Process for preparing an extract from EPILOBIUM SPP. with high oenotine content

Abstract

Abstract\nProcess for preparing an extract from EPILOBIUM SPP. with a high oenotine content\n\nThe present invention relates to a process for preparing an extract from Epilobium spp. with a high oenotine content.

Description

Process for preparing an extract from EPILOBIUM SPP. with high oenotine B content The present invention relates to a process for preparing an extract from Epilobium spp. with a high oenotine content. Industrial background Epilobium angustifolium L. is a perennial herbaceous plant belonging to the Onagraceae family. It has a straight, simple, smooth and glabrous stem and underground taproots. Its flowers are collected in pyramidal clusters of pink to purple. The fruits are elongated capsules that open after ripening and release the seeds of the plant. The seeds have a cob for wind dispersal. Epilobium is native to Europe and western Asia, where it grows wild in rocky soils in those regions (Gruenwald J. et al. PDR for herbal medicines, 4th Ed. Thomson, 2007). Epilobium phytocomplexes include three main classes of polyphenols: phenolic acids, flavonoids, and ellagitannins, the latter mainly in the form of macrocyclic ellagitannins such as oenotine B. Phytocomplexes, individual classes of polyphenols, and, to a greater extent, oenotine B, are associated with numerous health-promoting properties in this plant, such as anti-inflammatory and antioxidant functions, particularly in the urological (e.g., prostate enlargement, benign prostatic hypertrophy, urinary tract inflammation) and gastrointestinal tracts. areas (Granica S. et al., Phytochemistry, pharmacology and traditional uses of different Epilobium species (Onagraceae): A review. Journal of Ethnopharmacology, 156, 316-346, 2014); Schepetkin IA et al. Therapeutic potential of polyphenols from Epilobium angustifolium (Fireweed). Phytother Res. 30(8):1287-97, 2016). Oenotine B can be obtained from several plant species, not necessarily from E. angustifolium L. (US 2018 / 0256619). E. angustifolium L. is not included in the Eur. Ph. monographs; therefore, the herbal composition of this extract refers to infusions (traditional compositions in folk medicine) as well as those available in scientific records and on the market. Many of these extracts are aqueous (Lesuisse D. et al., Determination of oenothein B as the active 5-alpha-reductase-inhibiting principle of the folk medicine Epilobium parviflorum; Nat Prod. May;59(5):490-2, 1996; Assessment report on Epilobium angustifolium L. and / or Epilobium parviflorum Schreb, herba, EMA, 10 March 2015; FR 2712594; DE 3605250), but the use of other solvents such as methanol, ethyl acetate, butanol and ethanol is also reported (Deng L. et al. Evaluation of the therapeutic effect against benign prostatic hyperplasia and the active constituents from Epilobium angustifolium L. Journal of Ethnopharmacology. 232:1-10, 2019; Granica S. et al., Phytochemistry, pharmacology and traditional uses of different Epilobium species (Onagraceae): A review.Journal of Ethnopharmacology, 156, 316-346, 2014). The concentration of oenotine B obtained in aqueous extracts from Epilobium spp. is always lower than 10%. US 6528490 discloses the anti-inflammatory activity of E. angustifolium extracts with a content of less than 9.6% by weight. According to the report in US 2018 / 0256619, a preferred range of effective amounts of oenotine B varies between 2.5 and 14%, but the experimental data in the aforementioned document still point to an extract with an oenotine B content of 9.6% as described in US 6528490. Document FR 2712594 and related US patent 552594 disclose a process for extracting oenotine B from Epilobium parviflorum Schreb. with mixtures of water and water-soluble solvents, in particular acetone, and purification by reversed-phase HPLC. Document WO2017108907 discloses the relationship between different species of Epilobium and other plants. The importance of Oenotheine is proven by the references mentioned above. However: 1. According to current European regulations, it is currently not possible to add unpurified oenotine to food supplements in Italy (Ministerial Circular of 16 June 2016) and Europe (Catalog of Novel Foods), but it is possible to use extracts of E. angustifolium, titrated and standardized in this active principle. The extraction efficiency of this molecule in known processes is associated with a purification step. 2. The molecules in aqueous extracts do not contain phytocomplexes, since the yield of flavonoid extracts is low under these conditions. Most of the extracts available on the market lack standardization and the known extraction and purification processes do not provide an optimal solution that meets the need for extracts containing all the important bioactive components of Epilobium along with a high content of oenothein B. Oenothein B is an ellagitannin with antioxidant and anti-inflammatory activity. A large number of scientific studies indicate that oenothein B is able to restore proper prostate function. In particular, a recent clinical study (Esposito C. et al., “Epilobium angustifolium L. extract with high content in oenothein B on benign prostatic hyperplasia: A monocentric, randomized, double-blind, placebo-controlled clinical trial” Biomedicine & Pharmacotherapy 138 (2021) 111414) shows that E. angustifolium extract with a content of oenothein B higher than 15% by weight is significantly effective in the treatment of benign prostatic hyperplasia without renal or hepatotoxic effects. Therefore, there is a need for a process that allows the preparation of phytocomplex-enhancing extracts with high oenotine B content to achieve biological and physiological functions in dietary supplements and nutraceutical compositions. Explanation of the invention A process for extracting Epilobium species has now been discovered that provides extracts with oenotine B contents higher than 15% w / w, e.g. 15 to 35%, without the need for purification steps. The process of the invention can be successfully applied to various species of Epilobium, in particular E. angustifolium and E. parviflorum. The use of the herbal material (drug) in the flowering stage (herba cum floribus) is particularly preferred. The dry extract obtained by the inventive process with an oenotine B content higher than 15.0% has a very rich phytocomplex containing different classes of active ingredients; in particular the following: between 10 and 20% of phenolic acids (of which 50% are hydroxycinnamic acids), between 20 and 30% of flavonoids (mainly including flavonols) and between 40 and 60% of hydrolyzable tannins, a class of compounds to which oenotine B belongs. The inventive process for preparing an Epilobium spp. extract with an oenotine B content higher than 15% w / w has the following characteristics: - Extraction of the drug at least twice with a water:ethanol mixture in ratios ranging from 3:1 to 1:1 by weight; - Filtration of the percolated material, evaporation of the solvent, and drying of the resulting concentrated extract are the subsequent purification steps. The medicinal extraction is preferably repeated 3-6 times, more preferably 4-5 times, using fresh solvent each time. Extraction with a water:ethanol mixture is preferably carried out at a temperature in the range of 25 to 50 degrees Celsius for a period of time in the range of 1 to 3 hours at a pH in the range of 2 to 6, preferably at a pH of 2 to 3.5. The weight ratio of the drug (aerial parts of the plant) to the water:ethanol mixture is preferably in the range of 1:3 to 1:10. Drying of the water:ethanol extract concentrate can be accomplished by known methods, for example, by spray drying. According to a preferred aspect of the invention, the process further comprises the following steps: The aqueous extraction stage in the acidic solution of the drug under extraction with water and ethanol, filtration of the permeate, concentration of the liquid phase, drying, and addition of the dried aqueous extract to the dry extracts obtained from the previous process. Therefore, in accordance with this preferred aspect, the process of the invention is as follows: - A. Extraction at least twice with a water:ethanol mixture in ratios from 3:1 to 1:1 in acidic solution; filtration of the percolation material, evaporation of the solvent and drying of the resulting concentrated extract without additional purification steps; - B. Extraction with water in an acidic solution of the drug under extraction with water and ethanol, filtration of the permeate, concentration of the liquid phase, drying, and addition of the dry aqueous extract to the dry extracts obtained from step (a) as described above. The two fractions from steps A and B, with different metabolic profiles, are appropriately analyzed and combined to obtain a product with the desired concentration of oenotine B. Drying the concentrate from the aqueous extract with acidic solution can be accomplished by known methods, such as evaporation under reduced pressure. The inventive process does not involve purification and is thus easier and more cost-effective than conventional methods. Analysis of the titer of oenotin B is performed by HPLC-UV, which is a method comparable to that described in the following source: Kaškonienė V, et al. (2015). "Evaluation of phytochemical composition of fresh and dried raw material of introduced Chamerion angustifolium L. using chromatographic, spectrophotometric and chemometric techniques", Phytochemistry; 115:184-93 The method that allows the determination of the content of oenotine B in the dry extract is a chromatographic separation by HPLC-DAD using a reference standard. The sample is dissolved in a suitable solvent and, after filtration, is injected into the HPLC. The mobile phase is acid water and acid methanol, with a separation gradient on an RP-C18 column at a wavelength of 254 nm. The method specifically allows the determination of oenotine B without interference from other extractives. The method is linear in the concentration range of 0.1 mg / ml and 0.7 mg / ml and its accuracy allows the estimation of the average percentage recovery of all concentrations, which is in accordance with accepted criteria. Table 1 shows the results of the chemical profiling of the phytocomplex via RP-HPLC-PDA-ESI-MSn. The chromatogram is shown in Figure 1. Peak RT Compound ʎ Max (nm) m / z [MH] - Component 1 5.18 Hexose 236, 272 225 179 (100), 161 (5), 143 (5) 2 6.04 Disaccharide 264, 298 387 341 (100), 179 (5), 161 (10) 3 10.25 Gluconic acid 212, 299 195 129 (100), 159 (20), 177 (45), 75 (10), 99 (10) 4 12.32 Quinic acid 216, 292 191 127 (100), 173 (100), 85 (90), 111 (50), 93 (50) 5 15.88 Malic acid 214, 343 133 115 (100) 6 29.43 Gallic acid 221, 269 169 125 (100) 7 47.96 Oenotine B 213, 263 783 765 (100), 935 (20) 8 62.53 1-O-Caffeylquinic acid 210, 262, 321 353 191 (100), 179 (80), 135 (20) 9 69.56 3-O-Caffeylquinic acid 210, 262, 321 353 191 (100), 179 (15), 135 (5) 10 74.58 Dehydro catechin type B 216, 271 439 393 (100) 11 76.98 4-O-Caffeylquinic acid 210, 262, 321 353 179 (95), 173 (100), 135 (20), 191 (25) 12 83.61 Myricetin hexoside 204, 267, 377 479 316 (100), 287 (5), 179 (10) 13 85.18 Quercetin galloyl hexoside 265, 345 615 463 (100), 301 (20) 14 88.17 Quercetin-hexoside 226, 268, 285 463 301 (100) 15 89.81 Quercetin-3-O-pentoside 204, 258, 340 433 300 (100), 271 (5), 255 (5), 151 (5) 16 90.73 Kaempferol - hexoside 214, 233, 278 447 285 (100) 17 94.55 Kaempferol -3-O-rhamnoside 204, 264, 287 431 285 (100), 255 (5), 151 (5) 18 95.79 Kaempferol-p-cumaroylglucoside 227, 266, 315 593 447 (20), 285 (100), 175 (40) 19 101.45 Myricetin-3-O-glucuronide 204, 261, 357 493 317 (100) 20 103.96 Quercetin glucuronide 226, 255, 349 477 301 (100). The invention is explained in more detail in the following examples. Example 1 Process A: Extraction with a hydroalcoholic mixture In a 2-liter reactor, 50 g of the drug is introduced and 250 g of a hydroalcoholic solution in a water:ethanol ratio of 3:1 is added in an acidic solution. The extract mixture is heated to 50 °C and remains under percolation extraction for 2.5 hours. At the end of the first stage, the resulting liquid extract is removed and the drug is extracted three more times under the same conditions with the solvent of the extraction unit. At the end of the extraction, all the percolation products are filtered and combined and the solvent is evaporated under reduced pressure at a temperature of 50 °C to obtain a concentrated extract. Final drying is carried out by a spray dryer. The amount of dry extract obtained is 12.6 g. Process B: Water extraction At the end of step 1, 250 g of water in an acidic solution is added to the drug. The extract mixture is heated to 50°C and kept under percolation for 1 hour. At the end of the extraction, the percolation is separated from the drug, filtered and the solvent is evaporated under reduced pressure at a temperature of 50°C to obtain a concentrated extract. The remaining water is removed by drying under reduced pressure at 50°C to obtain a dry extract. The amount of dry extract is 2 g. Thus, the content of unfertilized oenotine is equivalent to 19.47% w / w. Example 2 Process A: Extraction or hydroalcoholic mixture In a 2-liter reactor, 50 g of the drug is introduced and 250 g of the hydroalcoholic solution in a water:ethanol ratio of 1:1 is added in the acidic solution. The extract mixture is heated to 50 °C and remains under percolation extraction for 1 hour. At the end of this first step, the resulting liquid extract is removed and the drug is extracted three more times under the same conditions with the solvent of the extraction unit. At the end of the extraction, all the percolation products are filtered and combined and the solvent is evaporated under reduced pressure at a temperature of 50 °C to obtain a concentrated extract. Final drying is carried out by a spray dryer. The amount of dry extract obtained is 13.4 g. Process B: Water extraction At the end of step 1, 250 g of water in an acidic solution is added to the drug. The extract mixture is heated to 50°C and kept under percolation for 1 hour. At the end of the extraction, the percolation is separated from the drug, filtered and the solvent is evaporated under reduced pressure at a temperature of 50°C to obtain a concentrated extract. The remaining water is removed by drying under reduced pressure at 50°C to obtain a dry extract. The amount of dry extract is 2 g. Thus, the content of unfertilized oenotheine is equivalent to 19.17% w / w. Comparative Example 3 Extraction with water In a 100 mL flask, 500 mg of the drug is introduced and 40 mL of aqueous solvent in acidic solution is added. The extract mixture is heated to 50 °C and extraction is continued for 15 minutes. The process is repeated in pairs and extraction is continued for another 15 minutes. At the end of this first step, the resulting liquid extract is filtered to remove the drug. The filtrate is evaporated under reduced pressure at a temperature of 50 °C to obtain a concentrated extract. Final drying is carried out by a spray dryer. The amount of dry extract obtained is equivalent to 99 mg. Thus, the content of unprocessed oenotine is equivalent to 7.86% w / w. Comparative Example 4 Extraction with a hydroalcoholic mixture In a 100 mL flask, 500 mg of the drug is introduced and 40 mL of 4:1 water:ethanol in a weakly acidic solution is added. The extract mixture is heated to 50°C and the extraction is continued for 15 minutes. The process is repeated in pairs and the extraction is continued for another 15 minutes. At the end of this first step, the resulting liquid extract is filtered to remove the drug. The filtrate is evaporated under reduced pressure at a temperature of 50°C to obtain a concentrated extract. Final drying is carried out by a spray dryer. Thus, the content of unfertilized oenotine is equivalent to 4.25% w / w.

Claims

Claims 1. A process for preparing an extract of Epilobium spp. with an oenothein B content higher than 15% w / w comprising: a) extracting the drug 3-6 times with a water:ethanol mixture in ratios ranging from 3:1 to 1:1 by weight; b) filtering the percolation, evaporating the solvent and drying the resulting concentrated extract without further purification steps; c) extracting the drug previously subjected to extraction with water and ethanol, with water in an acidic solution, filtering the percolation, drying and adding the aqueous extract to the dried extracts obtained from step (b).

2. The process according to claim 1, wherein the Epilobium spp. is selected from Epilobium angustifolium L. and Epilobium parviflorum Schreb.

3. The process according to one or more of claims 1 to 2, wherein the extraction is carried out with a water:ethanol mixture at a temperature in the range of 25 to 50°C for times between 1 and 3 hours.

4. The process according to one or more of claims 1 to 3, wherein the extraction is carried out with a water:ethanol mixture at a concentration in the range of 2 to 6.

5. The process according to any one of claims 1 to 4, wherein the medicament is herba cum floribus.

6. The process according to one or more of claims 1 to 5, wherein the weight ratio of drug to water:ethanol is in the range of 1:3 to 1:10 by weight.

7. Process according to one or more of claims 1 to 6, wherein the drying of the water:ethanol extraction concentrate is carried out by spray drying.

8. Process according to one or more of claims 1 to 7, wherein the drying of the concentrate from the extract with acidic deionized water is carried out by evaporation under reduced pressure.

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