Halophytic plant extract stabilised with a lipophilising agent, and process for the manufacture thereof

Abstract

Disclosed are extracts of halophytic plants such as Crithmum maritimum, stabilised with a lipophilising agent selected from hydrogenated vegetable oils, fatty alcohols, fatty acids, fatty acid monoglycerides, fatty acid diglycerides, fatty acid triglycerides, polyoxylglycerides and waxes. The extracts according to the invention are useful as active ingredients of pharmaceutical compositions or dietary supplements for human or veterinary use, which are useful for the treatment and / or in the prevention of disorders of the gastrointestinal system.

Description

[0001] HALOPHYTIC PLANT EXTRACT STABILISED WITH A LIPOPHILISING AGENT, AND PROCESS FOR THE MANUFACTURE THEREOF

[0002] Field of invention

[0003] The invention relates to a composition with low hygroscopicity comprising a halophytic plant extract and a lipophilising agent.

[0004] Background to the invention

[0005] Halophytes or halophytic plants constitute a group of plants which, due to specific morphological or physiological adaptations, are able to grow in highly saline soils; to ensure their optimum development, they require a NaCl concentration in the soil of about 1-2%. Due to their considerable drought resistance and their ability to develop and grow in soils not commonly used for agricultural purposes, said plants were traditionally eaten in Mediterranean countries in periods of food shortages or famine, and thus came to be included among the ingredients of the traditional cuisine known as the “Mediterranean diet”; halophytes such as Cichorium inlybiis. Cichorium spinosum. Crithmum maritimum and Portulaca oleracea. which are still eaten today, are not only part of a niche market in the agrifood sector, but also represent useful alternatives to traditional vegetables because they are classed as functional foods and can be grown in areas which, because of climate change and low possibilities of irrigation, are becoming highly arid and saline.

[0006] In addition to their traditional dietary use, halophytes are also used in the traditional medicine of Mediterranean countries; examples of the properties commonly attributed to said plants are antioxidant, detoxifying, anti-inflammatory, purifying, digestive, hepatoprotective, expectorant, diuretic, hypoglycaemic, antihyperlipidaemic and antipyretic properties (Petropuolos S. A. et al., Trends in Food Science & Technology, 2018, 74, 69-84).

[0007] Crithmum maritimum, commonly known as rock samphire, sea fennel or samphire, is a perennial halophytic species typical of the Mediterranean coasts, where it grows among rocks, on cliffs and on beaches.

[0008] Rock samphire was traditionally used as an ingredient of traditional dishes from several countries; in particular, the aerial parts of rock samphire can be eaten either raw or cooked, and can be used to make preserves or pickles.

[0009] Since ancient Greek times, rock samphire has been used as a medicinal plant for the treatment of gout and kidney disorders; rock samphire has also been used in traditional medicine for its anti-scurvy, digestive, carminative, purifying, diuretic and anthelmintic properties, for the treatment of influenza and inflammation, and as a wound-healing remedy.

[0010] The efficacy of rock samphire and the extracts thereof for traditional uses has been demonstrated by phytochemical analysis; rock samphire has a high content of vitamin C, which has antioxidant and antiscurvy properties, flavonoids, carotenoids and polyphenols, such as chlorogenic acid, quinic acid and caffeoylquinic acid, which have antiinflammatory, antibacterial and antiviral properties, etc.

[0011] Nowadays, rock samphire is mainly used in cosmetics, wherein its essential oil is employed in antiaging compositions; conversely, rock samphire extracts are rarely used in the nutraceutical field, probably due to their high level of hygroscopicity, which makes them difficult to store and / or formulate. Said formulation problems also arise in the development of extracts of other halophytes.

[0012] In view of their potential therapeutic applications, there is a need to develop halophyte extracts with low hygroscopicity, and compositions containing them.

[0013] Description of the invention

[0014] The applicant has developed halophyte extracts stabilised with a lipophilising agent, and compositions containing them; said extracts and compositions can be used for therapeutic, nutraceutical or dietary purposes.

[0015] A first aspect of the present invention relates to halophyte extracts stabilised with a lipophilising agent; the term “lipophilising agent” indicates an ingredient or mixture of ingredients whose structure contains at least one C12-C32 alkyl chain, which said alkyl chain can be straight or branched, saturated or unsaturated, and optionally substituted by, or alternate with, heteroatoms or groups containing heteroatoms, including, but not limited to, alcohols, ethers, and primary, secondary and tertiary amines. Typically, the stabilising or lipophilising agent is selected from the group consisting of hydrogenated vegetable oils, fatty alcohols, fatty acids, fatty acid monoglycerides, fatty acid diglycerides, fatty acid triglycerides, polyoxylglycerides and waxes. The extracts are obtained from the roots, leaves, flowers, buds, aerial parts and whole plants of halophytes selected from the group consisting of Arthrocnemum macrostachyum, Bassia spp., Beta vulgaris subsp. maritima, Cakile spp., Capparis spinosa, Cichorium spi nosum, Chenopodium spp. (Chenopodium album, Chenopodium quinoa). Crambe maritima, Cressa cretica, Crithmum maritimum, Cynara cardunculus, Descaurinia sophia, Diplotaxis spp., Eryngium maritimum, Halimione portulacoides, Inula crithmoides, Lepidium latifolium, Mesembryanthemum crystallinum, Nitraria spp. (Nitraria schoberi, Nitraria sibirica), Plantago spp. (Plantago coronopus, Plantago lanceolala), Portulaca spp. (Portulaca oleracea), Salicornia spp. (Salicornia herbacea), Salsola spp. (Salsola soda), Suaeda spp. (Suaeda frulicosa). Tetragonia decumbens, Tribulus ler resins., Tripolium pannonicum and Zygophyllum spp. (Zygophyllum fabago. Zygophyllum album).

[0016] A second aspect of the invention relates to a process for the preparation of a halophytic plant extract stabilised with a stabilising agent by extraction of parts of the plant, followed by the addition of a lipophilising agent.

[0017] A third aspect of the invention relates to pharmaceutical compositions and dietary supplements for human or veterinary use comprising extracts stabilised according to the present invention, and at least one excipient and / or pharmaceutically or nutraceutically acceptable carrier; the excipients and / or carriers are selected from the group consisting of diluents, disintegrating agents, binders, lubricants, coating agents, liquid carriers, sweeteners, flavourings, antioxidants, preservatives and mixtures thereof.

[0018] Said compositions can take the form of swallowable or chewable tablets, hard or softgel capsules, syrups, gelatins, powders or granulates, and can be taken orally.

[0019] In particular, the invention relates to the use of a Crithmum maritimum extract stabilised with a lipophilising agent, or formulations containing it, in the treatment and / or prevention of disorders of the gastrointestinal system; in particular in the treatment of functional dyspepsia and irritable bowel syndrome; more particularly in the treatment of functional dyspepsia and irritable bowel syndrome in a set of patients suffering from said disorders who exhibit post-prandial gastric bloating as their prevalent symptom. To clarify, the term “functional dyspepsia” indicates a disorder involving the upper gastrointestinal tract which is manifested by one or more of the following symptoms: nausea, premature satiety, post-prandial fullness, epigastric bloating, pain in the upper abdomen, rumbling stomach or belching; the term “irritable bowel syndrome” indicates a disorder involving the lower gastrointestinal tract which is manifested by one or more of the following symptoms: abdominal pain, colic, bloating, constipation and diarrhoea.

[0020] The invention also relates to a combined preparation, for simultaneous, separate or sequential use in the treatment and / or prevention of disorders of the gastrointestinal system, of a composition comprising a Crithmum maritimum extract stabilised with a lipophilising agent and a further pharmaceutical or nutraceutical agent.

[0021] With special reference to the first aspect of the invention, the stabilised extract is a Crithmum maritimum extract; in particular, said extract is characterised, by HPLC analysis, by a total hydroxycinnamic acid content not exceeding 5% and a total flavonoid content not exceeding 2%; preferably, the total hydroxycinnamic acid content ranges from 0.5- 3.5%, of which about 20-60%, preferably 30-55%, consists of chlorogenic acid, while the total flavonoid content ranges from 0.05-0.35%; even more preferably, the extract is characterised by a chlorogenic acid content ranging from 0.3-1.3%. To clarify, in the present application, the values of the extreme points of the numerical ranges shall be deemed to be included in the ranges.

[0022] Moreover, the1H-NMR profile of the stabilised extract demonstrates the presence of chlorogenic acid, quinic acid, malic acid, glucose and sucrose; in particular, using1H- NMR quantitation (internal standard dimethyl sulphone), the stabilised extract is characterised by a chlorogenic acid content ranging from 1.5-2.5%, a quinic acid content ranging from 3.5-4.5%, a malic acid content ranging from 3.5-4.5%, a glucose content ranging from 2.5-3.5%, and a sucrose content ranging from 3.5-4.5%

[0023] Typically, the stabilised extract is in powder form and is characterised by a total ash content, expressed as a percentage of the total weight of the stabilised extract, determined by the method according to the European Pharmacopoeia version 11 (para. 2.4.16), ranging from 15-25%, preferably 17-21%.

[0024] Typically, the stabilised extract comprises an amount of lipophilising agent ranging from 10-30%, preferably ranging from 10-28%, 12-25%, 15-22% or 16-20%.

[0025] Typically, the lipophilising agent is selected from the group consisting of hydrogenated cottonseed oil, hydrogenated palm oil, glyceryl stearate, glyceryl behenate, glyceryl palmitostearate, glyceryl myristate, stearic acid, palmitic acid, stearyl alcohol, cetyl alcohol, cetostearyl alcohol, lauroyl polyoxylglycerides, stearoyl polyoxylglycerides, carnauba wax, white wax, yellow wax and microcrystalline wax; preferably, the lipophilising agent is glyceryl dipalmitostearate or glyceryl behenate.

[0026] Typically, the stabilised extract has a moisture content, measured by Karl Fischer titration or 'H-NMR, not exceeding 5%; more preferably, the content ranges from 1-4%.

[0027] With special reference to the second aspect of the invention, the halophyte extraction process comprises the following steps: a) extracting the halophyte with a solvent A to give a solution A; b) concentrating solution A to give a solution B; c) adding a lipophilising agent to solution B to give a mixture C; d) removing the solvent from mixture C to give a composition according to the present invention.

[0028] Typically, in step a), the halophyte is Crithmum maritimum.

[0029] Typically, in step a) the solvent is selected from the group consisting of a straight or branched C1-C5 alcohol, ethyl acetate, acetone, dichloromethane, water and mixtures thereof; preferably, the solvent is water or a mixture of water and a straight or branched C1-C5 alcohol; more preferably, the solvent is water or a mixture of water and an alcohol selected from methanol, ethanol and isopropanol, wherein the water-to-alcohol ratio ranges between 95:5 and 1 : 1; even more preferably, the solvent is water or a 7:3 ethanokwater mixture. Step a) can optionally be repeated 1 to 3 times.

[0030] Typically, solution A is heated at a temperature ranging from 60-100°C for 1-6 hours; preferably at a temperature ranging from 70-90°C for 2-4 hours.

[0031] Typically, in step b), solution B is obtained by concentrating solution A by low- pressure distillation. Solution B is characterised by a dry residue ranging from 10-25%.

[0032] Typically, in step c), the suspension is maintained under stirring for a time ranging between 15 and 90 minutes at a temperature ranging from 60-100°C; preferably for 45-60 minutes at a temperature ranging from 80-90°C. Moreover, in step c), the lipophilising agent is added in an amount such as to obtain a ratio between the amount of dry residue of solution B, and the amount of lipophilising agent, ranging from 10: 1 to 1 :2; preferably, the ratio is 4: 1.

[0033] Optionally, after step b), the process comprises a step b’) wherein solution B is cooled to a temperature ranging between -10 and 10°C, preferably from 4-8°C, to obtain a precipitate B’; step b’) is typically followed by a step b”) wherein precipitate B’ is removed by filtration or centrifugation.

[0034] The process optionally comprises, after step d), a further step e) involving mixing the composition with SiCh or MgCCh; SiCh or MgCCh is added in the amount of 2% of the weight of the stabilised extract.

[0035] Without being bound by the theory, the addition of a lipophilising agent reduces the hygroscopicity of the extract; in fact, dispersing the lipophilising agent in the extract “encapsulates” the extract, thus preventing absorption of moisture from the external environment. The reduction in hygroscopicity, due to the presence of the lipophilising agent, is particularly advantageous; in fact, the high level of hygroscopicity of the halophyte extracts, especially Crithmum maritimum, makes them difficult to formulate and reduces their stability, limiting the possibilities for their use in pharmaceutical, nutraceutical, veterinary and dietary compositions. In support of said argument, a hygroscopicity test was conducted which, as specified in paragraph 5.11 of the European Pharmacopoeia, involves maintaining the test samples for 24 hours at 25°C under 80% relative humidity conditions; at time zero the Crithmum maritimum extract (Sample 1) without lipophilising agent appears as a granular, paste-like, sticky, yellowish-brown solid, while the stabilised extract (Sample 2) has the appearance of a whitish -yellow powder; at the end of the test, Sample 1 is completely liquefied, while Sample 2, although it has absorbed moisture, still has a partly powdery appearance. The amount of moisture at the end of the test is 39% for Sample 1 and 34.6% for Sample 2; although the difference in moisture content may not seem significant, as it is influenced by the extreme humidity conditions required by the test used, the stabilised extract according to the invention is particularly advantageous because the presence of the lipophilising agent eliminates viscosity problems. In fact, a flowability test conducted according to the European Pharmacopoeia (paragraph 3.9.36) demonstrated that due to the reduction in hygroscopicity, the stabilised extract according to the present invention is considerably more flowable than an unstabilised Crithmum maritimum extract. The test involves inserting samples into a funnel placed above discs containing holes; the hole diameter of said discs becomes gradually smaller, and the flowability of the samples is inversely proportional to the hole size. As can be seen from Table 1,

[0036] Table 1 the stabilised extract according to the invention is considerably more flowable than the extract without lipophilising agent, because it can pass through a hole with a diameter 3 times smaller. The paste-like, sticky, granular consistency of the extract without excipients not only not enables the sample to pass through a hole with dimensions smaller than 36 mm, but makes the test difficult to conduct, because said extract has difficulty in exiting the funnel, as it sticks to the walls due to simple absorption of ambient moisture.

[0037] Table 2 shows a comparison between the physical characteristics and hygroscopicity, measured by thin-layer absorption, of an aqueous extract of rock samphire and an extract stabilised according to the present invention.

[0038] Table 2

[0039] The reduction in water absorbed by the extract with added glyceryl behenate, amounting to about 26-27%, is particularly advantageous; in fact, the stabilised extract, after absorption of ambient moisture, can be easily repulverised, whereas the aqueous extract is packed and can no longer be pulverised.

[0040] Experimental section

[0041] Methods

[0042] HPLC analysis method

[0043] The HPLC analysis was conducted with an HPLC apparatus consisting of a quaternary pump, a thermostated autosampler, a UV / VIS detector, and a Zorbax SB-C18 column (1 =250 mm ; i.d.. = 4.6 mm, particle size 5 pm) and a mixture of solvents A (water + 0.01% (v / v) trifluoroacetic acid) and B (acetonitrile + 0.01% (v / v) trifluoroacetic acid) as mobile phase (gradient elution).

[0044] NMR analysis method

[0045] The 1H-NMR analyses were conducted with an Agilent 400 MHz NMR instrument; spectra recorded in D2O at the temperature of 30°C, one column

[0046] Thin-layer absorption

[0047] An amount of the product ranging from 3 to 5 g was distributed in a suitable glass container (Petri dish) to create the most uniform possible thin layer with a thickness of about 5 mm. The product was exposed to ambient temperature and humidity, measuring the weight increase due to absorption of ambient moisture at preset intervals. The measurements were continued until a situation of equilibrium was reached.

[0048] Example 1

[0049] 100 g of aerial parts of Crithmum marilimum. ground with a 4 mm mesh, were suspended in 1500 ml of 70% ethanol and stirred at 60°C for 2 hours. The liquid extract was separated by filtration through a Buchner funnel to obtain about 1 L of a clear solution. The residue was resuspended in 1500 ml of 70% ethanol and stirred at 60°C for 2 hours. The liquid extract was separated by filtration through a Buchner funnel to obtain about 1.5 L of a clear solution. The two solutions were combined (total volume about 2.5 L, dry residue 26-33 g) and concentrated under vacuum to obtain an aqueous suspension, 10-15% dry residue. The concentrate was cooled to 4-8°C and maintained at said temperature overnight to allow precipitation of an insoluble pitch residue. The supernatant was decantated and filtered through a paper filter to remove the insoluble particles drawn into the decanted liquid. Biogapress Vegetal 297 ATO glyceryl stearate (6-7 g) corresponding to 20% (of the total dry residue) was added to the clarified solution, and the resulting suspension was heated under vacuum to obtain a soft mass which was dried at 65°C (60- 70°C) under vacuum for 24 hours. The procedure described was repeated 3 times, and the following amounts of stabilised extract were obtained: 33.90 g, 33.25 g, 25.36 g.

[0050] Example 2

[0051] 15 kg of aerial parts of Crithmnum marilimum. ground with a 10 mm mesh, were loaded into a reactor, suspended in 225 L of 70% ethanol, and stirred at 60°C (55-65°C) for 2 hours. The liquid extract was separated by centrifugation to obtain about 180 L of a clarified solution. The solid residue was resuspended in 225 L of 70% ethanol and stirred at 60°C (55-65°C) for 2 hours. The suspension was centrifuged to provide about 220 L of a clarified solution. The two clarified solutions were combined (total volume about 400 L, dry residue 3.79 kg) and concentrated to obtain an aqueous suspension, 29 kg, dry residue 14% (10-15%). The concentrate was cooled to 4-8°C and maintained at said temperature overnight to allow precipitation of an insoluble pitch residue. The supernatant was decanted and filtered through a paper filter (porosity 17-30 pm) to eliminate the insoluble particles in suspension (dry residue of clear solution: 3.79 kg). 0.95 kg (20% of total dry residue) of Biogapress Vegetal 297 ATO glyceryl stearate was added to the clarified solution, and the resulting suspension was heated under gentle stirring, maintaining it at 80-90°C for 45-60 minutes. The suspension was concentrated under vacuum to obtain a soft mass that was dried at 65°C (60-70°C) under vacuum for 24 hours, to obtain 4.68 kg of dried extract. The extract was coarsely ground, mixed with 2% SiCh Syloid™ (batch 20B0149500), and ground through a 2 mm mesh to obtain 4.40 kg of stabilised extract; the process yield was 29.3%.

[0052] Example 3

[0053] 500 g of aerial parts of Crithmnum marilimum. ground with a 10 mm mesh, was loaded into a percolator and extracted with water at 90°C; the resulting extract was concentrated, and Compritol™ (glyceryl behenate) was added in an amount equal to 20% of the dry residue of the extract. Said mixture was heated to 80°C, then concentrated and dried under vacuum; the stabilised extract was obtained with a yield of 52% of the biomass.

[0054] The same process was repeated 4 more times, using 100 g of biomass and a reactor for the extraction. The extracts were obtained with a yield of 38-45% of the biomass.

Claims

CLAIMS1. An extract of a halophytic plant stabilised with a lipophilising agent.

2. The extract according to claim 1, wherein the halophytic plant is selected from the group consisting of Arthrocnemum macrostachyum, Bassia spp., Beta vulgaris subsp. maritima, Cakile spp., Capparis spinosa, Cichorium spinosum, Chenopodium spp., Chenopodium album, Chenopodium quinoa, Crambe maritima, Cressa cretica, Crithmum maritimum, Cynara cardunculus, Descaurinia sophia, Diplotaxis spp., Eryngium maritimum, Halimione portulacoides, Inula crithmoides, Lepidium latifolium, Mesembryanthemum crystallinum, Nitrar ia spp., Nitraria schoberi, Nitraria sibirica, Plantago spp., Plantago coronopus, Plantago lanceolata, Portulaca spp., Portulaca oleracea, Salicornia spp., Salicornia herbacea, Salsola spp., Salsola soda, Suaeda spp., Suaeda fruticosa, Tetragonia decumbens, Tribulus terrestris, Tripolium pannonicum and Zygophyllum spp. (Zygophyllum fabago, Zygophyllum album).

3. The extract according to claim 2, wherein the halophytic plant is Crithmum maritimum.

4. The extract according to any one of the preceding claims, wherein the lipophilising agent is selected from the group consisting of hydrogenated vegetable oils, fatty alcohols, fatty acids, fatty acid monoglycerides, fatty acid diglycerides, fatty acid triglycerides, polyoxylglycerides and waxes.

5. The extract according to claim 4, wherein the lipophilising agent is selected from the group consisting of hydrogenated cottonseed oil, hydrogenated palm oil, glyceryl stearate, glyceryl behenate, glyceryl palmitostearate, glyceryl myristate, stearic acid, palmitic acid, stearyl alcohol, cetyl alcohol, cetostearyl alcohol, lauroyl polyoxylglycerides, stearoyl polyoxylglycerides, carnauba wax, white wax, yellow wax and microcrystalline wax.

6. The extract according to any one of the preceding claims, wherein the amount of lipophilising agent is in the range of 10-30% of the total weight of the stabilised extract.

7. A process for preparing an extract according to any one of the preceding claims comprising extracting a halophytic plant, followed by adding a lipophilising agent.

8. The process according to claim 7, comprising the following steps:(a) extraction of a halophytic plant with a solvent A to give a solution A;(b) concentration of solution A to give solution B;(c) addition of a lipophilising agent to solution B to give a mixture C;(d) removal of the solvent from mixture C.

9. A pharmaceutical or veterinary composition comprising the extract according to any one of claims 1 to 6 and at least one pharmaceutically acceptable excipient and / or carrier.

10. A dietary supplement comprising the extract according to any one of claims 1 to6 and at least one pharmaceutically acceptable excipient and / or carrier.

11. An extract according to any one of claims 1 to 6 or a composition according to claim 9 or a dietary supplement according to claim 10 for use in the treatment and / or prevention of disorders of the gastrointestinal system.

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