PROCESS FOR THE PREPARATION OF A GINKGO BILOBA LEAF EXTRACT HIGHLY ENRICHED IN PROANTHOCYANIDINS AND FREE OF FLAVONOIDS AND TERPENIC TRILACTONES

Abstract

The invention relates to a method for preparing a Ginkgo biloba leaf extract highly concentrated in proanthocyanidins, and practically free of flavonoids and terpene trilactones. The invention also relates to the use of cheese fungal strains such as Penicillium Roquefortii PA and Geotrichum Candidum Geo 15, to catabolize high molecular weight proanthocyanidin polymers into small molecules in order to have beneficial effects on health.

Description

Title of the invention: Title

[0001] PROCESS FOR THE PREPARATION OF A GINKGO BILOBA LEAF EXTRACT HIGHLY ENRICHED IN PROANTHOCYANIDINS AND FREE OF FLAVONOIDS AND TERPENIC TRILACTONES

[0002] The invention relates to a process for preparing a Ginkgo biloba leaf extract highly concentrated in proanthocyanidins, practically free of flavonoids and terpene trilactones.

[0003] The invention also relates to the use of cheese fungal strains such as Penicillium Roquefortii PA and / or Geotrichum Candidum Geo 15, to catabolize high molecular weight proanthocyanidin polymers into small molecules in order to have beneficial effects on health.

[0004] The most well-known, widely used, and studied Ginkgo biloba leaf extract is EGb 761. This is a well-defined, standardized extract of Ginkgo biloba leaves containing approximately 24% flavone glycosides (predominant quercetin, kaempferol, and isorhamnetin) and 6% terpene trilactones (ginkgolides A, B, and C, and bilobalide). It was created by Dr. Willmar Schwabe Pharmaceuticals. The extraction process involving more than 20 operational steps is described in Schwabe's 1990 patent EP0431535A1

[0005] The main bioactive constituents found in EGb761 are flavonoids and terpene trilactones, hence the 24 / 6 specification. These are small molecules with a molecular mass of, for example, 610 daltons for rutin and 440 daltons for ginkgolide C. There is another class of compounds present in EGb761 that have molecular masses ranging from 1 to several hundred kilodaltons. These are the proanthocyanidins, also known as condensed tannins. EGb761 contains 7% proanthocyanidins. (Kulic, Planta Med 2022; 88: 398-404).

[0006] Proanthocyanidins are not directly bioavailable due to their high molecular weight, but they are catabolized by the human colonic microflora into low molecular weight phenolic acids that are known for their antioxidant properties (De'prez, J. Nutr. 130: 2733-2738, 2000. et al). Therefore, proanthocyanidins are a huge reservoir of antioxidant molecules. However, the authors, De' prez et al. did not identify the microbes responsible for the degradation

[0007] Proanthocyanidins obtained by selective capture and released on Sephadex LH-20 were analyzed as containing approximately 4% elemental nitrogen. Electrophoretic analyses of the proanthocyanidins on SDS-Page gels and revealed in a methylene blue solution showed a continuous streak between the markers protein molecules from 10 kilodaltons to over 250 kilodaltons without discrete bands. Taken together, these two observations indicate the presence of covalently bound peptides within the proanthocyanidin polymer. Therefore, catabolism of proanthocyanidins by colonic microflora can also release these small peptides.

[0008] The subject of the invention is an extract enriched in Ginkgo biloba proanthocyanidins (PEGb), free of flavonoids and terpene trilactones. The extract contains 30 to 70% proanthocyanidins, less than 2% flavonoids and less than 0.5% terpene trilactones. The extract preferably contains 45 to 55% proanthocyanidins, and is practically free of flavonoids and terpene trilactones.

[0009] The subject of the invention is a process for preparing a proanthocyanidin-enriched extract of Ginkgo biloba (PEGb) devoid of flavonoids and terpene trilactones as defined above. This procedure comprises a 2-step extraction of Ginkgo biloba leaves. In a first step, the dried leaves are exhaustively extracted with 95% ethanol to remove chlorophylls, flavonoids and terpene trilactones. In the second step, the leaves after extraction with 95% ethanol are subjected to a second exhaustive extraction with 50% ethanol or just with water to extract the proanthocyanidins. The 50% ethanol extract or the aqueous extract are then evaporated to dryness to obtain the proanthocyanidin-enriched extract of Ginkgo biloba (PEGb). The procedure is illustrated in Examples 1 and 2.

[0010] EGb761 is used for the treatment of mild to moderate cognitive impairment, tinnitus, and dementia. In the GuidAge study, which was a 5-year, double-blind, randomized clinical trial of EGb 761 for the prevention of Alzheimer's disease in elderly subjects with memory impairment, no definitive conclusions can be drawn about the effectiveness of the treatment. Other clinical trials supporting its use are often conflicting, sometimes positive, sometimes negative. One possible explanation may be that patients may or may not have the necessary fungi in the colonic myobiota to break down proanthocyanidins. Those who have them benefit; those who do not, may not.

[0011] A Google Scholar search for "Microbial degradation of condensed tannins" yielded only one result. A strain of Penicillium adametzi Zaleski was isolated from enrichment cultures with condensed tannins as the sole carbon source. (Grant, Science 193(4258), 1137-1139). Many French cheeses are ripened with Penicillium species, such as Penicillium Roquerfortii in Roquefort cheese and Penicillium Camamberti in Camambert cheese. The question is whether these Penicillium species used in the Cheese refining can also catabolize Ginkgo biloba proanthocyanidins.

[0012] The subject of the invention is the use of the PA strain of Penicillium Roquefortii and / or Geotrichum Candidum GEO 15 to decompose the proanthocyanidins of Ginkgo biloba as illustrated in example 3 and [Fig.l] (photo)

[0013] The subject of the invention is the use of the proanthocyanidin-enriched extract of Ginkgo biloba (PEGb) in association with Penicillium Roquefortii PA and / or Geotrichum Candidum GEO 15 incorporated together in pills or tablets or separately with the freeze-dried mushrooms as probiotics and taken together in order to have the beneficial effects on health of the degradation products of the proanthocyanidins.

[0014] The subject of the invention is the incorporation of the extract enriched in proanthocyanidins of Ginkgo biloba (PEGb) in the manufacture of cheeses using Penicillium Roquefortii PA or Geotrichum Candidum GEO 15 for ripening.

[0015] This may partly explain the health benefits of a Mediterranean-style diet characterized by cheese and wine consumption. Red wine is also a source of proanthocyanidins which is different from Ginkgo proanthocyanidins and may require different fungi for its breakdown.

[0016] Ginkgo Leaves: The extraction material was obtained from green Ginkgo biloba leaves, harvested in August 2022 in the commune of Gif-sur-Yvette, France, sun-dried (3 days) and ground into powder using a coffee grinder. The dried Ginkgo biloba leaves contained 4 to 12% proanthocyanidins. (Qa'dan, Pharmaceutical Biology, 49:5, 471-476)

[0017] Cheese Mushrooms: Here is the list of species / strains of mushrooms used in cheese ripening purchased from Agrodirect.fr

[0018] Freeze-dried Penicillium Camemberti -SAM3 Penicillium Roqueforti PA -1000 freeze-dried Penicillium Roqueforti PRB 18 - 5DCU _suspension Penicillium Album _suspension Penicillium candidum Snow_ freeze-dried Geotrichum Candidum GEO 15 - freeze-dried Ferment ALPD 50 dcu

[0019] Example 1

[0020] 50 g of dried Ginkgo biloba leaves were extracted 4 times, each time with 300 ml 95% ethanol-water for 1 hour at 50-70°C in a water bath and filtered through cotton. The 95% ethanol-water extract was discarded. The extracted leaves, which are now devoid of chlorophyll and beige in color, are then extracted 4 more times, each time with 300 ml of 50% ethanol-water for 1 hour. hour at 50-70°C in a water bath and filtered through a paper filter. The filtrates were then evaporated to dryness. 6.7 g of PEGb extract were obtained, a yield of 13.4% compared to the starting leaves. The amount of proanthocyanidins found in PEGb was estimated at 51%.

[0021] Example 2

[0022] 50 g of dried Ginkgo biloba leaves were extracted 4 times, each time with 300 ml of 95% ethanol for 1 hour at 50-70°C in a water bath and filtered through cotton. The 95% ethanol extract was discarded. The extracted leaves, which were now devoid of chlorophyll and beige in color, were then extracted 4 more times, each time with 300 ml of water for 1 hour at 50-70°C in a water bath and filtered through a paper filter. The aqueous filtrates were then evaporated to dryness. 5.7 g of PEGb extract were obtained, a yield of 11.4% compared to the starting leaves. The amount of proanthocyanidins found in the PEGb was estimated at 52%.

[0023] Example 3

[0024] 50 g of dried Ginkgo biloba leaves were extracted 4 times, each time with 300 ml of 95% ethanol for 1 hour at 50-70°C in a water bath and filtered through cotton. The 95% ethanol extract was discarded. The extracted leaves, which were now devoid of chlorophylls and beige in color, were then extracted 4 more times, each time with 300 ml of water for 1 hour at 50-70°C in a water bath and filtered through a paper filter. The aqueous filtrates were then concentrated to 250 ml to obtain a slightly cloudy, pale yellow aqueous suspension. 10 ml aliquots of the pale yellow suspension were pipetted into each of eight 20 ml capacity culture tubes. These were then sterilized in a pressure cooker and then cooled to room temperature to be inoculated with cheese fungi, 1 to 2 mg of freeze-dried strains, 1 drop of those in liquid suspensions. The culture tubes were labeled O, A, B, C, D, E, F, and G.Tube O is the control without inoculation and the others are as follows: . Tube O Control (Without inoculation) Tube A Penicillium Camemberti - SAM 3 - Freeze-dried Tube B Penicillium Roquerforti PA - Freeze-dried Tube C Penicillium Roquerforti PRB 18 - Suspension Tube D Penicillium Album - Suspension Tube E Penicillium candidum Neige - Freeze-dried Tube F Geotrichum Candidum GEO 15 - Freeze-dried Tube G Ferment ALDP

[0025] After inoculation, the tubes were screwed to simulate anaerobic conditions and left in the dark at room temperature of 18 to 22°C for 3 months. The colors of the tubes after 3 months are as follows: (See [Fig.l]. Photograph) Tube O Control (Without inoculation) Pale Yellow Tube A Penicillium Camemberti - SAM 3___ Pale Yellow Tube B Penicillium Roquerforti PA Black Tube C Penicilium Roquerforti PRB 18 Pale Yellow Tube D Penicillium Album - Suspension Pale Yellow Tube E Penicillium candidum Snow Pale Yellow Tube F Geotrichum Candidum GEO 15 Dark brown Tube G Ferment ALDP Pale Yellow Tube B, which turned black, shows that Penicillium Roquefortii strain PA can proliferate in the proanthocyanidin-rich medium. Tube C, which remained pale yellow, shows that Penicillium roquefortii strain PRB18 failed to proliferate in the proanthocyanidin-rich medium. Therefore, it can be seen that not only the Penicillium roquerfortii species is important, but also the strain. Tube F, which turned dark brown, shows that Geotrichum Candidum strain GEO 15 can also proliferate in the proanthocyanidin-rich medium. Tubes A, D, E and G which remained pale yellow show that the strains Penicillium Camemberti -SAM3, Penicillium Album, Penicillium candidum Neige and the ALP D ferment containing the bacteria Lactococcus and Lactobacillus failed to proliferate in the medium rich in proanthocyanidins

Claims

Claims

1. A proanthocyanidin-enriched extract of Ginkgo biloba leaves (PEGb) that is substantially free of flavonoids and terpene trilactones. The extract contains 30-70% proanthocyanidins, less than 2% flavonoids, and less than 0.5% terpene trilactones. The extract preferably contains 45-55% proanthocyanidins, and substantially free of flavonoids and terpene trilactones.

2. A process for preparing an extract as defined in claim 1, comprising a 2-step extraction of Ginkgo biloba leaves, step 1 of extracting Ginkgo biloba leaves several times with 95% ethanol to remove chlorophylls, flavonoids and terpene trilactones and step 2, exhaustively re-extracting the leaves from the first extraction with 50% ethanol or water to extract proanthocyanidins.

3. The use for the preparation of pills or tablets of the proanthocyanidin-enriched extract of Ginkgo biloba leaves (PEGb) as defined in claim 1 in combination with Penicillium Roquefortii PA and / or Geotrichum Candidum GEO 15 incorporated together in pills or tablets or separately with freeze-dried cheese mushrooms as probiotics and taken together to have the beneficial health effects of proanthocyanidin degradation products

4. The incorporation of the proanthocyanidin-enriched extract of Ginkgo biloba leaves (PEGb) as defined in claim 1 in the manufacture of cheeses which use Penicillium Roquefortii PA or Geotrichum Candidum GEO 15 for ripening.

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