Preparation process of Epilobium extract with high oenothein B content

Abstract

The present invention relates to a process for preparing an extract of Epilobium genus having a high oenothein B content.

Description

[Technical Field]

[0001] The present invention relates to a process for preparing an extract of Epilobium genus having a high oenothein B content. [Background technology]

[0002] cutting edge technology Epilobium angustifolium L. is a perennial herbaceous plant belonging to the Onagraceae family. It has simple, hairless, erect stems and rhizomes, and the flowers are collected in striking, pink to purple, pyramidal racemes. The fruits are elongated capsules that open at maturity to release the seeds, which have pappus hairs for wind dispersal. Epilobium is native to regions of Europe and Western Asia, where it grows wild in gravelly soils (Gruenwald J. et al. PDR for herbal medicines, 4th Ed. Thomson, 2007).

[0003] The Epilobium phytocomplex contains three major classes of polyphenols: phenolic acids, flavonoids and ellagitannins, the latter being mainly represented by macrocyclic ellagitannins such as oenothein B. The phytocomplex, individual classes of polyphenols, and, to a greater extent, oenothein B, are associated with many health-promoting properties of this plant, including anti-inflammatory and antioxidant activity, particularly in the urological area (e.g., prostatitis, benign prostatic hyperplasia, urinary tract inflammation), stomach, and intestinal 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). Oenothein B can be obtained from several plant species, not necessarily from E. angustifolium L. (US2018 / 0256619).

[0004] E. angustifolium L. is not included in the monograph of the European Pharmacopoeia, therefore the galenical preparation of this extract refers to the decoction (a preparation of traditional folk medicine) and to those available in the literature and on the market. Although most 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), the use of other solvents such as methanol, ethyl acetate, butanol, and ethanol has also been documented (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. (Al., Phytochemistry, pharmacology and traditional uses of different Epilobium species (Onagraceae): A review. Journal of Ethnopharmacology, 156, 316-346, 2014). The concentration of oenothein B obtained in aqueous extracts of Epilobium species is always less than 10%.

[0005] US6528490 discloses the anti-inflammatory activity of E. angustifolium extracts containing no more than 9.6% by weight of oenothein B. US2018 / 0256619 reports that the preferred range of effective oenothein B amounts varies from 2.5 to 14%, but the experimental data reported therein still refer to extracts with the 9.6% oenothein B content described in US6528490.

[0006] FR2712594 and the corresponding patent US552594 disclose a process for the extraction of oenothein B from Epilobium parviflorum Schreb with a mixture of water and a water-soluble solvent, in particular acetone, and purification by reversed-phase HPLC.

[0007] WO2017108907 discloses the association of various Epilobium species with other plants.

[0008] The importance of oenothein B is evidenced by the above references, however: 1) According to European regulations, purified oenothein B cannot currently be added to dietary supplements in Italy (Ministerial Circular of 16 / 06 / 2016) and Europe (i.e., Novel Food Catalogue), although the use of E. angustifolium extracts titrated and standardized with this active ingredient is permitted. The extraction efficiency of this molecule in known processes is related to the purification step. 2) The molecules present in the aqueous extract are not representative of the phytocomplex due to the low extraction yield of flavonoids under these conditions.

[0009] Most extracts on the market lack standardization, and known extraction and purification procedures do not provide an optimal solution to the need for an extract containing all of Epilobium's most important bioactive components, along with a high oenothein B content. Oenothein B is an ellagitannin with antioxidant and anti-inflammatory activities. Numerous scientific studies have shown that oenothein B can restore normal 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) demonstrated that E. angustifolium extracts with an oenothein B content of over 15% by weight were significantly effective in treating benign prostatic hyperplasia without toxic effects on the kidneys or liver.

[0010] Therefore, there is a need for a process that enhances the rich phytocomplex and allows the preparation of extracts with high oenothein B content to fulfill their biological and physiological functions in dietary supplements and functional food formulations. Summary of the Invention

[0011] A process for the extraction of Epilobium species has now been found that provides an extract having an oenothein B content higher than 15% w / w, for example 15-35%, without the need for a purification step.

[0012] The process of the present invention can be successfully applied to various species of Epilobium, in particular E. angustifolium and E. parviflorum. The use of plant material (drug) in the flowering stage ("herba cum floribus") is particularly preferred.

[0013] The dry extract obtained by the process of the present invention, with an oenothein B content of more than 15.0%, has a very rich phytocomplex consisting of different classes of active ingredients, in particular 10-20% of phenolic acids (50% of which are represented by hydroxycinnamic acids), 20-30% of flavonoids (mainly represented by flavonols) and 40-60% of hydrolyzable tannins (the class of compounds to which oenothein B belongs).

[0014] The process of the present invention for preparing an extract of Epilobium having an oenothein B content higher than 15% w / w comprises: - extraction of the drug at least twice with a water:ethanol mixture in a ratio ranging from 3:1 to 1:1; - filtration of the permeate, evaporation of the solvent and drying of the resulting concentrated extract without further purification steps; It is characterized by:

[0015] The drug extraction is preferably repeated 3-6 times, more preferably 4-5 times, using fresh solvent each time.

[0016] Extraction with a water:ethanol mixture is preferably carried out at a temperature in the range of 25-50° C. for a time in the range of 1-3 hours at a pH in the range of 2-6, preferably 2-3.5.

[0017] 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.

[0018] Drying of the water:ethanol extract concentrate can be done by any known technique, for example by spray drying.

[0019] According to a preferred embodiment of the present invention, the process further comprises a step of extraction of the drug extracted with water and ethanol with water at acidic pH, filtering the permeate, concentrating the aqueous phase, drying and adding the dried aqueous extract to the dried extract obtained from the previous process.

[0020] Thus, according to this preferred embodiment, the process of the present invention comprises: A. Extraction of the drug at least twice with a water:ethanol mixture at an acidic pH in a ratio ranging from 3:1 to 1:1, filtering the permeate, evaporating the solvent, and drying the resulting concentrated extract without further purification steps. -B. Extraction of the drug extracted with water and ethanol with water at acidic pH, filtering the permeate, concentrating the aqueous phase, drying, and adding the dried aqueous extract to the dried extract obtained from step A above. Includes.

[0021] The two fractions obtained from steps A and B, which have different metabolic profiles, are analyzed and mixed appropriately until a product with the desired concentration of oenothein B is obtained.

[0022] Drying of the concentrate from the acidic pH water extract can be done by any known technique, for example by evaporation under reduced pressure.

[0023] The process of the present invention does not involve purification and is therefore simpler and cheaper than known methods.

[0024] Analysis of oenothein B titer was performed by HPLC-UV (a method comparable to that described by Kaskoniene 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).

[0025] The method that allows the determination of the content of oenothein B in the dry extract is chromatographic separation by HPLC-DAD using a reference standard. The sample is solubilized in a suitable solvent, filtered, and then injected into the HPLC. The mobile phase is acidic water and acidic methanol, and the gradient separation is carried out on an RP-C18 column at a wavelength of 254 nm.

[0026] The specificity of the method allows the determination of oenothein B without interference from other extracted substances. The method is linear in the concentration range of 0.1 mg / mL and 0.7 mg / mL, and the precision allows the estimation of the mean recovery for all concentrations in accordance with the acceptance criteria.

[0027] Table 1 shows the results of chemical profiling of the phytocomplex by RP-HPLC-PDA-ESI-MSn. [Brief explanation of the drawings]

[0028] [Figure 1] FIG. 1 shows a chromatogram.

[0029] [Table 1]

[0030] The present invention is explained in more detail in the following examples.

[0031] Example 1 Process A: Extraction with water-alcohol mixture 50 g of the drug is introduced into a 2 L reactor, and 250 g of an aqueous alcohol solution with a water:ethanol ratio of 3:1 is added at an acidic pH. The extraction mixture is heated to 50°C and maintained for 2.5 hours for percolation extraction. At the end of this first step, the liquid extract obtained is removed, and the drug is extracted three more times with the same solvent under the same conditions. At the end of the extraction, all the percolate is filtered and combined, and the solvent is evaporated under reduced pressure at a temperature of 50°C until a concentrated extract is obtained. Final drying is carried out using a spray dryer. The amount of dry extract obtained is 12.6 g.

[0032] Process B: Water extraction At the end of step 1, 250 g of acidic pH water is added to the drug, and the extraction mixture is heated to 50°C and maintained for 1 hour for percolation extraction. At the end of the extraction, the percolate is separated from the drug, filtered, and the solvent is evaporated at a temperature of 50°C under reduced pressure until a concentrated extract is obtained. Residual water is removed by drying at 50°C under reduced pressure until a dry extract is obtained. The amount of dry extract obtained is 2 g.

[0033] The oenothein B content thus obtained is 19.47% w / w.

[0034] Example 2 Process A: Extraction with water-alcohol mixture 50 g of the drug is introduced into a 2 L reactor, and 250 g of an aqueous alcohol solution with a water:ethanol ratio of 1:1 is added at an acidic pH. The extraction mixture is heated to 50°C and maintained for 1 hour for percolation extraction. At the end of this first step, the liquid extract obtained is removed, and the drug is extracted three more times with the same solvent under the same conditions. At the end of the extraction, all the percolate is filtered and combined, and the solvent is evaporated under reduced pressure at a temperature of 50°C until a concentrated extract is obtained. Final drying is carried out using a spray dryer. The amount of dry extract obtained is 13.4 g.

[0035] Process B: Water extraction At the end of step 1, 250 g of acidic pH water is added to the drug, and the extraction mixture is heated to 50°C and maintained for 1 hour for percolation extraction. At the end of the extraction, the percolate is separated from the drug, filtered, and the solvent is evaporated at a temperature of 50°C under reduced pressure until a concentrated extract is obtained. Residual water is removed by drying at 50°C under reduced pressure until a dry extract is obtained. The amount of dry extract obtained is 2 g.

[0036] The oenothein B content thus obtained is 19.17% w / w.

[0037] Comparative Example 3 Water extraction 500 mg of the drug is introduced into a 100 mL flask, 40 mL of an aqueous solvent at acidic pH is added, the extraction mixture is heated to 50°C, and the extraction is maintained for 15 minutes. This process is repeated twice, and the extraction is continued for another 15 minutes. At the end of this first step, the liquid extract obtained is filtered to remove the drug. The filtrate is evaporated under reduced pressure at a temperature of 50°C until a concentrated extract is obtained. Final drying is performed using a spray dryer. The amount of dry extract obtained is 99 mg.

[0038] The oenothein B content thus obtained is 7.86% w / w.

[0039] Comparative Example 4 Extraction with water-alcohol mixture 500 mg of the drug is introduced into a 100 mL flask, 40 mL of water:ethanol = 1:4 at a weakly acidic pH is added, the extraction mixture is heated to 50 °C, and the extraction is maintained for 15 minutes. This process is repeated twice, and the extraction is continued for another 15 minutes. At the end of this first step, the liquid extract obtained is filtered to remove the drug. The filtrate is evaporated under reduced pressure at a temperature of 50 °C until a concentrated extract is obtained. Final drying is carried out using a spray dryer.

[0040] The oenothein B content thus obtained is 4.25% w / w.

Claims

1. 1. A process for preparing an extract of the genus Epilobium having an oenothein B content higher than 15% w / w, comprising: a) extracting plant material of the genus Epilobium 3 to 6 times with a water:ethanol mixture, the weight ratio of water:ethanol ranging from 3:1 to 1:1; b) filtering the liquid extract obtained in step a) and evaporating the solvent to obtain a concentrated extract, followed by drying the concentrated extract without further purification steps; c) extracting the extract residue of the plant material obtained in step a) with water at an acidic pH, filtering the liquid extract, concentrating the aqueous phase and then drying the concentrated extract; and d) adding the dry extract obtained in step c) to the dry extract obtained in step b). The process includes:

2. 2. The process of claim 1, wherein the Epilobium genus is selected from Epilobium angustifolium L. and Epilobium parviflorum Schreb.

3. 3. The process of claim 1 or 2, wherein the extraction with the water:ethanol mixture is carried out at a temperature ranging from 25 to 50°C for a time ranging from 1 to 3 hours.

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

5. 5. The process according to any one of claims 1 to 4, wherein the plant material is plant material in the flowering stage.

6. 6. The process according to any one of claims 1 to 5, wherein the weight ratio of the plant material to the water:ethanol mixture is in the range of 1:3 to 1:

10.

7. 7. The process according to any one of claims 1 to 6, wherein the drying of the concentrated extract with the water:ethanol mixture is carried out by spray drying.

8. 8. The process according to any one of claims 1 to 7, wherein the drying of the concentrated extract with water at acidic pH is carried out by evaporation under reduced pressure.

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