Use of a particular extract of propolis for combating the side effects of chemotherapy

Abstract

The invention relates to the use of a composition comprising at least one extract of propolis, having at least one of the following properties: an antioxidant value (ORAC) greater than or equal to 500 mmol TE/100 g of dry extract, a flavones and flavonols content greater than or equal to 5.5% by weight with respect to the total weight of dry material of the extract, a flavanones and dihydroflavanols content greater than or equal to 5% by weight with respect to the total weight of dry material of the product, in order to prevent and/or limit the side effects of chemotherapy.

Claims

1. A nutritional supplement or pharmaceutical composition comprising: a core comprising: at least one concentrated propolis extract powder, with said propolis extract powder having a total polyphenol content between 38.7% and 50% by weight in relation to the total weight of dry material of the extract powder and having each of the following characteristics: An antioxidant value (ORAC) that is between 698 and 750 mmol of TE/100 g of dry material of the extract powder, A content of flavones and flavonols that is between 5.5% and 10% by weight in relation to the total weight of dry material of the extract powder, A content of flavanones and dihydroflavonols that is between 5% and 10% by weight in relation to the total weight of the dry material of the extract powder product; wherein the propolis extract powder derives from a propolis collected by a grid method, and the propolis collected by the grid method has a wax content of less than 17%; and a gastro-resistant coating enveloping the core; wherein the composition is capable of preventing and/or limiting the side effects of chemotherapy in humans or animals.

2. The composition according to claim 1, wherein the propolis extract powder is a poplar propolis extract powder.

3. The composition according to claim 1, capable of limiting and/or protecting against the reduction in white blood cells, red blood cells, and blood platelets consecutive to injections of chemotherapy agents.

4. The composition according to claim 1, capable of protecting against and/or limiting the free-radical damage caused by the chemotherapy agents to organs.

5. The composition according to claim 1, capable of protecting against and/or limiting the free-radical damage caused by chemotherapy agents to the liver, the kidneys, and/or the heart.

6. The composition according to claim 1, capable of preventing and/or combating nausea, hair loss, nail loss, states of fatigue, weight loss, and depression in individuals undergoing chemotherapy treatment.

7. The composition according to claim 1, further comprising proteins, carbohydrates, lipids, vitamins, and/or minerals.

8. A method for substituting or adding to a meal, comprising administering the composition according to claim 1.

Description

EXAMPLE 1: EXAMPLE OF PROPOLIS EXTRACT

(1) The propolis that is used, in particular poplar propolis, is obtained by implementing the technique of grids, which makes it possible to obtain propolis with particular characteristics suitable for medical use in comparison to a propolis obtained by the scraping technique. The grid method makes it possible to obtain a propolis with a higher polyphenol level and a reduced percentage of wax.

(2) Preferably, the percentage of wax in the collected propolis is less than 21%, even more preferably less than 17%.

(3) Once the propolis is collected, it is treated by implementing an extraction method comprising the following steps: The propolis is mixed in an extractor with alcohol according to a ratio of 1/2.5 to 1/5 (w/v) for a given time period, The mixture then undergoes filtration so as to keep in liquid solution only the active ingredients of propolis: the polyphenols, A final clarification by gravitational decanting can optionally be carried out if necessary.

(4) This method makes it possible to produce a liquid extract that is loaded with active ingredients and is lacking in waxes and other impurities contained in the raw material.

(5) This liquid extract is then concentrated in terms of active ingredients by dealcoholization, with the parameters having to be adjusted to obtain a propolis extract that has the following characteristics: An antioxidant value (ORAC) that is greater than or equal to 500 mmol of TE/100 g of dry extract, A content of flavones and flavonols that is greater than or equal to 5.5% by weight in relation to the total weight of dry material of the extract, A content of flavanones and dihydroflavonols that is greater than or equal to 5% by weight in relation to the total weight of dry material of the extract, and Preferably a total polyphenol content that is greater than or equal to 30% by weight in relation to the total weight of dry material of the extract.

(6) A soft extract that is very concentrated in terms of active ingredients and that can be transformed into powder is then obtained.

(7) An example of a propolis extract that is obtained by the implementation of this method is a poplar propolis extract in powder form, having the following characteristics: An antioxidant value (ORAC) of between 500 and 750 mmol of TE/100 g of dry extract (mean value 698) A content of flavones and flavonols of between 5.5% and 10% by weight in relation to the total weight of dry material of the extract (mean 8.6%) A content of flavanones and dihydroflavonols of between 5% and 10% by weight in relation to the total weight of dry material of the extract (mean 7.1%) A total polyphenol content of between 30% and 50% (mean 38.7%).

EXAMPLE 2: COMPOSITION EXAMPLE

(8) A useful composition example according to the invention comprises: 65% of the extract of Example 1, and 10% carob powder, and/or 10% silica, and/or 15% of at least one other excipient (magnesium stearate).

EXAMPLE 3: COMPOSITION EXAMPLE

(9) A useful composition example according to the invention comprises: 75% of the extract of Example 1, and 5% carob powder, and/or 10% silica, and/or 10% of at least one other excipient (magnesium stearate).

EXAMPLE 4: COMPOSITION EXAMPLE

(10) A useful composition example according to the invention comprises: 75% of the extract of Example 1, and 25% kaolin.
Evaluation of the Physico-Chemical and Biological Differences of Different Propolis Extracts

(11) The characteristics of different propolis powders have been tested: Powder according to Example 1 Powders of the prior art (commercial-grade propolis powders): powder 1 comes from Aagaard, powder 2 from Aprolis, and powder 3 from Apimab.

(12) The dosages of total polyphenols and 2 flavonoid sub-fractions have been performed by spectrophotometry according to the methods that are described and validated by Popova et al. (2004), 15: 235-240 in Phytochemical Analysis.

(13) The antioxidant value is determined according to the ORAC method that is described by Ou et al. (2001), in JAFC, 49 (10), 4619-4626.

(14) The results are presented in the table below:

(15) TABLE-US-00001 Total Flavones and Flavanones and ORAC Polyphenols Flavonols Dihydroflavonols (mmol of (g/100 g) (g/100 g) (g/100 g) TE/100 g) Powder of 38.7 8.3 7.1 698 Example 1 Powder 1 14.1 1.5 4.7 98 Powder 2 34.4 7.2 7.2 480 Powder 3 12.3 1.9 3.5 177
In-Vitro Evaluation of the Effect of Propolis on the Side Effects of Chemotherapy
Measurement of the Reversion of the Chemotherapy Agent Toxicity

(16) Primary hepatocytes of rats were sampled and inoculated into a 96-well plate. 24 hours after the inoculation, the hepatocytes were treated with different doses of propolis (Example 1) for 24 hours. At the end of the 24 hours of pretreatment, the propolis is removed and the hepatocytes are treated with cytotoxic chemotherapy compounds (tamoxifen and cisplatin).

(17) The percentage of hepatic cells that are cytolyzed after a pretreatment with different concentrations of propolis and then subjected to a cytotoxic concentration of tamoxifen (25 M) was evaluated and presented in the table below:

(18) TABLE-US-00002 1 g/ml 3 g/ml 10 g/ml Hours Untreated of Propolis of Propolis of Propolis 0 4.09 3.54 4.03 4.04 2 4.04 3.61 4.08 4.20 4 4.44 4.02 4.41 4.19 6 5.67 5.54 5.21 4.65 8 7.03 6.92 5.68 4.80 10 8.75 8.29 6.32 4.91 12 10.22 9.78 6.78 4.96 14 11.71 10.82 7.11 5.01 16 12.76 11.89 7.46 5.03 18 13.85 12.89 7.68 4.89 20 14.46 14.16 7.89 4.85 22 14.94 14.61 8.21 4.90 24 15.47 15.28 8.26 4.98 26 15.87 15.85 8.54 5.01 28 16.41 16.09 8.80 5.13 30 16.60 16.51 9.00 4.87 32 17.30 16.90 9.20 4.68 34 17.94 17.46 9.20 4.70 36 18.22 17.42 9.44 4.45 38 18.67 18.08 9.74 4.54

(19) Likewise, the percentage of hepatic cells that are cytolyzed after a pretreatment at different propolis concentrations and then subjected to a cytotoxic concentration of cisplatin (10 M) is presented in the table below:

(20) TABLE-US-00003 1 g/ml 0.1 g/ml 0.3 g/ml of 10 g/ml Hours Untreated of Propolis of Propolis Propolis of Propolis 0 1.31 1.83 1.78 2.27 2.10 2 1.11 1.43 1.01 1.77 1.67 4 0.99 1.21 0.67 1.51 1.37 6 0.95 1.20 0.60 1.52 1.29 8 0.92 1.06 0.57 1.43 1.28 10 0.89 1.01 0.48 1.39 1.14 12 0.85 1.02 0.57 1.31 1.06 14 0.86 1.09 0.69 1.39 1.16 16 1.15 1.18 0.92 1.45 1.28 18 1.42 1.48 1.08 1.52 1.29 20 1.90 1.77 1.52 2.07 1.74 22 2.82 2.30 1.83 2.63 1.87 24 4.29 3.33 2.98 3.45 2.30 26 5.82 4.59 3.96 4.14 3.33 28 7.68 6.10 4.81 5.12 3.98 30 9.70 7.53 6.44 6.83 5.58 32 12.48 9.05 7.98 8.08 6.27 34 15.01 11.10 9.25 9.71 7.66 36 16.84 12.10 10.28 10.63 8.37 38 18.49 13.46 11.43 11.43 9.22 40 20.46 15.04 11.93 12.96 9.71 42 21.74 15.78 12.68 14.38 11.02 44 23.61 17.33 13.59 14.53 11.10 46 25.68 18.65 14.18 15.71 12.14 48 27.07 19.45 14.71 16.95 12.34 50 28.48 20.76 15.36 16.89 12.94 52 30.37 22.09 16.65 17.26 13.28

(21) Tamoxifen and cisplatin bring about a dose-dependent cytolysis of hepatocytes, where the increase in the dose induces an increase in cells cytolyzed with a complete cytolysis of between 6 and 48 hours for the highest dose.

(22) It is noted that a pretreatment with a propolis extract according to the invention has a hepatocytic protective action against tamoxifen. It imparts protection against chemotherapy treatment that is comparable to a protective hepato compound of known reference: oltipraz.

(23) It is also noted that the propolis extract according to the invention is able to reduce the toxic effects of cisplatin.

(24) In-Vivo Evaluation of the Effect of Propolis on the Side Effects of Chemotherapy

(25) Wistar rats of approximately 200 g were used during this in-vivo experiment, at a rate of 8 animals per group. The animals received orally either the vehicle (gr control) or propolis (Example 1) at 12.5 mg of powder/Kg of body weight for 5 consecutive days before the injection of a chemotherapy agent (epirubicin, cyclophosphamide, 5-FU and taxotere), or the chemotherapy agents alone. The animals are sacrificed 21 days after the injection of the chemotherapy agent. The weight and the different parameters of the blood formula will be tracked in these animals on days D+1, D+6 and D+14 and D+21 post-chemotherapy injection. The contents of endogenic antioxidants (glutathione=GSH) and the hepatocytic lipid peroxidation (MDA) levels will be evaluated on D+21.

(26) Change in Weight of the Animals of the Different Groups.

(27) TABLE-US-00004 Variation between D + 1 D 5 D + 1 D + 6 D + 14 D + 21 and D + 21 Gr Control 151.6 164.5 Gr Epirubicin 150.2 156.1 156.1 149.5 155.2 0.9 Gr Epirubicin + Propolis 157 163.5 168.6 171.5 178.4 +14.9 Gr Cyclophosphamide 160.4 167.2 157.7 164 152.3 14.9 Gr Cyclophosphamide + Propolis 155.6 162.6 165.6 169.2 181.4 +18.8 Gr 5-FU 153.5 157.6 153.7 147.9 143.7 13.9 Gr 5-FU + Propolis 153.6 158.6 163.4 167.7 169 +10.4 Gr Taxotere 151.6 156.9 164.6 170.7 175.5 +18.6 Gr Taxotere + Propolis 151.7 158.7 165.6 173.5 180.4 +21.7

(28) It is noted that the animals that are treated with the chemotherapy agents see their weight decrease rapidly after the injection (except for the taxotere). The pretreatment with the propolis extract according to the invention (Example 1) restores the treated animals' weight gain curves.

(29) Change in Red Blood Cells of the Animals of the Different Groups.

(30) TABLE-US-00005 Variation between D + 1 D 5 D + 1 D + 6 D + 14 D + 21 and D + 21 Gr Control 6.6 6.2 Gr Epirubicin 6.73 5.95 5.04 4.72 3.07 48% Gr Epirubicin + Propolis 6.52 6.03 5.83 5.33 4.81 20% Gr Cyclophosphamide 6.51 5.94 4.23 3.80 3.22 46% Gr Cyclophosphamide + Propolis 6.69 6.24 5.84 5.28 4.60 26% Gr 5-FU 6.51 6.56 4.19 3.05 2.94 55% Gr 5-FU + Propolis 6.67 6.40 5.99 5.15 5.31 17% Gr Taxotere 6.39 6.23 5.18 4.18 2.95 52% Gr Taxotere + Propolis 6.47 6.23 5.95 4.92 4.61 26%

(31) It is known that one of the side effects of the chemotherapy agents is their ability to destroy the blood lines often leading to aplasia in individuals. The results confirm that the injection of agents brings about a very distinct and significant reduction in red blood cells48, 45, 55, and 52% on the 21.sup.st day post-injection for epirubicin, cyclophosphamide, 5-FU and taxotere, respectively. Regardless of the agent, the propolis extract according to the invention brings about a significant 50% reduction in the loss of red blood cells.

(32) Change in White Blood Cells of the Animals of the Different Groups.

(33) TABLE-US-00006 Variation between D 5 D 5 D + 1 D + 6 D + 14 D + 21 and D + 21 Gr Control 6.83 8.38 Gr Epirubicin 6.79 3.55 2.32 12.54 3.63 46% Gr Epirubicin + Propolis 6.65 5.76 4.71 8.40 5.26 21% Gr Cyclophosphamide 6.30 3.33 2.96 10.20 3.19 49% Gr Cyclophosphamide + Propolis 6.41 6.01 5.18 9.36 4.63 28% Gr 5-FU 6.49 3.91 2.74 10.82 3.86 40% Gr 5-FU + Propolis 6.65 5.77 5.18 9.53 5.67 15% Gr Taxotere 6.44 4.41 4.29 10.18 3.13 51% Gr Taxotere + Propolis 6.50 6 5.34 9.28 4.26 33%

(34) Regarding the white blood cells, the chemotherapy agents bring about a drop on the 6.sup.th day, followed by a very distinct rise on the 14.sup.th day, which in turn is followed by a new drop on the 21.sup.st day. Overall, the pretreatment with the propolis extract according to the invention limits the drop in white blood cells on the 6.sup.th day and on the 21.sup.st day.

(35) Variation of the Contents of Plasmatic Creatinine in the Animals of the Different Groups.

(36) TABLE-US-00007 Variation between D 5 and D 5 D + 1 D + 21 D + 21 Gr Control 6.17 6.33 Gr Epirubicin 7.13 10.89 11.39 +60% Gr Epirubicin + Propolis 7 9.3 9.4 +34% Gr Cyclophosphamide 6.38 7.25 10.14 +59% Gr Cyclophosphamide + Propolis 6.38 6 6.13 4% Gr 5-FU 7.25 9 10.19 +41% Gr 5-FU + Propolis 8.25 7.29 8.81 +7% Gr Taxotere 7.60 8.91 11.53 +52% Gr Taxotere + Propolis 7.81 7.80 8.90 +14%

(37) The dosage of the plasmatic creatinine, a reflection of the functionality of the kidneys, shows that the injection of different chemotherapy agents brings about a more or less marked elevation of this parameter at D+1 and D+21, reflecting a renal attack. The pretreatment with the propolis extract according to the invention reduces plasmatic creatinine and even restores it to its base level.

(38) Variation of the Contents of Plasmatic Glutathione in the Animals of the Different Groups.

(39) TABLE-US-00008 Variation between D 5 and D 5 D + 21 D + 21 Gr Control 20.92 Gr Epirubicin 23.86 12.83 46% Gr Epirubicin + Propolis 22.80 20.35 11% Gr Cyclophosphamide 26.39 23.81 10% Gr Cyclophosphamide + Propolis 22.79 23.20 +2% Gr 5-FU 22.14 17.65 20% Gr 5-FU + Propolis 22.71 24.67 +8% Gr Taxotere 22.88 16.83 26% Gr Taxotere + Propolis 23.61 23.61 0
Variation of the Contents of Plasma MDA in the Animals of the Different Groups.

(40) TABLE-US-00009 Variation between D 5 and D 5 D + 21 D + 21 Gr Control 46.08 Gr Epirubicin 46.61 85.45 +83% Gr Epirubicin + Propolis 45.91 58.62 +28% Gr Cyclophosphamide 43.78 79.10 +48% Gr Cyclophosphamide + Propolis 44.49 52.26 +17% Gr 5-FU 44.49 62.85 +41% Gr 5-FU + Propolis 41.66 47.31 +13% Gr Taxotere 45.90 86.16 +88% Gr Taxotere + Propolis 43.07 63.56 +47%

(41) The chemotherapy agents also have an impact on the primary intracellular antioxidant: glutathione (GSH) and on oxidative damage through the formation of diene conjugates, which is a reflection of the lipid peroxidation of the cellular membranes (the MDA) on the plasmatic level. These two measurements are complementary since normally a correct level of GSH makes it possible to maintain a relatively low level of MDA.

(42) It is noted that the injection of epirubicin, 5-FU, and taxotere brings about a very distinct significant decrease in the level of plasma GSH on the 21.sup.st day (and a little less distinct for the cyclophosphamide). In parallel, the MDA levels are multiplied by 2 for all of the chemotherapy agents on the 21.sup.st day. The pretreatment with the propolis extract according to the invention (Example 1) makes it possible to keep the GSH level at its base level regardless of the chemotherapy agent that is used. At the same time, the MDA levels of the animals pretreated with the propolis extract according to the invention (Example 1) are significantly reduced compared to the groups that are treated with the chemotherapy agents alone.