**5. Extraction technologies and innovation products**

#### **5.1. Extraction technology**

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

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

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

50 mg/ml—10 ml/day (children 1–3 year

50 mg/ml—15 ml/day (children 4–5 year

500 mg propolis (2 capsules/day) 500.0 [117]

20 drops, 3× /day ˜350.0 [120]

**converted to human adult (mg/day)**

2482.27 [116]

2876.71 [116]

265.20 [116]

**Reference**

**Type of study (oral route) Dosage Dosage**

old)

old)

each

Pilot clinical trial—recurrent stomatitis 500 mg propolis/day 500.0 [118] Propolis for wound healing 500 mg propolis/day 500.0 [119] Clinical trial with asthmatic patients 1 sachet with 260 mg propolis/day 260.0 [115]

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,

Pilot clinical trial with asthmatic volunteers 2–3 tablets/day with 88.4 mg propolis

have a safe dose of 1700 mg or 1.7 g/day of propolis for an adult.

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

of prostaglandins E2, F2, and leukotriene D4.

Double‐blind study with children—prevention

Double‐blind study with children—prevention

Pilot clinical trial with healthy volunteers—

Pilot clinical trial with Brazilian propolis for

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

treatment of Helicobacter pylori

with no adverse effects.

**4.2. Clinical studies**

with fever.

to respiratory infections

to respiratory infections

prophylactic study

Propolis is a very complex material depending on the vegetation present in the area visited by bees. Besides the propolis source, the extraction process associated with solvents used will definitely provide a completely different extract [20]. It is well established that chemical compounds possess several particularities such as solubility, volatility, partition coefficient oil/ water, pKa, and therefore, different solvents will probably extract different compounds [122]. Depending on the temperature and equipment necessary, volatile compounds can be lost and then, it is common to use cold procedures such as maceration or percolation, and when it is necessary to use higher temperatures, it is important to be careful in order to preserve every compound of interest.

Park and Ikegaki [122] studied propolis extracts obtained from water with 96% ethanol solution as solvents. The results demonstrated that propolis extract obtained with 80% ethanol grade showed higher absorption at 290 nm. Using ethanol solution at 60%, higher quantities of isosakuranetin, quercetin, and kaempferol were extracted, while pinocembrin and sakuranetin were better extracted with ethanol solution at 70% and kaempferide, acacetin, and isorham‐ netin were most extracted with ethanol solution 80%. More expressive antimicrobial activities were found in propolis extracts from 60 to 80% of ethanol solution extraction and higher antioxidant results came from propolis extract obtained with ethanol 70–80%.

The most common extraction process for propolis to oral administration is the alcoholic (70%) extraction using maceration, percolation and/or turboextraction. The ratio of propolis raw material:extract used is completely variable and usually, in Brazil, 1:3–4, i.e., 1 part of propolis raw material may offer 3 or 4 parts of extract, considering the production of a liquid extract with at least 11%w/v of propolis dry matter. Of course, this ratio may vary and it is completely dependent on the quality of propolis raw material used.

Jorge et al. [46] studied green propolis extracts obtained from four different locations in São Paulo and Minas Gerais States, using the same extraction process, i.e., hydroalcoholic solution 70% with maceration for 30 days. Although all samples evaluated were Brazilian green propolis using the same extract, different results were found for drupanin, baccharin, and artepillin C during the same month, in spite of seasonal differences. Therefore, it is possible to conclude that, using the same floral source, solvent and process extraction, different regions, and seasonal variations also offer a different chemical composition. Interestingly, these differences did not affect the safe of propolis ingestion [111].

Besides maceration, percolation or turboextraction, Trusheva et al. [123] compared ultrasound extraction and microwave‐assisted extraction with the maceration process. The careful analysis of the results obtained with each process demonstrated that statistical differences were found for total phenolics and propolis total extractable matter for ultrasound process (30 min) and microwave (2 · 10 s) when compared to maceration extraction. For ultrasound, higher total phenolics were found (52 3%) in comparison to maceration (43 2%) while the reduced total extractable matter (53 3% versus 55%). In turn, microwave offered reduced values for total phenolics (40.4 0.6% versus 43 2%) and expressively higher amounts of the total extractable matter (75% versus 55%). Flavonoids analysis did not show important differences among the procedures evaluated. It is important to consider the time of extraction in each process, since maceration takes around 72 h, and ultrasound was effective with 30 minutes and microwave 2 · 10 s. Another important thing is to define the objective of the extraction: for analytical purposes, it is more practical and cheaper to use ultrasound or microwave, however, for industrial scale, these latter may not be easily implemented.

Propolis water extract was also obtained by some authors [12, 17, 98, 124] using completely different procedures, demonstrating some interesting biological activities that had been previously studied for propolis alcoholic extract, such as anti‐inflammatory [12], inhibition of inflammatory angiogenesis [98], and antiviral [17]. Although the demonstration of these interesting results, chemical characterization was poorly explored in the manuscripts, except by Urushisaki et al. [17] that presented the caffeoylquinic derivatives as the most important compounds of this extraction process; however, the manuscript does not present the extraction process used. De Moura et al. [98] performed the extraction from propolis raw material properly crushed in water maceration (500 ml) with temperature around 70°C (30–60 min), two fractions were obtained from the same propolis raw material, followed by filtration. The filtrate was then lyophilized. A similar procedure was used by others too. Nafady et al. [124] in turn, used an innovative process with ‐cyclodextrin as encapsulate agent. To obtain this extract, 10 g of crushed propolis was dispersed in 1 l of water containing 10 g of ‐cyclodextrin previously dissolved. The inconvenient of this procedure is the elevate costs involved in the acquisition of ‐cyclodextrin besides the limitation of the propolis concentration obtained in the final product. Machado et al. [12] proposed the extraction using hydroalcoholic solvent (70%) as usual, however, the solvent was evaporated and after a hydrolysis step the propolis soft extract was then resuspended in water, in this last case, the obtained extract demonstrated similar chemical results of the alcoholic extract in the moment of preparation.
