**3.** *Apis mellifera***—Propolis**

 Propolis is a resinous substance, collected and transformed by the *Apis mellifera*  bees, used in the hives as sealant. The vegetation, climate, and other factors influence the characteristics and composition of the propolis of each region. Studies have shown that propolis is composed of more than 300 (three hundred) substances, among them the most important are flavonoids and their phenolic compounds. Its biological properties include anticancer, antioxidant, anti-inflammatory, antibiotic, and antifungal activities [12, 13].

 Worker bees of the *A. mellifera* species remove propolis from the shoots and resinous secretions present in the trees. By using their jaws and their legs, they mix these secretions with the wax synthesized by them. The mandibular glands of the workers secrete 10-hydroxydecanoic acid, which enables the finishing of propolis. Factors such as plant ecology of the region where the propolis was collected and even the genetic variability of the queens also influence the chemical composition of propolis. According to CONAPIS (information body of CONAP, National Council of Apiculture Ltda.), the ideal propolis is the type produced in regions where there is as little environmental pollution as possible, away from urban centers and factories that emit pollutants [14].

Almeida et al. [15] reported that propolis is a complex mixture of resinous, gummy, and balmy substances collected by *A. mellifera* bees. It has a bactericide, antimicrobial, antioxidant, anti-inflammatory, immunomodulatory, hypotensive, anesthetic, anti-cancerous, anti-HIV, and anti-carious biologic function. These biological activities are related to their chemical components present in the propolis of the Amazon region.

Ishida et al. [14] analyzed the ethanolic propolis extracts from four propolis samples (E1–E4) from Manaus (Brazilian Amazon) by HPLC/DAD/ESI–MS/MS and GC/EIMS. The main components of E2 and E4 were polyprenylated benzophenones: 7-epi-nemorosone, 7-epi-clusianone (major E4 constituents), xanthochymol, and gambogenone (major E2 constituents), making up a chemical profile so far unreported for Brazilian propolis. Aristhophenone, methyl insigninone, 18-ethyloxy-17-hydroxy-17, 18-dihydroscrobiculatone B, and derivatives of dimethyl weddellianone A and B, propolones, and a scrobiculatone derivative were detected as minor constituents. Triterpenoids (b-amyrins, b-amyrenone, lupeol, and lupenone) were ubiquitous and predominant in E1 and E3. The extracts E2 and E4 were highly active against the cariogenic bacteria *S. mitis*, *S. mutans*, and *S. salivarius*. E2 was more active than E4, probably due to a higher content of 2-epi-nemorosone, while the latter was more abundant in dihydroxylated compounds.

 By histological analysis of the extracts in subcutaneous connective tissue in rats, a propolis solution for cavity cleansing and its toxicity was investigated through hemolytic and *Artemia franciscana* tests. Fifteen male rats were selected and randomly distributed into three experimental time intervals (07, 30, and 45 days), in which each animal received the four groups of treatment in rounds: Group I, Propolis I; Group II, Propolis II; Group III, calcium hydroxide water; and Group IV, 2% CLX; the sides of the tube were the control group. As regards biocompatibility, the results showed that all materials presented a significant reduction in inflammatory infiltrate and an increase in collagen fiber thickness values (**Figure 7**). In decreasing order of biocompatibility, the use of the following materials may be suggested: calcium hydroxide-water, 2% chlorhexidine®, Propolis I, and Propolis II. In the cytotoxicity test using *Artemia franciscana*, the propolis extract showed high toxicity in the tested concentrations, and in the hemolytic activity test, the Propolis I extract showed more activity than Propolis II. Therefore, the present study suggested the use of propolis as a cavity cleansing solution for shallow and medium cavities similarly to the use of 2% chlorhexidine® [15].

In continuing studies of the application of biomaterials in dentistry, the response of inflammatory periodontal disease (PD) induced in rat periodontal tissue was histologically evaluated after the use of 0.1, 1, and 10% aqueous suspensions of propolis (SAP) for subgingival irrigation. A total of 84 Wistar rats (*Rattus norvegicus*) were distributed into the following experimental groups: Group I (n = 14, 0.1% SAP), Group II (n = 14, 1% SAP), Group III (n = 14, 10% SAP), Group IV (n = 14, 5% Tween 80 solution), Group V (positive control, n = 14, with induced and untreated PD), Group VII (n = 14, 2% CLX), and Group VI (n = 84, negative control, non-induced and unprocessed contralateral teeth of Groups I, II, III, IV, V, and VII). After induction of PD by the ligation technique in the cervical portion of the mandibular left first molar for 15 days, the periodontal pocket was irrigated three times (first, fourth, and seventh days). The animals were sacrificed at 15 and 30 days after the treatment. The results suggested that SAP demonstrated an effective inflammatory response in the short term (15 days) and in the concentration of 0.1% was associated with the presence of dense gingival fibers, blood vessels

#### **Figure 7.**

*(A) Anatomical shape of rat molar (H.E. 40x): (RM) mesial root, (RD) distal root, (F) furca, (GM) mesial gingiva, and (GD) interproximal gingiva. (B) Furcation region of the tooth treated with aqueous suspension of 0.1% propolis at 30 days (H.E. 200x): (E) epithelium of the exocytosis purse-string, (F) moderate collagen fibers, (O) bone tissue, and (arrow) moderate, chronic, and focal inflammatory infiltrate.* 

without congestion, absence of dental resorption and bone loss, and may be an alternative treatment of PD. However, future studies are required to demonstrate the biological feasibility of the use of propolis as an adjuvant to periodontal therapy.
