**4.** *Libidibia ferrea*

## **4.1 Plant species and its applicability**

Among the vast biodiversity of medicinal plants, *Libidibia ferrea*—popularly referred to as Jucá or ironwood—is another plant from the Brazilian biome noted for several therapeutic properties [20]. Characterized as belonging to the Leguminosae family tree, its scientific name is *Libidibia ferrea* (Mart. ex Tul.) L.P.Queiroz *var. ferrea*, ex *Caesalpinia ferrea* Mart. ex Tul. (International Plant Names Index, 2009) [36].

The fruits (pods) of this species are used for the treatment of diabetes and cancer prevention, in addition to wound healing [37]; the roots have been documented as having antipyretic effects, being used in the treatment of diarrhea, and having anticancer properties [38, 39]; and the bark has been used for treatment of enterocolitis and rheumatism [40]. Therefore, these parts of this species have shown antiinflammatory [36], antifungal [19], antihistaminic, antiallergic, anticoagulant [41], antiproliferative, cytoprotective, and antimutagenic effects [42]. Furthermore, *L. ferrea* has been used for biosynthesis of silver nanoparticles (AgNPs), thereby preserving its antimicrobial activity, reducing its toxic effects on human cell lines and increasing its practical use without the impact on the environment. AgNPs are one of the most important nanomaterials among several metallic nanoparticles that are involved in biomedical applications [23].

In dentistry, *L. ferrea* has demonstrated potential antibacterial activity against oral microorganisms [20, 21], in addition to anti-inflammatory and analgesic properties [41, 43]. Nevertheless, several tests are still necessary to improve new dental products and prove their efficacy when used in the oral cavity, with the aim of using them in the dental clinic.

### **4.2 Preliminary studies—standardization of the extract**

Initially, taking into consideration the vast biodiversity of medicinal plants of the Amazon region (Brazil), which have been used empirically due to their antibacterial action, these species have been screened with regard to their antimicrobial activity against microorganisms isolated from dental biofilm [44].

Since the research began, there has been constant concern to ensure the legitimacy of plant species used to obtain the study extracts. The species were collected from official research institutions (Brazilian Agricultural Research Corporation, EMBRAPA; Federal University of Amazonas, UFAM; and Brazilian Institute of Environment and Renewable Natural Resources, IBAMA) and were stored at the Lauro Pires Xavier Herbarium in the Systematic and Ecology Department/Federal University of Paraíba, according to the Genetic Heritage Component Sample Access and Delivery Authorization (No. 044/2004–IBAMA/MMA). From this initial screening, both *L. ferrea* and *C. multijuga* extracts exhibited antimicrobial activity against *S. sobrinus, S. mutans, S. mitis, S. sanguis*, and *Lactobacillus casei* strains and inhibited microbial adherence of these tested strains (**Table 2**) [44].

Sequentially, studies have shown *L. ferrea* fruit extract inhibited the in vitro growth of the following oral pathogens (*C. albicans*, *S. mutans, S. salivarius, S. oralis*, and *L. casei*) on planktonic cells and multispecies biofilm models, supporting the use of this extract for the treatment of oral infections [19]. On the other


#### **Table 2.**

*Diameter of the inhibition halos (mm) of the substances against the microorganisms.* 

hand, *L. ferrea* stem bark extract exhibited better antimicrobial activity than fruit extract when tested against the same oral microorganisms in planktonic cells [45].

 Dental biofilm is a dense, whitish, noncalcified aggregate of bacteria, with desquamated epithelial cells and food debris creating conditions for an imbalance of resident oral microflora, favoring the destruction of hard and soft tissues by the development of oral pathologies such as caries and gingivitis. Recently, an *L. ferrea*  extract was standardized according to the current Brazilian legislation with regard to pH, sedimentation, density, and stability, along with microbiological tests of the extract. The microbial test was used to verify the presence of *Staphylococcus aureus*, *Pseudomonas aeruginosa*, fungi, yeasts, coliforms, and minimum inhibitory concentrations of *S. mutans* and *S. oralis* strains. Thus, this *L. ferrea* extract was shown to have antibacterial activity against the oral microorganisms tested and satisfactory stability and quality, enabling the formulation of a mouthwash using this extract to control dental biofilm [20].

#### **4.3 Standardization of the orabase and mouthwash formulations**

After the preliminary studies of the *L. ferrea* extract, orabase and mouthwashes based on this extract were analyzed.

 The use of oral antimicrobial formulations as an adjunct treatment to mechanical means of dental biofilm and gingival inflammation control has been well established [46]. However, acceptance of the use of plant-based oral products still faces obstacles due to a lack of quality control, since the profile of the end-product constituents has implications in phytotherapeutic efficiency and safety [47]. Thus, the Brazilian ANVISA [48] has established that all phytotherapeutic medication must be submitted to formulation stability tests. Production operations must follow operational procedures with clearly defined and approved standards, in conformity with the notification or registration of traditional phytotherapeutic products with the competent sanitary agency. The final objective is to obtain products that are within the quality standards demanded.

Therefore, Marreiro et al. [20], in a preliminary study, evaluated the antimicrobial activity of aqueous extracts of the fruits, stem bark, and an orabase formulation of *L. ferrea* against biofilm microorganisms by the agar diffusion and broth microdilution methods and evaluated cytotoxicity by hemolysis assay on fibroblast cell

#### *Biopharmaceutical Products and Biomaterials of the Amazon Region Used in Dentistry DOI: http://dx.doi.org/10.5772/intechopen.82056*

 culture. This study endeavored to find an alternative material as a way to guarantee the supply of raw vegetable matter independent of the seasonality of the fruits. The microorganisms used for determining the MICs were *S. salivarius*, *S. mutans*, *S. oralis*, *L. casei,* and *C. albicans*. The extract of the stem bark and the orabase formulation of *L. ferrea* showed antimicrobial activity against microorganisms of the biofilm and were not toxic when tested on erythrocytes and in cell cultures (**Figure 8**).

 In addition to these results, Venâncio et al. [21] evaluated the in vitro pharmacological stability of a phytotherapeutic mouthwash based on *L. ferrea* extract with regard to the microbiological parameters of control, organoleptic characteristics, sedimentation, pH, and density. Using methods in accordance with the legislation, the study determined the total number of microorganisms and *Salmonella* sp., *Escherichia coli*, *Pseudomonas aeruginosa*, and *Staphylococcus aureus*; stability characteristics (color, odor, brightness, and consistency); sedimentation test (centrifuge); the pH measurement (pH meter); and density evaluation (pycnometer). The results demonstrated the *L. ferrea* mouthwash was shown to be free of contamination for the tested microorganisms and was within the standards of safety demanded for its use.

 In addition to the reported studies, with the aim of finding another purpose for its use, Matos [49] evaluated a formulation of *L. ferrea* orabase for use in the healing of oral ulcers. The overall aim of this study was the physical-chemical quality control of an orabase ointment formulation of *L. ferrea*. For physicochemical evaluation, centrifugal tests, pH, mass, relative density, microbiological assessment, and organoleptic character of contaminant tests were performed. The physical conditions tested were storage at room temperature (±25.9°C), room temperature, protected from light (±28.8°C), and air conditioning (±23.7°C); the experimental time intervals were 0, 30, and 60 days. The results showed that in the centrifugation test, phase separation was observed at all times and in all storage environments. In the pH test, only the formulation stored at room temperature obtained a lower pH value, mean (1.95) after 60 days of manipulation. The density test showed the mean value of 0.809 g/cm3 when tested at time 0 and after 30 days of formulation values under air conditioning (0.746 g/cm3 ), room temperature (0.702 g/cm3 ), and room temperature, protected from light, dark (1.022 g/cm3 ). The evaluation of contaminants showed that there was no bacterial growth in any environment and experimental time, but macroscopically, the increase in cotton wool colonies compatible with fungal colonies was observed in the formulation stored under air conditioning in the time interval of 30 days and under all storage locations in the time interval of 60 days. In the organoleptic assessment, the ointment showed changes at 60 days after formulation. Based on the results, the formulation tested maintained the best stability of the tested characteristics when stored at room temperature in the dark. However, after 60 days of storage, the formulation presented chemical and physical instability and growth of contaminants.

#### **Figure 8.**

*Hemolysis assay: Triton™ X-100 (positive control). \*\*\* Statistically significant in comparison with the positive control (P < 0.05).* 
