Login| Sign Up| Help| Contact|

Patent Searching and Data


Title:
PROCESS OF EXTRACTION FROM APPLE THINNINGS OR APPLE PROCESSING RESIDUES, AND EXTRACTS THUS OBTAINED
Document Type and Number:
WIPO Patent Application WO/2021/079304
Kind Code:
A1
Abstract:
Disclosed is a process for the preparation of extracts of unripe apples or apple processing residues comprising grinding steps, enzyme treatments, pressing, filtration, adsorption chromatography and concentration. The extracts obtained contain active substances useful to reduce the appetite of individuals suffering from superfluous fat accumulation and for the treatment of obesity and sarcopenia in children and the elderly.

Inventors:
BOMBARDELLI, Ezio (Via Gabetta 13, Gropello Cairoli, IT)
LOMBARDO, Giuseppe (Zona Industriale Località Chiusi Strada Provinciale SNC, Bianco, IT)
TRUNFIO, Giuseppe (262, Torregrotta, IT)
Application Number:
IB2020/059923
Publication Date:
April 29, 2021
Filing Date:
October 22, 2020
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
HERBAL E ANTIOXIDANT DERIVATIVES SRL ED IN FORMA ABBREVIATA H&AD SRL (Strada Provinciale SNC, Bianco, IT)
International Classes:
A23L19/00; A23L19/15; A23L27/12; A61K36/73
Attorney, Agent or Firm:
MINOJA, Fabrizio (Via Plinio 63, Milano, IT)
Download PDF:
Claims:
CLAIMS

1. A process of preparation of extracts of unripe apples or apple processing residues, which comprises: a) grinding of unripe apples or apple processing residues; b) treatment of the ground mass with cellulase and pectinase at 15-35°C for 10-60 minutes in an acid medium; c) continuous pressing at pressures ranging between 100 and 300 bars; d) drying of the solid mass to a paste-like consistency and heating the liquid to temperatures ranging between 50 and 80°C, preferably 70°C, for a time of 10-30 minutes. e) treatment of the liquid from step d) with pectinase at 50-70°C; f) filtration and adsorption chromatography of the filtrate, eluting with ethanol or with 9: 1 ethanol: water mixtures; g) concentration of the eluate and addition of the concentrate to the dried solid from step d); h) pulverisation or micronisation of the mixture from step g).

2. The process according to claim 1 wherein the apples belong to the varieties Gala, Fuji, Bella del Bosco, Rosa mantovana, Annurca, Elstar, Golden Delicious, Red Delicious, Granny Smith, Rennet or Canadian Reinette.

3. The process according to claim 2 wherein a mixture of cultivars is used.

4. The process according to one or more of claims 1 to 3 wherein step a) is carried out in the presence of citric acid.

5. The process according to one or more of claims 1 to 4 wherein the filtration of step f) is carried out by centrifugation, passage through a filter press or microfiltration through a membrane with a cut-off of 5000 Daltons.

6. The process according to one or more of claims 1 to 5 wherein the adsorption chromatography of step f) is carried out on XAD7 polystyrene absorption resin.

7. Unripe apple extract obtainable by the process of claims 1-6.

8. An extract according to claim 7 having a content, as a percentage of the weight of the dried extract, of 0.1-0.5% abscisic acid, 5-9% phloridzin and phloretin and 4-8% ursolic acid.

9. Pharmaceutical or nutraceutical compositions comprising the extracts of claims 7- 8

10. Compositions according to claim 9 further comprising one or more of the following ingredients:

- extracts of Vitis vinifera, Cyclanthera pedata, Rosmarinus officinalis, Salvia officinalis, Zingiber officinalis, Vaccinium myrtillus, Taraxacum officinalis, Citrus aurantium var. bergamia (bergamot orange), Citrus reticulata (mandarin), Leuzea carthamoides, Cyclanthera pedata, Bacopa monnieri, Pisum sativum , Centella asiatica, kidney beans, lupin, citrus fruit pressing residues, sesame, almonds, rice husks, buckwheat; amino acids; vitamins; trace elements; lipoic acid; phosphoserine; phospholipids; creatine; creatinine.

11. The compositions of claims 9-10 for use in the treatment of obesity and sarcopenia in children and the elderly.

Description:
PROCESS OF EXTRACTION FROM APPLE THTNNINGS OR APPLE

PROCESSING RESIDUES, AND EXTRACTS THUS OBTAINED

The present invention relates to the preparation of extracts obtained from unripe apples collected as thinnings in industrial apple production or from industrial apple processing residues such as peel, cores and fibrous matrix, and the formulations containing them. Description of the invention:

Apples are traditionally considered to be synonymous with health and well-being. Recent scientific evidence has confirmed the nutritional and health value of apples, correlated with their content of vitamins (C, PP, Bl; B2, A), mineral salts, pectin, tannins, polyphenols, triterpenes and other compounds responsible for properties beneficial to the cardiovascular, immune, metabolic, muscle and respiratory systems.

For example, it has been reported that eating two apples a day of the Annurca variety gives rise to a significant mean reduction in total cholesterol and LDL cholesterol levels, and an increase in HDL cholesterol, after only eight weeks (Tenore, G. C. et al, Journal of the Science of Food and Agriculture, 97(7), 2107-2115, 2016). Although other varieties contain different amounts, their polyphenol content suggests that cholesterol- reducing properties are not the sole province of the Annurca variety.

The chemopreventive potential of the polyphenols contained in three apple varieties (Annurca, Red Delicious and Golden Delicious) was studied by Scafuri B et al. in Scientific Reports, volume 6, Article number: 32516 (2016). Moreover, polyphenols are not the only active constituents contained in apples.

Other classes of compounds, such as pentacyclic terpenes (ursolic acid) which stimulate protein synthesis, phytohormones such as abscisic acid, and dihydrochalcones (phloretin and phloridzin) with hypoglycaemic activity, are equally important in determining the health-giving properties of apples. The content of said substances is highest in unripe apples, which are available in large amounts from apple thinning operations, an essential practice to ensure not only the production of apples of suitable size, but also to guarantee the following year’s flowering. Thinnable apples constitute up to 60-65% of the fruit present on the tree before ripening. After thinning, the sour apples with a size of 1-3 cm are left on the ground or used as biomass for biofuel production. Their use has also been proposed in the animal feed industry, despite the presence of large percentages of tannins and polyphenols, about 10 times higher than in ripe apples, with possible anti-nutritional effects.

Extracts from unripe apples have been described for use as active ingredients of nutraceutical compositions. For example, KR100331578, CN102210471, KR20100112226 and EP657169 disclose processes for the extraction of polyphenols from unripe apples using various techniques (pressing, enzymolysis, extraction, chromatography and the like).

Description of the invention:

A process for the preparation of extracts from unripe apples or apple processing residues has now been found which provides an improved extract compared with those described to date, which mainly focus on polyphenol content alone. If a suitable sequence of steps is followed, and both the juice and the solid residue obtained from a pressing step after enzymatic treatment are mixed, the resulting extract contains, in addition to polyphenols, pentacyclic triterpenes such as ursolic and oleanolic acid, amyrin, sterols, abscisic acid, phloridzin glucoside and phloridzin xyloside, and phloretin.

The fraction of polyphenols, present as such or in the form of glycosides, contains catechin, epicatechin, dimer Bl, dimer B2, procyanidin Cl, tetramers, pentamers and hexamers and measurable oligomeric amounts up to dodecamers, quercetin, quercitrin, rutin, chlorogenic acid and the isomers thereof, and hydroxycinnamic acid. The process according to the invention comprises: a) grinding unripe apples and/or apple processing residues; b) treating the ground product with cellulase and pectinase at 15-35°C for 10-60 minutes in an acid medium; c) continuous pressing at pressures ranging between 100 and 300 bars; d) drying the solid part until it attains a paste-like consistency and heating the liquid to temperatures ranging between 50 and 80°C, preferably 60°C or 70°C, for a time of 10-30 minutes. e) treating the liquid from step d) with pectinase at a temperature ranging between 50 and 70°C; f) filtration and adsorption chromatography of the filtrate, eluting with ethanol or 9:1 ethanol: water mixtures; g) concentrating the eluate and adding the concentrate to the solid dried product from step d); h) pulverising or micronising the mixture resulting from step g).

Alternatively, before step b), the ground product is heated in a shell and tube heat exchanger or by an equivalent technique to temperatures ranging between 75 and 90°C for a time ranging from 5 to 15 minutes to inactivate the hydrolytic enzymes, and then cooled to 50°C, followed by addition of cellulase and pectinase.

The unripe apples which can be used according to the invention can belong to any variety. The varieties Gala, Fuji, Bella del Bosco, Rosa mantovana, Annurca, Elstar, Golden Delicious, Red Delicious, Granny Smith, Rennet and Canadian Reinette are preferred, especially a mixture of at least two, and more preferably at least three, of said varieties.

As an alternative or in addition to unripe apples, the peel, cores and fibrous matrix, and apple processing waste in general, can be used to prepare gelatins, juices, purees and the like. By-products of industrial processing involving separation of the peel, with the corresponding heat shock, must be used within not more than three months after the fruit is picked due to the high level of degradability of the active ingredients.

The varieties are mixed directly at the time of picking, and stored at a temperature ranging between 0 and 4°C, preferably 2°C. At said temperature, after thorough washing with an acidulous solution, they are coarsely ground in the presence or absence of vitamin C or organic acids such as citric, tartaric or malic acid or the like, to inactivate the oxidases.

The material is then heated to a temperature ranging between 15 and 35°C, preferably 25°C, and an enzymatic mixture consisting of cellulase and pectinase is added (with a mechanical mixer) for a time ranging between 10 and 60 min, preferably 15 min; the material is then continuously pressed at pressures ranging between 100 and 300 bars, preferably 200 bars.

Alternatively, grinding is conducted with simultaneous treatment of the biomass at temperatures ranging between 75 and 90°C, in order to inhibit oxidases and hydrolytic enzymes completely, for a time ranging between 5 and 15 minutes. After cooling to 40- 60°C, preferably 50°C, treatment with cellulase and pectinase is carried out.

The pressed material (solid part) is sent for drying, while the juice is rapidly pasteurised at temperatures ranging between 50 and 80°C, preferably 70°C, for a time ranging between 10 and 30 minutes.

The liquid is cooled to 50-70°C and pectinase is added until a solution is obtained, which is filtered by centrifugation or passing through a filter press, or undergoes microfiltration through a membrane with a cut-off of 5000 Daltons. Initial heat shock is followed by pressing in accordance with the preceding process.

The filtrate is absorbed on a column containing XAD7 polystyrene absorption resin or equivalent and washed with the minimum amount of deionised water until the substances not absorbable by the resin have been completely eliminated.

The resin is eluted with 95% ethanol or a 9/1 v/v mixture of ethanol/water, and the eluate is concentrated to a reduced volume and added to the previously dried material. The mixture of the two fractions is then dried and micronised.

The extract obtainable from the process contains flavanols (catechin, epicatechin and procyanidins A2 and B2 in monomeric, trimeric or tetrameric form), flavonols (quercetin and quercetin-glycosides), flavones (apigenin), dihydrochalcones (phloretin, phloridzin and glycosides), hydroxycinnamic acids (p-cumaric acid, caffeoylquinic acid), triterpenes (ursolic, oleanolic, maslinic, annuric and boswellic acids), abscisic acid and the glycosides thereof and indoleacetic acid.

Some of the characteristic ingredients are listed below, as a percentage of the dry weight of the extract: chlorogenic acid 6 to 9%, preferably 6%; abscisic acid, the esters and glycosides thereof, between 0.1 and 0.5%; pentacyclic triterpenes expressed as ursolic acid, between 4 and 8%, preferably 7%; phloridzin and phloretin glycosides between 5 and 9%, preferably 6%; procyanidin B1 and procyanidin B2 between 2.5 and 5%; polyphenols between 45 and 70%, preferably 50%; quercetin glycosides between 5 and 15%.

The chalcones phloridzin and phloretin influence the epigenetic processes (genetic changes not encoded in the DNA sequence), which play an important part in the gene expression of breast cancer cells. The chalcones also interact with sodium-dependent glucose transport in the intestine and then, by reducing glucose absorption, reduce the postprandial blood glucose peak, with favourable effects for the treatment and prevention of diabetes.

Polyphenols like quercetin inhibit the sulphotransferases involved in the metabolism of thyroid hormones, steroids and catecholamines.

Procyanidins are potent antimutagens, act on inflammation at inflammasome level, possess antiproliferative activity, and act at epigenetic level and on innate immunity. Said substances are also useful in preventing cognitive decline and the states of depression typical of the elderly.

The extracts according to the invention can be added to foods, or can be formulated in pharmaceutical or nutraceutical compositions or medical aids according to conventional techniques, as described, for example, in “Remington’s Pharmaceutical Handbook”, Mack Publishing Co., N.Y., USA. Examples of oral formulations are tablets, dragees, soft and hard gelatin capsules, cellulose capsules and bars.

Particularly suitable forms of administration are tablets, capsules or bars enriched with fibres, especially Citrus aurantium var. bergamia fibres, which are extremely rich in polyphenols that act on the lipid and carbohydrate metabolism. According to a further aspect, the compositions according to the invention may contain other phytotherapeutic compounds or extracts with synergic or complementary activity.

In particular, the compositions may contain one or more of the following ingredients: extracts of Vitis vinifera , Cyclanthera pedata , Olea europea, Rosmarinus officinalis , Salvia officinalis , Zingiber officinale , Vaccinium myrtillus , Taraxacum officinalis , Citrus aurantium var. bergamia /bergamot orange/, Citrus reticulata /mandarin), Leuzea carthamoides, Bacopa monnieri, Pisum sativum, Centella asiatica, kidney beans, lupin, citrus fruit pressing residues, sesame, almonds, rice husks, buckwheat; amino acids; vitamins; trace elements; lipoic acid; phosphoserine; phospholipids; creatine and creatinine.

The extracts according to the invention are particularly useful to reduce the appetite of individuals suffering from superfluous fat accumulation, and for the treatment of obesity and sarcopenia in children and the elderly. The combination with protein-rich plant sources such as kidney beans, lupins, citrus fruit pressing residues, sesame, almonds, rice husks and buckwheat is particularly indicated for the treatment of sarcopenia. The compositions are also useful, due to their immunoregulatory effect, for the prevention of the most common inflammatory disorders.

The examples set out below further illustrate the invention. Example 1 - Preparation of matrix enriched with active ingredients of sour apples.

500 kg of sour Golden Delicious, Fuji, Bella del Bosco and Rosa mantovana apples in approximately equal ratios, picked a month after flowering and refrigerated for a month at a temperature of 2°C (times required for harvesting and storage), then thoroughly washed in a mixer with an aqueous solution containing 0.05% citric acid, is coarsely ground in a hammer mill, and the pulp, with the addition of a pectinase solution (0.1%), is heated in a linear tunnel set to 25°C for 20 min. The mass continuously enters the presses, wherein it undergoes forced filtration at a pressure of 200 bars. The pressing residue is dried in a fluid-bed dryer until the water has been removed and the residue amounts to 3% of the biomass. 14 Kg of yellow/green residue is obtained. The pressed juice undergoes subsequent enzymatic treatment with pectinase and centrifugation until a clear solution is obtained. Said solution is passed over 20 L of XAD7 absorption resin and washed with demineralised water until the substances not trapped by the resin have been completely eliminated. At the end of the elution with water, the resin is eluted with 95% ethanol until the trapped matter has been completely recovered, monitoring the eluate by TLC; the partly water-alcohol solution is concentrated to a small volume, and added to the dried pressing residue; the resulting mass, after thorough homogenisation of the powder, is dried until the water has been removed, and micronised.

A product with the following characteristics is obtained: green-beige powder containing 1.2% chlorogenic acid, 0.6% dimeric procyanidins, 10% total polyphenols, 2.15% phloridzin and 2.6% tetracyclic triterpenes expressed as ursolic acid (percentages of the weight of the extract).

Example 2 - Preparation of matrix enriched with active ingredients of sour apples.

500 kg of sour Golden Delicious, Fuji, Bella del Bosco and Rosa mantovana apples in approximately equal ratios, picked a month after flowering and refrigerated for a month at a temperature of 2°C, is thoroughly washed and coarsely ground in a hammer mixer, and the pulp is immediately heated in a suitably sized shell and tube heat exchanger at 80°C for a run time ranging between 10 and 15 minutes; the pulp is cooled to 50°C and, after the addition of a pectinase solution (0.1%), is maintained in a linear tunnel at the same temperature for 20 min. The mass continuously enters the presses, wherein it undergoes forced filtration at a pressure of 200 bars. The pressing residue is dried in a rotary dryer at a temperature ranging from 500 to 600°C until the water has been removed and the residue amounts to 3% of the biomass, which takes 3-5 min. 18 Kg of yellow/green residue is obtained. The pressed juice is centrifuged until a clear solution is obtained. As in Example 1, said solution is passed over 20 L of XAD7 absorption resin and washed with demineralised water until the substances not trapped by the resin have been completely eliminated. At the end of the elution with water, the resin is eluted with 95% ethanol until the trapped matter has been completely recovered, monitoring the eluate by TLC; the partly water-alcohol solution is concentrated to a small volume, and added to the dried pressing residue; the resulting mass, after thorough homogenisation of the powder, is dried until the water has been removed, and micronised.

A product with the following characteristics is obtained: green-beige powder containing 2.1% chlorogenic acid, 0.85% dimeric procyanidins, 16% total polyphenols, 2.1% phloridzin, 1.1% quercetin derivatives and 4.1% tetracyclic triterpenes expressed in ursolic acid (percentages of the weight of the extract).

Example 3 - Preparation of 20 g bars of apple residue according to Example 2, with the addition of group B vitamins and protein synthesis catalysts.

The bars are prepared according to the equipment and the experience of the workforce . Each 20 g bar contains:

Dried sour apple product according to Example 2 5 g

Bacopa monnieri extract 100 mg

Phospholipids 20% phosphatidylserine 150 mg

Phosphoryl serine 150 mg

Vitamin B6 25 mg

Acetylglutamine 100 mg

Glucomannan 3 g Citrus bergamia , micronised pulp 5 g

Calcium caseinate 3 g

Puffed rice 5 g

Orange flavouring with dark chocolate coating.

Example 4 - 20 g dessert bars

The bars are prepared according to the equipment and the experience of the workforce. Each 20 g bar contains:

Dried sour apple product according to Example 1 3 g

Bacopa monnieri extract 100 mg

Centella asiatica extract 25 mg a-lipoic acid 150 mg

Phospholipids 20% phosphatidylserine 150 mg

Phosphoryl serine 50 mg

Vitamin B6 25 mg

Acetylglutamine 100 mg

Asparagine 60 mg

Creatine 60 mg

Arginine 50 mg

Zinc aspartate 30 mg

Chromium picolinate 10 mg

Selenium 50 m

Glucomannan 3 g

Citrus bergamia , micronised pulp, 15% protein 5 g

Calcium caseinate 2 g

Puffed rice 5 g

Orange flavouring with dark chocolate coating.

Example 5 - Preparation of 30 g bars of apple residue according to Example 1, with the addition of group B vitamins and protein synthesis catalysts. Dried sour apple product according to Example 1 3 g

Leuzea carthamoides extract, 20% ecdysones 100 mg

Phosphoryl serine 150 mg

Vitamin B6 25 mg

Acetylglutamine 500 mg

Glucomannan 3 g

Citrus bergamia, micronised pulp 5 g

Pisum sativum , micronised seed 10 g

Puffed rice 5 g

Asparagine 60 mg

Creatine 60 mg

Arginine 50 mg

Zinc aspartate 30 mg

Chromium picolinate 10 mg

Orange flavouring with dark chocolate coating.

Example 6 - Preparation of 30 g bars of apple residue according to Example 1, with the addition of group B vitamins and extract of Cyclanthera pedata and Taraxacum officinale for metabolic syndrome.

Dried sour apple product according to Example 1 3 g

Cyclanthera pedata extract 100 mg

Taraxacum officinale extract 150 mg

Vitamin B6 25 mg

Acetylglutamine 500 mg

Glucomannan 3 g

Citrus bergamia, micronised pulp 5 g

Puffed rice 5 g

Asparagine 60 mg

Creatine 60 mg Arginine 50 mg

Zinc aspartate 30 mg

Chromium picolinate 10 mg

Orange flavouring with dark chocolate coating.

Example 7 - Clinical trial on elderly patients suffering from type 2 diabetes characterised by hyperglycaemia, slight memory loss, moderate hypertension and capillary fragility.

21 patients suffering from diabetes received crossover treatment (Table 1) with a placebo or the formulation according to Example 3.

The patients’ case histories, taken after screening on the basis of the inclusion and exclusion criteria according to the protocol, showed the following baseline profile:

In addition to blood glucose and the main lipid parameters, the isoprostanes in the urine were measured as vascular oxidation index.

Table 1 Patients’ characteristics

Age 70 ± 2 68 - 73

BMI 25.7 20 -30

TC (mg/dL) 223 ± 8 190 - 270

HDL 45± 3 32 - 59

TG (mg/dl) 208± 3 60 - 320

Blood glucose (mg/dl) 170± 3 98 -270

HbAlc (%) 7.9± 0.3 5.6 -11.3

21 patients treated for 4 weeks with a 4-week wash-out

Placebo Formula of Example 3

Age 70 ± 2 68 - 73

BMI 25.7 25.1 ns

TC (mg/dL) 225 ± 4 198 ± 9* HDL 48± 2 51± 1.5 * TG (mg/dl) 208± 3 145± 31*

Blood glucose (mg/dl) 170± 3 121± 12 HbAlc (%) 7.9± 1.8 7.4± 1.8**

The 8-iso-PGF2a isoprostane determined in the urine ranged from 63.8± 31 pmol/mmol creatinine on admission to 59± 22 after treatment. The comparison with the placebo compared with the admission data ranged from 64.8± 27.1 for the placebo to 55.2± 17.1. Urinary excretion of 8-iso-PGF2a8-iso-PGF2a8-iso-PGF2a during the wash out period between the two crossover treatments amounted to 62.82± 27.1

The above data demonstrate the efficacy of the formulation according to the invention in the treatment of excess weight, type 2 diabetes and obesity.