**4. Safety aspects**

#### **4.1. Nonclinical studies**

Although propolis has been used for centuries around the world demonstrating to be safe, several scientific studies have been done in order to evaluate propolis safety by oral or topical route. Here we intend to present Brazilian propolis studies done in animals and, the studies found in humans, as clinical trials are not so numerous, despite the clinical trials did not focus on safety, we are presenting them in order to compare the dosages previously used and documented in humans.

Sforcin et al. [111] evaluated some biochemical parameters of animals treated with several different types of propolis aiming to study propolis safety and the differences in the propolis source could interfere in the results. The authors determined total proteins, glucose, urea, creatinine, triglycerides, cholesterol, cholesterol‐HDL, aminotransferases, and lactic dehydro‐ genase (LDH). The results demonstrated that all parameters were under standard values for the species studied and the propolis sources did not affect the results.

Reis et al. [95] evaluated the safety of propolis standardized extract (EPP‐AF®), a Brazilian propolis composition that presents more than 50% of green propolis, by oral route in mice, in an acute model. DL50 was determined to be 3000 mg/kg after 24 h of treatment, and dosages under this value did not demonstrate intoxication signs in the animals. In the subchronic protocol (30 days) done in Wistar rats, there were no differences in the food and water intake, animals' weight and diuresis. Hematological and biochemical analysis did not show statistical differences between the treated (propolis 650 mg/kg) and placebo group. All parameters were in accordance with reference standards for the species studied. Microscopic analysis of all tissues did not show any differences with the placebo group, and it was not possible to detect any lesions, hemorrhages or cells infiltration, demonstrating the safety of oral administration of Brazilian propolis up to 650 mg/kg during 30 days of ingestion.

Mani et al. [112] evaluated the safety of Brazilian propolis in distinct treatments: (i) rats treated with 1, 3, and 6 mg/kg/day during 30 days; (ii) rats treated with 1 mg/kg/day of propolis alcoholic or aqueous during 30 days, and (iii) rats treated with 1 mg/kg/day during 90 and 150 days, demonstrating that all levels of seric cholesterol, HDL‐cholesterol, total lipids, triglycer‐ ides, aminotransferases (AST), and lactic dehydrogenase (LDH) of propolis treated group were similar to the control group. The authors suggested that Brazilian propolis in the dosages used during the period of treatment were safe (**Table 4**).


\* Conversion considering adult weight around 70 kg.

\*\* Results of our group and not published yet.

**Kind of propolis Biological properties References** Brown Antigenotoxicity [43, 44]

72 Superfood and Functional Food - An Overview of Their Processing and Utilization

Green Antibacterial [8–10, 46, 47, 49]

Red Antibacterial [24, 56–59]

**Table 3.** Biological activities presented in Section 3—summary.

**4. Safety aspects**

**4.1. Nonclinical studies**

documented in humans.

Antimicrobial [38, 41, 44, 45] Antioxidant [42, 75]

Anticancer [102] Antifungal [51–53] Anti-inflammatory [12, 15, 16, 87]

Antimutagenic, [96]

Antiulcerogenic [94] Antiviral [17, 54, 55] Immunomodulatory [12, 88] Inhibition of angiogenesis [98] Preservative [60]

Anticancer [33] Anticariogenic [24] Antifungal [24, 52, 59] Anti-inflammatory [24, 80] Antioxidant [24, 33] Antiproliferative [24] Immunomodulatory [24]

Although propolis has been used for centuries around the world demonstrating to be safe, several scientific studies have been done in order to evaluate propolis safety by oral or topical route. Here we intend to present Brazilian propolis studies done in animals and, the studies found in humans, as clinical trials are not so numerous, despite the clinical trials did not focus on safety, we are presenting them in order to compare the dosages previously used and

Sforcin et al. [111] evaluated some biochemical parameters of animals treated with several different types of propolis aiming to study propolis safety and the differences in the propolis source could interfere in the results. The authors determined total proteins, glucose, urea,

Antioxidant [27, 42, 75, 76, 103]

**Table 4.** Safety non‐clinical results for propolis administration for oral route.

According to Dobrowolski et al. [114], LD50 for different sources of propolis varied from 2 to 7.3 g/kg in mice, suggesting a safe dose for humans of 1.4 and 70 mg/day (when using safety factor of 1000). In conclusion, considering Brazilian propolis LD50 as 17,073 mg/day for humans [95], the application of a safety factor of 10 suggested by FDA guidelines, we would have a safe dose of 1700 mg or 1.7 g/day of propolis for an adult.

#### **4.2. Clinical studies**

Khayyal et al. [115] evaluated propolis extract activity in asthmatic patients with oral admin‐ istration of 260 mg of propolis/day, for 2 months. The results demonstrated reduced night attacks (2.5 attacks/week for 1/week) and improved ventilatory functions, as a consequence of a decrease of TNF‐, ICAM‐1, IL‐6, and IL‐8, and an increase of 3· of IL‐10, besides a decrease of prostaglandins E2, F2, and leukotriene D4.

Cohen et al. [116] evaluated 430 children aged 1–5 year old. Treated group (*n*=215) received a mixture of echinacea (50 mg/ml), propolis (50 mg/ml), and vitamin C (10 mg/ml), during 12 weeks, and compared to the placebo group. Children aged 1–3 year old received 5.0 ml, 2·/day, orally while children aged 4–5 year old received 7.5 ml. They were benefits in the incidence and severity of respiratory tract infections, with a decrease of 55% in the number of sick children, 50% in the incidence of respiratory diseases, and 60% decrease in the number of days with fever.


**Table 5.** Clinical trials done with propolis administrated for oral route.

Brätter et al. [117] evaluated the oral administration of 500 mg of propolis for 13 days in healthy volunteers focusing on the evaluation of the immune response (TNF‐α, IL‐6, and IL‐8). There was an increased ability in the cytokines secretion, however, without plasmatic levels. Then, prophylactic administration of propolis depends on the immune system reactivity and time, with no adverse effects.

Samet et al. [118] tested the oral administration of 500 mg of propolis in a randomized, placebo‐ controlled double‐blind study, in which it was possible to demonstrate the benefits of propolis treatment in the repeated stomatitis, especially important in cases of resistance to treatment.

Finally, Zedan et al. [119] evaluated the administration of 500 mg of propolis/day in 45 patients aiming to offer an alternative treatment to cutaneous healings. The study compared propolis with echinacea and placebo, and propolis demonstrated to be more efficient than the other groups, especially in usual and superficial healings (**Table 5**).

Jasprica et al. [121] studied the antioxidant effects of propolis (propolis soluble in water and maltodextrin, 0.65 g of propolis, presenting 2.5% of flavonoids, equivalent to 16.25 mg expressed as galangin, Specchiasol, Italy) when administered in healthy volunteers (*n*=47, women and men), 3 doses/day (total daily dose of 48.75 mg of flavonoids) for 15 and 30 days, with the following parameters under investigation: superoxide dismutase, glutathione peroxidase, and catalase, malondialdehyde, total cholesterol, low‐ and high‐density lipopro‐ tein cholesterol, triglycerides, glucose, uric acid, ferritin and transferrin, and all routine red blood cell parameters. Interestingly, only men with 30 days of treatment presented differences in malondialdehyde (decrease), superoxide dismutase activity (increase), and a few changes in some parameters of red blood cell were detected.

Considering all the previously presented studies, it is possible to suggest that propolis dosages ranging from 260.0 mg to 2.87 g, which have already been used in humans can be considered safe. The biological results observed also varied much. Considering that propolis around the world is largely used as a supplement or functional food, it is reasonable to assume that dosages within this range will probably be safe, since none of the articles suggested any damage or complications for the volunteers. Regarding the antioxidant evaluation proposed by Jasprica et al. [121] in humans and the literature available until now, it is likely that the propolis dosage used was very high for this purpose. Some data previously published suggested that propolis can have a "pro" or "anti" action and, because several *in vitro* and *in vivo* studies demonstrated antioxidant actions at certain does, it is possible that the best result may be achieved using dosages around 500 mg/day successfully, but further investigation is needed.
