**2. Methods**

#### **2.1 Research sites**

Within the oil palm production basins of Cameroon, three main sites were chosen to drive this study namely Sanaga Maritime Division in 2013–2014, Ngwéi (2016–2018) and Ekondo-Titi (2016) Subdivisions (**Figure 1**). The diversity of the biophysical environment favours the cultivation of a wide variety of food (cassava, maize, millet, macabo, rice, etc.), and cash (sugar cane, cotton, palm oil, rubber,

## *Environmental Impacts of the Oil Palm Cultivation in Cameroon DOI: http://dx.doi.org/10.5772/intechopen.97862*

cocoa, etc.) crops (Tchindjang et al. 2015–2016). Regarding oil palm, it develops preferentially in the coastal area qualified as the "elaeisfarming" belt of Cameroon (**Figure 1**). Administratively, oil palm plantations and concessions are set up in the maritime facades of the southern, coastal and southwestern regions [10, 15, 16].

As shown by Tchindjang et al. [17] and Ndjogui [18], oil palm belt offers suitable conditions for the development of oil palm: low altitude (less than 500 m); sufficient rainfall (more than 1800 mm/year); favorable temperature between 22 and 30°C; low thermal amplitude; rich and deep soils; etc. Agro-industries are also common in this area and constitute the major producers of palm oil (**Table 1**). At the edge of this "elaeisfarming" belt, there are a few small marginal farms both on the vast southern Cameroonian plateau and in the Western Highlands where oil palm could be grown with limited success.

#### **Figure 1.**

*"Elaeiscultivation" areas, oil pam farms and industries of Cameroon.*

**Table 1.**

*Summary of the research sites.*


**1. Introduction**

The genus *Momordica* L., with around 59 species, is one of the most abundant genera in the Cucurbitaceae family. *Momordica charantia* L. is a taxon extensively studied for its antidiabetic properties [1]. Two varieties have been taxonomically identified: *M. charantia* L. (var. *muricata* (Willd.) Chakrav. and var. *charantia*) [1]. The *muricata* variety produces small size fruits, whereas *charantia* typically produces larger fruits [1]. Several phenotypes of *M. charantia* var. *charantia* appear in nature, displaying noteworthy differences in fruit size and shape. The most common fruit from the Indian cultivar has a greenish color, a narrower shape, and a serrated surface. *M. charantia* var. *charantia* L. is commonly consumed in traditional Asian cuisines and is well-known for its unique taste and flavor [2,3].

Cucurbitane-type triterpenes are the most representative sub-class of compounds in *M. charantia*, and some investigations suggest that they are responsible for their antidiabetic property [4,5]. More than 270 cucurbitane-type triterpenes have been isolated from different plant organs from 1980 to date. However, these compounds are found in very

#### **Citation:** Perera, W.H.;

Shivanagoudra, S.R.; Pérez, J.L.; Kim, D.M.; Sun, Y.; K. Jayaprakasha, G.; S. Patil, B. Anti-Inflammatory, Antidiabetic Properties and In Silico Modeling of Cucurbitane-Type Triterpene Glycosides from Fruits of an Indian Cultivar of *Momordica charantia* L. *Molecules* **2021**, *26*, 1038. https://doi.org/10.3390/molecules 26041038

Academic Editors: Ilias Muhammad and Nanayakkara Dhammika Received: 18 January 2021 Accepted: 12 February 2021 Published: 16 February 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

low concentration and their composition has been reported to vary between cultivars. Our comprehensive survey of the literature indicates that the Indian phenotype has received less attention than the Chinese or Sri Lanka phenotypes. Octonorcucurbitacins A, B and C (342 Da) are the smallest cucurbitane-type compounds reported from *M. charantia,* whereas momordicoside T (1110 Da) the largest one with four sugar units, one glucose linked at position C-25, two glucose units, and one xylose attached at C-3 as follows: -Glc[Glc(1- 4)]Xyl(1–4) [4–6]. Additionally, several aglycones, mono, di, and tri-glycoside triterpene derivatives have also been isolated from bitter melon. The glycosidic moieties attached to these triterpenes typically consist of D series of allose, galactose, glucose and xylose monosaccharide units, mainly at positions C-23 and C-25 of the side chain, and C-3 and C-7 of the A and B rings, respectively [4,7–9].

Many purified compounds, as well as crude extracts, have been screened in response to various ethnomedicinal claims, concluding that bitter melon may play a potential role in the management of various chronic diseases by working as an antidiabetic, antioxidant, antiviral, antiobesity, and anticancer agent among others [8–14]. Moreover, some studies have been conducted in fruit, seeds, and leaves showing hypoglycemic activity in both diabetic animal and human models [15,16]. Thus, *M. charantia* var. *charantia* stands out as a promising natural alternative to reduce the risk and/or manage diabetes mellitus type II [17].

Diabetes is a chronic disease that affects millions of people worldwide. Therefore, it is vital to find new natural prevention and management strategies for this disease and related complications. Results from our previous studies indicated that cucurbitane-type triterpenoids isolated from Chinese cultivar might modulate biological activities involved in the pathogenicity of diabetes [18,19]. Due to these molecules structural complexity, much more work is warranted to test biological activities and better understand the structure-activity relationship. Therefore, herein we describe the isolation and structure elucidation of a new cucurbitane-type triterpene together with six known compounds from acetone extract of the Indian cultivar of *M. charantia*. The anti-inflammatory activity of purified compounds using lipopolysaccharide (LPS)-activated RAW264.7 macrophage cells, inhibition of the α-amylase, and α-glucosidase enzymes were presented. Furthermore, the molecular interaction of purified compounds with α-amylase and α-glucosidase enzymes was also studied using *in silico* molecular docking.


**5. Diabetes mellitus and insulin resistance**

Diabetes is an additional cardiovascular risk factor that needs to be addressed in children. Historically, diabetes mellitus type 1 was considered of main importance in children. With increasing obesity, diabetes mellitus type 2 is becoming more prevalent [25]. Other etiologies, causing diabetes mellitus in children, are presented in **Table 3** [26].

#### **Table 3.** *Causes of diabetes mellitus in children.*

#### *Cardiovascular Risk Factors in Children DOI: http://dx.doi.org/10.5772/intechopen.99729*

Regardless of the type of diabetes mellitus, hyperglycemia leads to impaired cardiovascular function, which was demonstrated in type 1 diabetes mellitus with impaired carotid artery structure and function, and decreased elastic properties of the aorta, already in children [27]. Diabetes mellitus is associated with a two-fold increase in the risk of CVD with a premature cardiovascular mortality and further risk increment when other cardiovascular risk factors coexist [28]. Early onset of diabetes mellitus further worsens cardiovascular risk [28].

Studies in the last decades indicate that insulin resistance is the predecessor of type 2 diabetes mellitus and has been associated with obesity, metabolic syndrome, hypertension and heart disease. It is defined as decreased tissue response to insulin and its cellular actions, commonly associated with obesity, however, not always, and not all obese have insulin resistance. Clinically, methods for insulin resistance measurement are scarce and, in many cases, limited to the research environment, however, one of the consequences of insulin resistance is chronic compensatory hyperinsulinemia, which can be demonstrated [29].

The diagnosis of both, diabetes mellitus and insulin resistance, is based on clinical symptoms, blood glucose monitoring, oral glucose tolerance test, and additional optional investigations, such as autoantibodies associated with diabetes or insulin levels [30]. In diabetes mellitus type 1 insulin therapy should be initiated immediately with recommendation of eventual insulin pump application in all small children, in patients with dawn phenomenon, severe hypoglycemia events or severe blood glucose fluctuations, glycated hemoglobin (HbA1c) values outside target range despite intensified conventional therapy, incipient microvascular or macrovascular secondary disease, limitations of the quality of life, in children with great fear of needles, pregnant adolescents and competitive athletes [30]. Treatment of type 2 diabetes mellitus and preceding insulin resistance is similar to treatment in adults. Weight loss and lifestyle change is the cornerstone of initial management. Patients may also be treated with oral agents, most appropriately starting with metformin, the only registered oral agent in children with diabetes mellitus type 2, and some may require administration of insulin to achieve glycemic control [31, 32].


**4.1. Aerobactin**

Aerobactin is a low-weight molecule and a hydroxamate siderophore with a higher Fe3+ binding stability in acidic environments and is maximally produced at low pH [44, 53]. This siderophore extracts Fe3+ from host iron-binding proteins and is taken up through an outer membrane receptor protein [44]. However, aerobactin has many advantages over other siderophores and is formed from the condensation of two lysine molecules and one citrate catalyzed by an enzyme named aerobactin synthase [13, 25, 30].

#### **4.2. Enterobactin**

Enterobactin is another specialized highly prevalent catecholate siderophore which is less soluble and less stable than aerobactin [53–55]. But this siderophore has a higher iron affinity and can deferrate transferrin more rapidly than aerobactin in aqueous solution [13, 54]. However, iron is released from enterobactin through the hydrolysis of this siderophore [13]. Besides these, enterobactin may afford UPEC the ability to colonize within an iron-limiting environment such as the urinary tract [56]. But this siderophore has a limitation that it can be inactivated by host proteins such as serum albumin and siderocalin [25].

The capsule provides protection against engulfment and complement-mediated bactericidal effect in the host, also including antimicrobial resistance and antiserum activity [1, 48]. Certain capsulars, such as K1 and K5, prevent a proper humoral immune response of the infected host by showing a molecular mimicry to tissue components [1]. The K1 polysaccharide, a linear α2–8-linked sialic acid homopolymer, has a very important role in IBC development as well

Virulence Factors of Uropathogenic *E. coli* http://dx.doi.org/10.5772/intechopen.79557 15

Lipopolysaccharide (LPS) is an integral component of the cell wall and consists of the highly conserved lipid A-core and repeating O-antigen subunits that differ greatly between strains based on the sugar residues and their linkage patterns within the repeating subunits [37, 61]. LPS is very well known to activate host response and to induce nitric oxide and cytokine (IL-1, TNF-α) production which enhances the inflammatory response [1, 15]. It also induces the synthesis of specific antibodies to the somatic antigen and exerts an immune-adjuvant effect that promotes the humoral immune response to other antigens of the pathogen. However, certain antigenic types of LPS are also involved in resistance of the pathogen to the killing effect of the normal human serum [46]. According to study upon animal models, acute renal failure due to LPS depends on the systemic response to LPS and does not depend on expression of functional LPS receptor, TLR4, in the kidney. But it is not clear whether LPS plays a role in mediating a renal failure and acute allograft injury in patients with ascending UTIs [1].

Flagellum is an organelle that is responsible for bacterial motility and plays a role in the initial adhesion phase of biofilm formation [1, 62]. A recent study showed that motility is involved in the migration of the infection from the bladder to the kidneys [63]. About 70–90% of all urinary tract infections is caused by flagellated UPEC, and pathogenesis involves contact between the bacteria and epithelial cell surface of the urinary tract [1]. However, flagellar motility enhances the ability of *E. coli* by adaptive responses to attractive or repellent environ-

Toll-like receptor 4 (TLR4) in the epithelial cells of the mammalian bladder can recognize lipopolysaccharides (LPS) of bacterial cell wall, and the downstream signaling cascade produces IL-6 and IL-8, of which IL-8 is well known as an important chemoattractant for neutrophils. Urinary levels of IL-6 and IL-8 are measurable in UPEC-infected human and murine models. There is another pathway parallel to this one that is responsible for increased levels of IL-6 and IL-8 in urine. Upon TLR-4 activation by LPS, intracellular level of cAMP is increased and results in of Ca2+ influx. Later, cAMP response element-binding protein (CREB) becomes phosphorylated.

as in the multiple stages of UTI pathogenesis [27, 50].

**6. Lipopolysaccharide**

**7. Motility**

mental stimuli [15].

**8. Mechanism of immune escape**

### **4.3. Yersiniabactin**

Yersiniabactin, a mixed-type siderophore, is widespread in *Enterobacteriaceae* including *E. coli* and encoded on the high-pathogenicity island [53]. Yersiniabactin has a high iron affinity and produced yersiniabactin-Fe3+ complex binding to the iron molecule which recognizes the specific bacterial outer membrane TonB-dependent receptor and Fyu (Psn). The iron molecule is released from yersiniabactin in the cytosol with the help of membrane-embedded proteins [57]. In addition, this siderophore increases resistance to copper stress by chelating Cu2+ [10].

#### **4.4. Salmochelin**

Salmochelin is a glucosylated derivative of enterobactin which is not recognized by siderocalin and thus escapes from the host immune response [53]. However, siderocalin, neutrophil gelatinase-associated lipocalin is also known as lipocalin 2 that binds enterobactin and prevents its uptake [53, 56]. To overcome this, enterobactin is modified to salmochelin by glucosylation via the action of glucosyltransferase and is not recognized by lipocalin 2 [56]. However, a recent study found that salmochelin siderophore receptor IroN is involved in the invasion of urothelial cells, and thus IroN may play both an iron uptake receptor and an internalization factor in the establishment of urinary tract infections [26].

#### **4.5. Hemin uptake system**

There is another iron acquisition system called hemin uptake system including ChuA and Hma, which involves direct upregulation of haem receptors. This system uptakes free iron during UTIs, and several studies found its role in bacterial growth and biofilm formation [48, 58, 59]. ChuA expression is regulated by other regulatory proteins, for instance, in uropathogenic *E. coli* strain 536, increase in RfaH level induces the expression of ChuA [60]. But the other receptor Hma functions independently of ChuA, and a residue, Tyr-126, is necessary for its function. However, both ChuA and Hma contribute to haem utilization which is required for the maximum kidney colonization [51].