flavanone contained in every citrus fruit especially, with good concentrations in sweet oranges [15] and orange juice [16]. Naringenin-7-*O*-rutinoside (narirutin) is also found in orange juices with traces of

**Figure 2.** *Structures of citrus flavonoid subclasses.*

hesperetin-7-*O*-rutinoside-3′-*O*-glucoside, 4′-*O*-methyl-naringenin-7-*O*-rutinoside (didymin) and eriodictyol-7-*O*-rutinoside (eriocitrin) [17].

Naringenin, structurally recognized as 5,7,40 -trihydroxyflavanone, is present and largely concentrated in citrus fruits [18]. Naringenin can occur as aglycone and/ or glycosides where naringin and narirutin are most abundant. Naringin also known as naringenin-7- neohesperidoside has a characteristic bitter flavor because of the glucose component. It is the main flavonoid found in grape and sour oranges [19], with varying naringin contents according to their varieties. However, other citrus species such as tangelo (*C. reticulata x C. paradisi*), lemon, sweet oranges and lime have a low content of naringin. Narirutin, also known as naringenin-7-rutinoside, is a major naringenin glycoside. It is very abundant in grapefruit although not as much as naringin. At some detectable levels, narirutin is also found in citrus fruits such as sweet oranges, tangor, tangelo and tangerine [20]. During processing, the naringenin content of these fruits may be affected due to the liberation and degradation of associated flavanones because of their thermo-responsive nature [21].

Hesperetin together with the glycosides is found in citrus fruits [22]. Its glycosides form is more dominant than the aglycone. Hesperidin and neohesperidin are the most widely distributed glycosides and are conjugates of rutinose and neohesperidose, respectively. Hesperidin exists in large amounts in sweet oranges, grapefruits, limes, lemons, tangerine and tangor (*C. reticulata* × *C. sinensis*) [22]. Neohesperidin is highly present in a significant amount in tangelo and sour orange.

Polyphenols in citrus have a lot of beneficial effects. Their free radical scavenging capacity has been reported *in vitro* [23] while their potential to activate the endogenous antioxidant defense system has been described. The activation was found to coincide with depletion in the level of reactive oxygen molecules such as nitric oxide (NO. ), hydrogen peroxide (H2O2) and other biological markers of oxidative stress. In addition, the involvement of overexpression of the transcription factor Nrf2 [24, 25] has been linked to the mediated stimulation of the antioxidant response element. Studies by [26] showed the upregulation of haem-oxygenase (HO-1) and a decrease in the activity of xanthine oxidase (XO), a superoxide radical generating enzyme by hesperidin and hesperetin.

Citrus polyphenols also possess immunomodulatory activities [26]. The molecular targets involve the lowering of the levels of pro-inflammatory cytokines *interleukin*-1β (IL-1β), *interleukin*-2 (IL-2), *interleukin*-6 (IL-6), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) which is probably facilitated through the weakening of overactive immune cells as is demonstrated by the fall in glial fibrillary acidic protein (GFAP) level and reduction of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) which controls the transcription of chemokines and inflammatory mediators [27, 28]. Their ability to ameliorate mitochondrial dysfunction has been described [29] as this can increase the stability and function of mitochondrial respiratory chain complexes (I–IV).

## **3. Multidrug resistance: a looming global crisis**

MDR can be defined as insensitivity to administered drugs despite earlier responsiveness to them [30, 31]. Microorganisms such as viruses, fungi, bacteria and parasites can be resistant to antimicrobial drugs, leading to unsuccessful treatment and resulting in a continuous spread of infections [32]. MDR promotes difficulty in disease management by elevating the likelihood of the spread of resistant pathogens. *Inhibition of Multidrug Resistance by Polyphenolic Phytochemicals of Citrus Fruits DOI: http://dx.doi.org/10.5772/intechopen.107903*

**Figure 3.** *Effects of multidrug resistance.*

This declines the efficacy of treatment, thereby extending the time of infection in patients (**Figure 3**). Further, the adoption of expensive therapies is also triggered by the rise in cost implications of treatment attributed to MDR as the pathogens would have turned out to be insusceptible to the accessible drugs. In addition, there is suppressed immune function and decreased drug bioavailability due to the inflated rate of drug metabolism [32].

The emergence of infectious microorganisms having a considerable number of resistant species and the ability to counter barriers imposed by drugs during treatment has unfolded over the years, and this is quite alarming. It's well documented that virtually all the known and capable infecting agents (fungi, bacteria, viruses and parasites) have employed a high level of MDR with an increased death rate [32]. Many deadly diseases such as HIV, influenza, tuberculosis, pneumonia, malaria and yeast infections, caused by these infecting agents, have been identified as major causes of death in recent times [33]. This is a global concern as MDR continues to pose a serious risk to the health of the public. For instance, there has been a decreased chance of managing tuberculosis owing to its resistance to antibiotic treatment. Likewise, pneumonia is an infection that has become untreatable because of the development of resistance to cephalosporin in response to elongated-spectrum -lactamases-mediated mechanism [33], hence making all accessible treatment with - lactam antibiotics useless. Drug resistance during HIV treatment has also resulted in antiretroviral therapy failure. Protozoan parasites responsible for malaria are showing resistance to most of their effective anti-malaria drugs such as chloroquine and artemisinin [34] which have demanded their replacement with novel drugs, thereby increasing healthcare expenses and contributing to the global economic burden. MDR explains why some microbes are sometimes irresponsive to treatment with standard drugs, thus, stretching treatment duration and further raising the healthcare costs, especially for those who are not capable of such expenses [35].

A series of mechanisms to survive, be resistant and subdue the effects of drugs and exposure to drugs has evolved in microorganisms. Notably, the cell walls of these microorganisms play a vital role in their viability. They strategized by undergoing mutation in their chromosomes [36], and this could result in the modification in the cell membrane structure translating to reduced penetrability and drug absorption into the cell [30]. In addition, active target binding sites for drugs may become unavailable. Though drugs for treating viral infection normally select the DNA

polymerase of the virus due to their ability to reverse the transcriptase activity and prevent viral replication, drug-resistant variants of microorganisms usually undergo mutations where the reverse transcriptase is located on the polymerase gene [36]. This influences the cooperation between the drug and the enzyme.

Another mechanism of MDR in microorganisms which can affect a drug's access to the target sites is by overexpressing the target enzymes which can cause modification in some metabolic pathways, generating other target molecules and tampering with protein production [37]. This process can negatively affect the entry of drugs to the designated sites [32].

Microorganisms also exhibit MDR by inactivating or enzymatically degrading antimicrobial drugs by hydrolysing their ester or amide bonds. Other apparent ways by which drugs can be transformed by microorganisms include phosphorylation, adenylation, hydroxylation, acetylation and glycosylation [38]. They can alter the activity of antimicrobial drugs and hinder their connection with the target sites [38]. They can also induce the expression of P-glycoprotein and multidrug-resistant proteins, an action that affects the fluidity and porosity of their membranes, causing ATP-dependent outflow of the antimicrobial drugs and reducing their intracellular level [39].

One of the key challenges in subduing the existence of cancer is MDR. Some mechanisms in cancer have been linked to MDR [40]. They include target changes in enzymes such as DNA topoisomerases [41], mitotic arrest [42], interference in DNA repair [43] and apoptosis impairment or genes involved in apoptosis and necrosis [44].

A common biochemical mechanism of MDR is drug efflux via ATP-binding cassette transporters (ABC transporters). The exaggerated expression of these transporters has been connected with chemoresistance in cancer cells [45]. There are 48 members in the family in humans [46], and the most extensively studied include P-Glycoprotein (P-gp), multidrug resistance protein 1 (MRP1) and breast cancer resistance protein (BCRP). They are largely present in healthy cells of mammalian tissues for the translocation of small compounds. Their presence in the epithelial cells of the intestine and kidney proximal tubules and endothelial cells of blood capillaries has been documented [46, 47]. Expression of ABC transporters by cells can occur if anticancer drugs, which are also substrates of these transporters, are regarded as foreign. In addition, cancer cells that have no resistance to anticancer agents initially can also acquire the potential by overproducing the transporters to counter the effect of toxic substances. Eventually, this triggers an elevated efflux, lowering intracellular drug concentration triggers an elevated efflux, lowering intracellular drug concentration [48] that is deficient in killing a cancer cell.

The phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway is another signaling pathway that promotes drug efflux through the expression of ABC transporter. The pathway encourages the proliferation of tumor by activating NF-κB which can rescue the cancer cells from cell death [49], and suppression of caspase-3 activity, thereby inhibiting apoptosis. This has been considered an underlining mechanism for MDR in tumor cells.

## **4. Multidrug resistance inhibition mechanisms of Citrus polyphenols**

#### **4.1 In Cancer**

The existence of MDR has been a notable challenge during chemotherapeutic treatment. Extensive studies have been carried out on potential MDR reversers, and

## *Inhibition of Multidrug Resistance by Polyphenolic Phytochemicals of Citrus Fruits DOI: http://dx.doi.org/10.5772/intechopen.107903*

several inhibitors of P-gp have been identified among the accessible drugs. However, their toxicity profiles and drug-interaction effects have propelled researchers to explore novel compounds with mild toxicity and lesser side effects [50].

Currently, the discovery of non-toxic, more efficacious and low-cost compounds originating from natural sources to tackle MDR is receiving recognition. The consumption of citrus fruits has been observed to be useful for the reduction of MDR usually experienced during cancer chemotherapy (**Table 1**). The concentration range 6.71–11.43 μM of nobiletin (also known as 5,6,7,8,3′,4′-hexamethoxyflavone) found in orange juice [55] was discovered to be sufficient for inhibition of P-glycoprotein function as documented by [56]. This polymethoxyflavonoid has been identified as a substrate of P-glycoprotein and can competitively connect at its drug-binding site. This action leaves little or no space for related compounds to attach to the transporter, thus resulting in a declined activity of ABCB1 transporter. Part of its inhibitory mechanism against MDR is on P-glycoprotein function which is dependent on ATP hydrolysis. It is known that hydrolysis of ATP is tightly coupled to the transportation of compounds by ABC transporters, with stimulation of ATPase activity [57]. Since the energy consumed by ABCB1 transporter is derived from ATP hydrolysis, nobiletin lowers the ATPase activity [58], thereby modulating its drug transport activity. This is one of the mechanisms of reversing the overexpression of ABCB1 during MDR.

The enhancement of the intracellular accumulation of drugs during chemotherapy is another mechanism of MDR inhibition by citrus polyphenols. Nobiletin at 50 μ M was discovered to have the capacity to increase daunorubicin accumulation in KB-C2 cells [56] and the absorption of vinblastine in Caco-2 cells [59] together with ABCB1 transfected LLC-GA5-COL300 cells at 20 μ M [60] demonstrating the possible P-gp inhibition action of nobiletin. Further, nobiletin at concentrations of 0.5–9 μM remarkably raised the sensitivity of ABCB1 involved in the overexpression of A2780/T and A549/T cell lines to chemotherapeutic agents [58]. Also, tangeretin is


#### **Table 1.**

*Mechanisms of action of some citrus polyphenols in the inhibition of MDR in cancer and bacteria cells.*

shown to be an effective MDR modulator by increasing the awareness of cancer cells to doxorubicin. Another citrus polyphenol, hesperidin, likewise utilizes the sensitivity tool in cancer cells with resistance, thereby decreasing the expression of P-gp [61]. Its inhibitory effect in overcoming MDR in cancer cells is significant and higher than nobiletin [62].

The initiation of apoptosis is another mechanism citrus polyphenols employ in preventing the occurrence of MDR during chemotherapy without developing toxicity. It involves modulation of the PI3K/Akt pathway which is beneficial in preventing drug resistance in many cancer models. Hesperitin has no report of significant toxicity to normal cells [63], and its anti-tumor activity occurs through interaction with various carcinogenic signaling pathways and promoting mitochondrial apoptosis pathways. [64] described the promotion of apoptosis in gastric cancer by hesperetin. The mechanism involves the inhibition of the PI3K/AKT signaling pathway through the upregulation of phosphatase and tensin homolog (PTEN) expression, thus hampering cell proliferation. The PI3K/AKT signaling pathway is considered a very important and crucial pathway that regulates the development/occurrence of MDR in cancer cells and other events such as cell cycle progression and apoptosis [65, 66]. PI3K in its active state facilitates the phosphorylation of AKT at Thr 308 and Ser 473, leading to its partial or full activation, respectively [67]. The activated AKT then partakes in controlling the inhibition of apoptosis which involves direct phosphorylation of apoptotic signal proteins or regulation of the activity of transcription factors [68].

Further, apoptosis stimulation by citrus polyphenols can lead to the migration of BCL2-associated X (BAX) on the outer membrane of mitochondria, which is a crucial key step to initiate apoptosis [69]. This sensitizes the mitochondrial permeability transition pore (MPTP) to liberate cytochrome C from the mitochondria into the cytoplasm to stimulate caspases, thus initiating cancer cell apoptosis [70]. During this process, mitochondria release apoptosis-inducing factor (AIF) into the cytoplasm, which moves to the nucleus, where it facilitates chromatin condensation, resulting in cell death [51]. This is in support of the study carried out by [71] where hesperitin in combination with cisplatin significantly caused a surge in the expression levels of PTEN and cytochrome C with a concomitant reduction in the levels of phosphorylated AKT (p-AKT) and CyclinD1. In addition, hesperetin induced a remarkable increase in AIF, BCL2-associated X, (BAX), caspases 3 and 9 and suppressed B-cell lymphoma 2 (BCL2) level [72].

Aside from the suppression of the kinase pathways for pro-apoptotic action against MDR in cancer cells, hesperidin also induces apoptosis by triggering the accumulation of reactive oxygen species (ROS) and sensitization of signal-regulating kinase 1/Jun N-terminal kinase (ASK1/JNK) pathway [73]. The high level of ROS, adenosine triphosphate (ATP) and calcium has a participatory role in the initiation of apoptosis by hesperidin in cancer cells via the sensitization of the mitochondrial pathway [74].

The arrest of cell cycle at G0/G1 phase through suppression of cyclin D1, cyclin E1 and cyclin-dependent kinase of p21, 2 (Cdk2) at the protein level with a corresponding increase in the expression levels p53 E1 is also another strategic means that hesperidin uses to change anti-apoptosis scenario in cancer cells [75].

Also, reduction of the expression of Nrf2 is known to be notoriously engaged in the modulation of drug resistance [76]. The combination of apigenin with doxorubicin during chemotherapy has been considered effective against chemoresistance treatment. Recent *in vitro* studies have also revealed that polyphenols can overcome drug resistance in cancers by inhibiting efflux pumps that extrude anticancer drugs, increasing the level of drug absorption and cell apoptosis and reducing cancer proliferation [77, 78].

#### **4.2 In Bacteria infections**

The existence of some particular structural features in flavonoids is believed to enhance their pharmacological effects, thereby describing a correlation between the flavonoid structure and its antimicrobial properties [79, 80]. For instance, a high abundance in hydroxyl groups is responsible for the increased antioxidant effects of flavonoids because of an increase in the available sites for quenching radicals and chelation of metal ions. However, high-level hydroxylation is not favorable for flavonoid lipophilicity, thus limiting their inflow across the cell membranes of the pathogen. Hence, flavonoids such as hesperetin and naringenin that are lipophilic could invade the lipid bilayer membrane, causing modifications in fluidity and accessibility of the membrane [81].

A notable number of studies have identified antimicrobial and anti-virulence as mechanisms utilized by some bactericidal flavonoids to curtail MDR through the use of specific molecular targets in microorganisms. The inhibition of the vital functions of response regulator-like transcription factor HsrA [82] such as virulence, maintaining the availability of nutrients, involvement in response to oxidative stress and cell division by these flavonoids have been described [52, 83, 84].

This can occur by blocking the interaction of HsrA with DNA at its C-terminal effector domain as observed with hesperitin [82] while other recognized molecular targets in microorganisms such as enzymes [53, 54, 85] can also be acted upon.

Another mechanism to combat the alarming increase in MDR encountered with microorganisms such as Helicobacter pylori whose infection is a risk factor for developing gastric cancer is via anti-urease activity and structure–activity relationship has proved to be beneficial. This comes to play by generation of hydrogen bonds with the amino acid residues of H. pylori urease, which is an indispensable inhibitory strategy employed by flavonoids such as quercetin. This shows the critical impact of the presence of hydroxyl groups in the structure of quercetin against *H. pylori* enzymes [86].

Inflammation has been associated with MDR that exists during infection with *H. pylori*. Cytotoxin-associated gene A (CagA) and vacuolating cytotoxin A (VacA) are considered to be very important factors during the inflammation process. Cag A is transported to host cells via the secretion system that introduces the toxin into gastric epithelial cells [87]. This triggers the activation of NF-kB with subsequent modulation of the expression of pro-inflammatory cytokines, such as IL-8, TNF-a and IL-1b [88]. Hence, there is induction of apoptosis, vacuolation and stimulation of the p38 MAPK signaling pathway in the host cell. However, nobiletin has demonstrated protection against the inflammation process linked with MDR in *H. pylori* infection by downregulating the expression of pro-inflammatory cytokine [89]. Some of the mechanisms include a decrease in the mRNA levels of IL-8, TNF-α and IL-1b, decrease in lipid peroxidation, inhibition of vacA expression and suppression of CagA and VacA translocation to its target cells by lowering the expression of secretion components [54] and downregulation of the p38 MAPK signaling pathway [90].

The decline in the expression of the efflux pump gene hefA [91] is another mechanism citrus polyphenols utilize to reduce MDR during infections. HefA is a gene that encodes a TolC-like outer membrane channel tunnel protein and connects with translocases in the inner side of the membrane to make efflux systems partake in drug resistance [92]. The decrease in its expression increases the inhibitory capacities of antibiotics and their effectiveness in multidrug-resistant strains of bacteria [91].

Also, cell shape transformation that entails a morphological transition from spiral to coccoid forms by *H. pylori* is usually inhibited by citrus flavonoids to avoid the increase in antimicrobial resistance associated with this pathogen. This has been proven in myricetin [93].
