HK1118702B - Production of glucosamine from plant species - Google Patents

Description

Production of glucosamine from plant species

The present invention relates to a process for the preparation of a crude plant material having a glucosamine content equal to or greater than 0.5% (wt) of dry matter of the plant.

Background

Use of glucosamine

The use of pure glucosamine in the treatment of arthrosis is widely described in the patent as well as in the scientific literature, where it is usually combined with other compounds or extracts from various natural sources. Pure glucosamine is added as glucosamine hydrochloride or glucosamine sulfate and results from shellfish hydrolysis. For example, WO2000/0074696 describes "herbal compositions for treating inflammation or degeneration of joint tissue comprising glucosamine and an herbal composition of tripterygium wilfordii, ligulum lucidum and/or oremycin schmidti for treating inflammation or degeneration of joint tissue" wherein pure glucosamine is mixed with a botanical product. Other patents relate to compositions of plant sugars as dietary supplements (EP1172041 or EP923382) in which glucosamine is derived from chitin, i.e. also from shellfish hydrolysis.

The use of glucosamine as an anti-osteoarthritic has been extensively studied over the last decade. Glucosamine is suspected to be the only active compound against joint diseases such as osteoarthritis (only recently symptomatic therapy, such as non-steroidal anti-inflammatory drugs, has been found to be effective).

Glucosamine has also been shown to prevent cartilage degradation by inhibiting the production of MMPs (matrix metalloproteinases), such as MMP1, MMP3, and MMP 13. Interestingly, glucosamine is also associated with the aging process of the skin, which is mainly characterized by a continuous loss of elasticity and a loss of moisturization of said skin. Skin aging is manifested by major structural and compositional changes. Most notably, aged skin has less collagen and glycosaminoglycans than young skin. Glycosaminoglycan molecules produced by the skin include hyaluronic acid (poly-d-glucuronic acid-d-acetyl-d-glucosamine), chondroitin sulfate, and dermatan sulfate. Skin cells produce greater amounts of hyaluronic acid with exfoliation. Hyaluronic acid has a large hydration capacity.

Inhibition of MMP-1 is linked to inhibition of polysaccharide/collagen degradation and, therefore, to skin aging: MMP-1 can be induced by UV light and is considered to be a marker for skin aging. In US2002/119107, the invention is based on the selective inhibition of MMP-1, which claims topical compositions for protecting human skin from collagen degradation. US2004/037901 claims a regimen for inhibiting the negative signs of the effects of skin aging comprising an extract from rosemary plant that inhibits the expression of metalloproteases.

Glucosamine has been shown to significantly improve skin dryness and exfoliation. Glucosamine increases the moisture content of the skin and improves smoothness. These findings indicate that long-term intake of glucosamine is effective in improving moisture content and smoothness of skin.

Oral supplement containing glucosamine has been shown to cause a reduction in the number of visible wrinkles (34%) and fine lines (34%) in the group of women ingesting the supplement. The use of an oral supplement containing glucosamine, minerals and various antioxidant compounds can improve the appearance of visible wrinkles and fine lines.

US6413525 describes a method for substantially exfoliating skin. In particular, the invention relates to a composition for topical application containing an amino sugar in the form of N-acetylglucosamine: after exfoliation exposes young skin cells, they produce a greater amount of hyaluronic acid, which is a glycosaminoglycan composed of chains of alternating repeating molecules of D-glucuronic acid and N-acetyl-D-glucosamine. N-acetyl-D-glucosamine is known to be the rate limiting factor in the production of hyaluronic acid in living cells. Topical application of glucosamine aids in the sustained production of hyaluronic acid.

Other topical compositions containing N-acetyl-D-glucosamine are also disclosed, for example, in JP59013708 (softening and moisturizing skin) or in us patent No.5866142 (compositions for exfoliating skin).

Sources of glucosamine

Glucosamine, 2-amino-2-deoxy-D-glucose, is a naturally occurring derivative of fructose and is an essential component of glycoproteins and proteoglycans, an important component of many eukaryotic proteins. This is the basic component of mucopolysaccharides and chitin. Glycosaminoglycans (mucopolysaccharides) are large complexes incorporated into connective tissue, skin, tendons, ligaments, and cartilage.

Industrial sources of glucosamine

Industrial glucosamine is a pure compound obtained from the acidic hydrolysis of chitin (complex sugar derived from N-acetyl-D-glucosamine) from shellfish. For example, us patent 6486307 describes an improved process for the acidic hydrolysis of chitin: a process for preparing glucosamine hydrochloride from chitin by grinding chitin to an extremely fine size and digesting it with concentrated hydrochloric acid.

Glucosamine can also be made from the enzymatic hydrolysis of shellfish. For example, U.S. patent 5998173 describes a novel process for the direct preparation of N-acetyl-D-glucosamine from chitin by hydrolyzing chitin of shellfish shells using the whole chitinase family of enzymes.

Patents have also been filed which protect microbial fermentation processes in which cultivated microorganisms biosynthesize glucosamine. For example, US6372457 describes methods and materials for the preparation of glucosamine by fermentation using genetically modified microorganisms.

All of these methods involve the preparation of pure extracted glucosamine that competes with shellfish extracts.

In WO2005/053710, it has been found that glucosamine can be formed from several crude plant materials following a special drying process, thus obtaining glucosamine contents of 150 to 1000 mg per kg dry weight.

Summary of The Invention

In a first aspect, the present invention describes a novel process for obtaining glucosamine from plants by adding glucosamine precursors after harvesting of the plant material and before or during the heating process, thereby obtaining a plant raw material with a glucosamine content higher than 0.5% (5 g/kg dry weight) of dry matter. The present invention thus enables a much higher glucosamine content in plant material than previously described in the prior art (e.g. as reflected in WO 2005/053710). Less crude plant material or plant extract is therefore required to achieve the doses of glucosamine active described in the literature. Therefore, the process is more useful on an industrial scale.

The above precursors can be added after the harvesting of fresh or previously dried plant material or in the derived plant extract, during the heating of the plant material or derived plant extract, or during the preparation of an aqueous extract from the plant material.

Detailed Description

In the present description, the expression "heating" (and the derivative "heated") must be understood as a heating process at 70-110 ℃ for more than 10 hours, preferably for less than 1 week. This heating process can be described as a drying process. The heating process may also include liquid impregnation that occurs under the same temperature and time conditions, instead of drying.

In the present description, "free glucosamine" must be understood as non-polymeric glucosamine.

In the present description, "high amounts of glucosamine" must be understood as amounts of glucosamine higher than the traces of glucosamine, greater than the amounts in the corresponding fresh (not dried) material and higher than any content described in the literature or in the patent. It is to be understood that glucosamine is present in an amount higher than 5 g/kg dry matter of plant raw material, preferably higher than 20 g/kg dry matter of plant raw material, most preferably higher than 40 g/kg dry matter of plant raw material.

In this specification, "plant" and "plant material" are considered synonyms. By "plant", "plant material" or "plant extract" it must be understood any plant material capable of producing glucosamine according to the heating method of the invention, and any type of plant extract obtained by any extraction procedure known to the skilled person from said plant material capable of producing glucosamine according to the heating method of the invention. For example, a plant comprising an amount of glucosamine can be a dried or rehydrated plant material that has undergone the method of the invention. The plant extract comprising an amount of glucosamine may be an aqueous solution extracted from said plant subjected to the method of the invention.

For the first object of the present invention, the plant or plant extract is processed according to the present invention to contain a high amount of natural free glucosamine.

In a preferred embodiment, the plant or plant extract is from any part of the plant, such as a leaf, tuber, fruit, seed, root, grain (grain) or cell culture. After the controlled heating process of the plant raw material, the plant or plant extract may be in the form of a dried lyophilized extract or glucosamine-rich fraction of the leaves, roots and/or fruits, depending on the source of the plant or fresh plant.

Selecting a plant or plant extract according to its ability to produce free glucosamine by the method of the invention; in particular, it may be selected from plant species containing sucrose, fructose or inulin, such as chicory (Cichorium), Daucus, Helianthus (Helianthus), Beta (Beta).

In a most preferred embodiment, the plant material or plant extract may be derived, for example, from chicory (Cichorium intybus)) roots, carrots (carrot carota), tubers of jerusalem artichoke (Helianthus tuberosum), sugar beet (Beta vulgaris) roots.

In one embodiment, fresh plant material or plant material may first be completely or partially dried and then rehydrated, after which both steps may be processed according to the new invention to obtain plant material with a high glucosamine content.

In a preferred embodiment, fresh plant material is used.

As disclosed in WO2005/053710, the drying process is one way to obtain large amounts of glucosamine in plants: using the drying process described in WO2005/053710, a content of about 500 mg per kg dry matter of chicory root, 100 mg per kg dry matter of carrot root, or 50 mg per kg dry matter of Jerusalem artichoke tuber or beetroot can be obtained.

Depending on the plant species and plant organs, the fresh or dried or rehydrated plant starting material is heated using a liquid dipping or drying process at a temperature below 110 ℃, preferably 70 to 110 ℃, most preferably 70 to 91 ℃ or less for more than 10 hours, preferably less than 1 week, preferably 10 to 120 hours, for example 12 to 50 hours. If the temperature and/or heating time is too low and/or too short, the production of glucosamine is not efficient or very low, which results in an economically unfeasible process. Conversely, if the temperature and/or heating time is too high and/or too long, glucosamine is produced, but then gradually degraded.

Thus, the temperature and time are chosen so as to obtain a glucosamine content of at least 5 g glucosamine per kg dry matter of the corresponding plant material subjected to the heating process.

The most preferred example involves drying in an oven at 85 ℃ for 48 to 72 hours.

According to the invention, the same process is used, but with the difference that the plant material or plant extract is first contacted with the glucosamine precursor. As a result, the obtained amount of glucosamine is much higher than in WO 2005/053710. In fact, according to the invention, the glucosamine content of the plant is higher than 10 g/kg dry matter of chicory roots and higher than 15 g/kg dry matter of carrot roots or beetroot.

The precursors of glucosamine used according to the invention are compounds that enable the sugar-nitrogen compound condensation that is required for the formation of glucosamine to take place. They preferably consist of ammonium salts. Examples of such ammonium salts are ammonium nitrate, ammonium sulfate, ammonium acetate, ammonium dihydrogen phosphate or glutamine, etc. Preferred precursors of glucosamine are ammonium sulfate and ammonium nitrate, which have shown surprisingly good results in the process of the present invention.

In a preferred embodiment, the precursor is added to the freshly harvested material shortly before the heating process. The skilled person will know how to select the amount of glucosamine precursor on the plant material or plant extract, but in the most preferred embodiment the final amount of ammonium sulphate is between 1 and 8%, preferably 4% of the fresh weight of the plant raw material.

In a most preferred embodiment, the glucosamine precursor is added to the plant material or plant extract as a solution applied by spraying or by soaking. For example, 200 ml of aqueous solution (4M) is sprayed for 5 to 30 minutes or soaked in the same solution for several minutes to several hours. These examples are not to be construed as limiting the invention in any manner. That is, one skilled in the art will recognize many variations in this example to cover a wide range of processing and mixtures to rationally adjust the natural yields of the compounds of the invention for various applications.

For the final process leading to glucosamine production, a suitable method for preparing plant material without addition of precursors is described in WO 2005/053710: the plant material is harvested, cut and dried in an oven or in an industrial dryer at a temperature below 110 ℃, preferably 80 to 105 ℃, most preferably 91 ℃ or less for more than 10 hours, and preferably less than 1 week, preferably 10 to 120 hours, for example 12 to 50 hours, depending on the plant species and plant organs. While not wishing to be bound by theory, it is believed that it is preferred that the plant material is, for example, cut into slices or cubes, preferably having a maximum width of 5 mm. The inventors do believe that this is important for the present invention to achieve optimal thermodynamic exchange.

After harvesting, the addition of glucosamine precursors before or during the heating process can significantly increase the above reaction, from several hundred mg glucosamine/kg dry weight without precursors to at least 5 g glucosamine/kg dry matter of the corresponding plant material.

The process of the present invention directly produces glucosamine in free form. Without wishing to be bound by theory, it is believed that at least half of the glucosamine produced by the process is believed to be in free form, and even that nearly all of the glucosamine produced is in free form. Indeed, according to the methods of the present invention, it is believed that at least 50%, at least 70%, and even at least 90% of glucosamine is produced in free form. This is another advantage of the present invention over known techniques for preparing glucosamine, where a hydrolysis step is mandatory to release free glucosamine from complex molecules such as chitin, glycoproteins or proteoglycans.

Pre-extraction of the starting material with ammonium sulfate prior to the heat treatment produced an extract that was more enriched in glucosamine (example 8).

Glucosamine can first be extracted from plant material, alone or in combination with other compounds, such as inulin or Fructooligosaccharides (FOS).

Examples

The following examples are illustrative of some of the products and methods of making them that fall within the scope of the invention. They are not to be considered as limiting the invention in any way. Modifications and variations of the present invention are possible. That is, those skilled in the art will recognize many variations in these examples to cover a wide range of formulations, ingredients, processing, and mixtures to rationally adjust the natural yields of the compounds of the invention for various applications.

Example 1: fresh sugar beet

And (3) drying: after harvesting, 200 g (fresh weight) of beet (Beta vulgaris) roots were cut into 1 × 1 × 1 cm squares. Spray 25 ml ammonium sulfate (NH) on all surfaces of the dice₄)₂SO₄Solution, 4M (Prolabo cat # 21332.293), the cubes were then dried in an oven at 91 ℃ for 56 hours.

And (3) analysis:

extraction of glucosamine:

2 g of ground and specially dried beetroot are extracted with 20 ml of water at room temperature for 1 minute. The solution was filtered by Schleicher&Filtration on Schultz (n ℃ 597), or centrifugation. The purification step of the solution was carried out using a cation exchange column (Oasis cartridge WATERS, model MCX, cat # 186000776). The basic compound trapped on the matrix is washed with MeOH/NH₄OH 2% (v/v) elution. After filtration, aliquots were used for direct injection on LC system (DIONEX).

Separation:

analysis was performed using an ion exchange PA1 column (4 x 250 mm) and DIONEX DX 500 apparatus with HPAE/PED system.

The procedure is as follows:

elution (%)

Time (minutes)	H2O	0.1M NaOH	0.25 NaOH	Review the following
					0	85	15	0	Balancing
60	85	15	0
					60.1	0	0	100	Washing machine
70	0	0	100
					70.1	85	15	0	Balancing
90	85	15	0

Flow rate: 1 ml/min. Injection volume: 20 microliter. The standard is as follows: glucosamine from Sigma (catalog number: G4875)

Under these conditions, glucosamine has a retention time of about 11 minutes and is easily detected for further quantification in properly processed sugar beet extracts. In this example a concentration of 16 g/kg dry weight was quantified by this method, instead of less than 300 mg/kg without precursor and less than 1 mg/kg without heating process.

Confirmation of glucosamine Presence:

three different quantification techniques have been evaluated in order to confirm the presence of glucosamine in sugar beet plant extracts.

Thin Layer Chromatography (TLC)

Pure glucosamine and plant extracts were analyzed on HPTLC (high Performance thin layer chromatography) silica gel plates (Merck, Cat. No. 1.05642) using ethyl acetate/MeOH/water (50/50/10; V/V/V) as eluent. After elution, the plate was sprayed with an acetic acid solution containing the ketone 1% ninhydrin and heated at 120 ℃ for 10 minutes. One pink/blue dot is shown for the reference and extract at the same rate factor (Rf).

Chemical degradation

In the presence of ninhydrin, oxidative deamination with glucosamine occurs, which results in the release of arabinose that is readily detected by conventional sugar LC analysis. The presence of arabinose in the control and chicory extracts was clearly confirmed.

Derivatization of glucosamine (derivitization)

Purification compounds and plant extracts were derivatized using reverse phase chromatography using a pre-column with phenyl isothiocyanate and UV detection (. lamda. 254 nm) as described by Zhongming et al, "Determination of nucleotides in raw materials, analysis form and plasma use pre-column with UV HPLC. in J. of pharmacy. and biomed. analysis, 1999(20), 807-814".

The corresponding peaks of derivatized glucosamine as well as of the pure compound were detected in chicory extracts.

Mass spectrometric analysis

Plant extracts were analyzed by electrospray mass spectrometry in positive ionization mode to confirm the presence of glucosamine. The mass spectrometer was a time-of-flight instrument (LCT from Micromass with Z-spray interface). Standard glucosamine generates an ion at m/z 180.0887. Such ionic fragments are found in the plant extract under analysis.

Example 2: fresh carrot (Daucus carota) root

200 g (fresh weight) of carrot roots are cut into 1X 1 cm cubes and then sprayed with the ammonium sulphate solution of example 1. The cubes were then dried in an oven at 91 ℃ for 37 hours. Extraction and analysis were performed as in example 1, resulting in a glucosamine concentration of 15 g/kg dry weight, instead of less than 190 mg/kg dry weight without precursor and less than 1 mg/kg without heating process.

Example 3: fresh chicory (Cichorium intybus) root

200 g (fresh weight) of chicory roots are cut into 0.5X 0.5 cm cubes. The cubes were soaked in ammonium sulphate solution (4M) for 30 minutes, or sprayed with ammonium sulphate solution, or mixed with ammonium sulphate solution at 85 ℃ for 8 hours. The cubes were then dried in an oven at 91 ℃ for 40 hours. The extraction and analysis was performed as in example 1, resulting in a glucosamine concentration of 43 g/kg dry weight, instead of less than 900 mg/kg dry weight without precursor and less than 10 mg/kg without heating process or in commercially dried chicory roots.

Example 4: dried chicory root

Fresh chicory roots were cut into cubes (1X 1 cm), dried using the current procedure (inlet air 115 ℃ C., 2 hours in a fluid bed dryer) and then stored at room temperature. When glucosamine generation is desired, the dried cubes are soaked in sodium sulfate solution (4M) for 1 hour, the cubes are rehydrated and the precursor is introduced. The cubes were then dried in an oven at 85 ℃ for 43 hours. Extraction and analysis were performed as in example 1, resulting in a glucosamine concentration of 11 g/kg dry weight, instead of less than 190 mg/kg dry weight without precursor and less than 1 mg/kg without heating process.

The invention can therefore be used for plant material which has been stored in dry form for a prolonged period of time.

Example 5: dried powder chicory root

1.5 g of the dried powder was suspended in an Erlenmeyer flask with 100 ml of ammonium sulfate (3M), shaken vigorously and incubated for 30 minutes at 80 ℃. The solution was then dried in an oven at 85 ℃ for 50 hours. Extraction and analysis were performed as in example 1, resulting in a glucosamine concentration of 110 g/kg dry weight.

This is another example of plant material that the present invention can therefore be used for over long term storage in dry form.

Example 6: dried chicory root in its entirety

Freshly harvested 10 whole chicory roots were stored at ambient temperature for three days. The whole root was then soaked in ammonium sulphate solution (4M) for 24 hours. The roots were then cut into 0.5X 0.5 cm cubes and then processed (dried) as in example 3 to yield a glucosamine concentration of 44 g/kg dry weight.

Example 7: fresh chicory root dices treated in liquid medium

100 g of fresh chicory roots are cut into dices (0.5X 0.5 cm), suspended with 200 ml of ammonium sulfate (4M) in an Erlenmeyer flask which is open or plugged with a cellulose stopper and shaken vigorously. The solution was then heated in an oven at 85 ℃ for 60 hours. Extraction and analysis were performed as in example 1, resulting in a glucosamine concentration of 30 g/kg fresh weight. It is therefore also possible to apply the invention under wet heating conditions.

Example 8: glucosamine-rich extract

100 g of fresh chicory roots are cut into dices (0.5X 0.5 cm), suspended in an Erlenmeyer flask with 200 ml of ammonium sulfate (4M) and shaken vigorously. The solution was then filtered and the resulting eluate was dried in an oven at 85 ℃ for 60 hours. Extraction and analysis were performed as in example 1, resulting in a glucosamine concentration of 100 g/kg dry weight.

Claims (7)

1. Method for producing glucosamine from plants, wherein fresh plant material, rehydrated dried plant material or plant extract is heated at a temperature between 70 and 110 ℃ for more than 10 hours but less than 1 week, characterized in that an ammonium salt is added to the fresh plant material, rehydrated dried plant material or plant extract before or during the heating step, wherein the plant species belongs to the genus Cichorium, Daucus, Helianthus or beta.

2. The method of claim 1, wherein the ammonium salt is ammonium sulfate.

3. A method according to claim 1 or 2, wherein the plant is chicory, carrot, Jerusalem artichoke or beet.

4. A process according to claim 1 or 2, wherein the plant material or plant extract is from chicory root, carrot, jerusalem artichoke tuber or beetroot.

5. A method according to claim 1 or 2, wherein the plant or plant extract comprises at least 5 g glucosamine per kg dry weight of plant material.

6. A method according to claim 5, wherein the plant or plant extract comprises more than 20 g glucosamine/kg dry weight of plant material.

7. A method according to claim 6, wherein the plant or plant extract comprises more than 40 g glucosamine/kg dry weight of plant material.