Research on the Anti-inflammatory Action of Propolis Tincture Gel

MITITELU Magdalena1#, NICOLESCU Teodor Octavian2#, NICOLESCU Florica4#, NEACȘU Sorinel Marius1#, POLL Alexandru3#, MOROȘAN Elena1#, IONIȚĂ Ana Corina1#, UDEANU Denisa Ioana1#

1Clinical Laboratory and Food Hygiene Department, Faculty of Pharmacy, ”Carol Davila” University of Medicine and Pharmacy, 6, Traian Vuia Street, 020956, Bucharest (ROMANIA)

2 Organic Chemistry Department, Faculty of Pharmacy, ”Carol Davila” University of Medicine and Pharmacy, 6, Traian Vuia Street, 020956, Bucharest (ROMANIA)

3Anatomy Department, Faculty of Dentistry, ”Carol Davila” University of Medicine and Pharmacy, 17-23, Calea Plevnei, 060015, Bucharest (ROMANIA)

4Toxicology Department, Faculty of Pharmacy, ”Carol Davila” University of Medicine and Pharmacy, 6, Traian Vuia Street, 020956, Bucharest (ROMANIA)

Authors emails: magdamititelu@yahoo.com, nicolescu.teodor@gmail.com, florica.nicolescu@gmail.com, neacsu_23@yahoo.es, morosan_elena@yahoo.com, ana_corina_ionita@yahoo.com, denisa.udeanu@umfcd.ro 

#Author Contributions: All authors have equally contributed to the work reported

Abstract

     The anti-inflammatory action of the gel with propolis tincture 3 and 5% was investigated by two experimental methods that aimed at how they influence the inflammatory exudate from the experimental model of edema induced in rat paw with various edematous substances, with the possibility of focusing on theirs mechanism of action.

Keywords: propolis tincture, anti-inflammatory action, gel with propolis tincture

Introduction

      Propolis is a sticky, resin-colored substance, a product of digestion of bees, harvested from the buds and bark of several species of trees, used by them to protect their hive.

       Natural medicine uses propolis to cure various ailments including inflammation, viral diseases, ulcers, and superficial burns.

       Propolis is a complex mixture of substances represented by resins, waxes, essential oils, pollen and various other organic compounds. Over 300 constituents were identified during chemical analyzes: polyphenols, benzoic acid and derivatives, alcohol and cinnamic acid and their derivatives, sesquiterpene and aliphatic hydrocarbons, benzaldehyde derivatives, sesquiterpene alcohols, ketones, sterols, amino acids and minerals. The high content of flavonoids and the rich composition of nutrients turn propolis into an adjuvant, or even a natural remedy for the prevention and treatment of many diseases [1,2].

      Propolis tincture is one of the most popular forms in which bee propolis can be found. Propolis is a basic ingredient in creams, ointments and other dermatological products due to its anti-inflammatory and antimicrobial properties, having good results in treating acne. Its popularity is due to its many uses, in various forms, for treating various diseases. Propolis is also used to stimulate the immune system, treat gastrointestinal problems and acts as an antioxidant and anti-inflammatory agent [3,4].

      Well known and intensively studied in recent decades, propolis has a complex chemical composition (phenolic compounds and flavonoids, essential oils) and a very wide range of biological actions, in studies conducted on human subjects highlighting the positive effects of ingestion of propolis solutions on oxidative status and lipid spectrum, aspects that also contribute to reducing the risk of cardiovascular disease [5,6,7].

      Regarding the quality of bee products used as such or processed in various pharmaceutical forms, they must meet a series of quality and safety conditions in use [8,9,10,11]. Contaminants (heavy metals, polycyclic aromatic hydrocarbons, pesticide residues, microorganisms) are primarily targeted, for which European regulations impose certain maximum permitted limits [12,13,14].

Methodology

     30% Propolis tincture is prepared by adding in a dark bottle 30 g of crude propolis and 70 ml of alcohol of 75-80 degrees. Seal well and shake every day for 3 weeks. After 3 weeks, strain and store in dark containers.

      Propolis tincture was incorporated into 1% carbopol gel in a concentration of 3 and 5%. The base for incorporating the propolis tincture, carbopol hydrogel, was prepared from: carbopol 940 1g, glycerol 5g, triethanolamine 2.5g, preservative solution up to 100g.

     The carbopol is triturated with glycerol and then 80 g of preservative solution is added and homogenized. The mixture was neutralized with 2.5 g of gradually added triethanolamine and then made up to 100 g with preservative. Leave to stand for 24 hours for complete gelation.

      The tests were performed by two experimental methods of acute inflammation: edema induced in rat paw with 10% kaolin suspension and 6% dextran solution [15,16].

      By injecting kaolin into the rat’s paw, the formation of prostaglandins is stimulated, causing local inflammation and edema. Dextran-induced edema is mainly due to the release of histamine and serotonin and is called anaphylactoid edema.

      Edema was induced by intraplantar injection of 0.1 mL of 10% kaolin suspension and 0.2 mL of dextran solution.

      Four batches of 10 male rats, Wistar, weighing 170 ± 15 g, were used for each edematous agent. One batch was the control batch, one batch was treated with 3% propolis tincture gel, one batch was treated with 5% propolis tincture gel and a batch was treated with 4% phenylbutazone gel.

      The animals brought from the farm were kept in laboratory conditions for 2 days, in order to get used to the new habitat and the diet.

       The edematous agent was administered to all animals. The test preparation (uniform application, in a thin layer ~ 0.25 g gel) was applied on the paw in which the edema was induced.

       The evaluation of the anti-inflammatory effect of the gels studied was made against the preparation with 4% phenylbutazone (on a similar basis), applied to the paw with edema, under the same conditions as previously exposed.

       Determinations were made against control groups (untreated individuals).

       The volume of the rat paw was measured plethysometrically, after intraplantar injection of the edematous agent, and further plethysometric measurements were performed at intervals: 2 hours, 4 hours, 6 hours, 24 hours (for the edematous agent 10% kaolin suspension) and at intervals: 30 minutes, 60 minutes, 90 minutes, 120 minutes after induction of edema (for the edematous agent 6% dextran solution).

       The mean value of anti-inflammatory edema (expressed in mL), standard error and percentage of edema inhibition were calculated for each batch, according to the formula:

Edema inhibition % = (1-X substance / X control) x 100

where: X substance represents the average value of the edema produced by the test substance;

 X control represents the average value of the edema produced in the control in the same time interval from the administration of the edemogenic agent.

 Statistical evaluation of the results was done by the t test (Student) [17].

Results

      The gel with 3% propolis tincture showed the most intense anti-inflammatory effect 2 hours after the induction of edema, the inflammation produced with 10% kaolin suspension being reduced by 33.19%.

       The gel with 5% propolis tincture showed the most intense anti-inflammatory effect 2 hours after the induction of edema, the inflammation produced with 10% kaolin suspension being reduced by 42.85%.

       The phenylbutazone gel showed the most intense anti-inflammatory effect 2 hours after the induction of edema, the inflammation produced with 10% kaolin suspension being reduced by 68.57% (Table 1, Fig. 1).

Table 1. Anti-inflammatory effect of propolis tincture gels studied on inflammatory edema produced with 10% kaolin suspension

Tested product Edema 2 h (mL)

( ± SD)

 Edema 4 h (mL)

( ± SD)

 Edema 6 h (mL)

( ± SD)

 Edema 24 h (mL)

( ± SD)
Control 0.238 ± 0.01 0.276 ± 0.03 0.340 ± 0.02 0.293 ± 0.01
Gel with 5% propolis tincture 0.136 ± 0.04** 0.173 ± 0.01** 0.229 ± 0.03** 0.201 ± 0.02**
Effect % -42.85 -37.31 -32.64 -31.39
Gel with 3% propolis tincture 0.159 ± 0.01** 0.189 ± 0.01** 0.231 ±0.02** 0.211 ± 0.03**
Effect % -33.19 -31.52 -32.05 -27.98
Phenylbutazone gel 0.076 ± 0.01** 0.103 ± 0.03** 0.135 ± 0.02** 0.104 ± 0.04**
Effect % -68.57 -62.65 -60.05 -64.50

Legend ± SD = average ± standard deviation; **p<0.05.

Fig. 1.  Graphical representation of the action of propolis tincture gels studied on inflammatory edema produced with 10% kaolin suspension

In the experimental model of edema produced with 6% dextran solution, the gel with 5% propolis tincture showed the most intense anti-inflammatory effect 90 minutes after the induction of edema (46.82%).

The gel with 3% propolis tincture showed the most intense anti-inflammatory effect after 60 minutes from the induction of edema, the inflammation produced with 6% dextran solution being reduced by 37.00%.

Phenylbutazone gel showed the most intense anti-inflammatory effect 30 minutes after the induction of edema, the inflammation produced with 6% dextran solution being decreased by 72.41% (Table 2, Fig. 2).

Table 2. Anti-inflammatory effect of propolis tincture gels studied on inflammatory edema produced with 6% dextran solution

Tested product Edema 30 min(mL)

( ± SD)

 Edema 60 min(mL)

( ± SD)

 Edema 90 min(mL)

( ± SD)

 Edema 120 min(mL)

( ± SD)
Witnesses   0.203 ± 0.02     0.227 ± 0.02 0.252 ± 0.03 0.265 ± 0.03
Gel with 5% propolis tincture 0.114 ± 0.01**     0.128 ± 0.01** 0.134 ± 0.01** 0.151 ± 0.01**
Effect % – 43.84 -43.61 -46.82 – 43.01
Gel with 3% propolis tincture 0.129 ± 0.01** 0.143 ± 0.02** 0.163 ± 0.01** 0.177 ±0.03**
Effect % – 36.45 – 37.00 – 35.31 – 33.73
Phenylbutazone gel 0.056±0.04** 0.065 ± 0.01** 0.074±0.02** 0.074 ± 0.01**
Effect % – 72.41 -71.36 -70.63 -72.07

Legend ± SD = average ± standard deviation; **p<0.05.

Fig. 2 Graphical representation of the action of propolis tincture gels studied on inflammatory edema produced with dextran 6% solution

Conclusions

      By testing the anti-inflammatory action of the gels under study, it was found that the gel with 5% propolis tincture has significant anti-inflammatory effects on both the edema produced by the 10% kaolin suspension and the edema produced by the 6% dextran solution. This is due to the complex composition of propolis tincture.

       In the case of edema produced with 10% kaolin suspension, the most intense anti-inflammatory effect occurred 2 hours after the induction of edema for all gels studied as well as for phenylbutazone.

        In the case of edema produced with 6% dextran solution, the most intense anti-inflammatory effect was noted 90 minutes after induction of edema for 5% propolis tincture gel, 60 min after induction of edema for 3% propolis tincture gel and after 30 min for the phenylbutazone gel.

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