TWI533875B - Rutin-rich extract and method of making same - Google Patents

Description

Eucalyptus extract and preparation method thereof

A method for obtaining a rutin-rich extract from a plant and a eucalyptus-rich extract of Uncaria elliptica thus obtained.

Mutin is a natural flavonoid used as a starting material for Troxerutin, which increases the tolerance of microvessels and reduces the permeability of venous blood vessel walls. Clinical studies have shown that treatment of patients with rutinu derivatives (venoruton) leads to improved post-thrombotic syndrome. See Panminerva Medicine, 53, page 13, 2011. Another study found that rutin is one of the most effective antithrombotic agents. See The Journal of Clinical Investigation, 122, No 6, 2012. As a starting material for pharmaceuticals, ruthenium is becoming more important.

In order to extract and purify rutin from a variety of plant sources (for example, Sophora Japonica (Asia), Fava d'anta (Brazil) and Ellipticus (Brazil)), water and methanol solvents are typically used. However, in fact, the eucalyptus extracts from such plant sources have significant disadvantages, such as rich brown color and high iron content (compared to eucalyptus extracts from other sources). This brown is attributed to the pigment in the plant.

It is very difficult to eliminate this brown and reduce the iron content by conventional extraction and/or purification methods because iron is shown to be tightly incorporated into the pigment. Attempts have been made to overcome these disadvantages. However, these attempts have made it difficult to reduce the iron that has been incorporated into the pigment while maintaining a good muskene yield.

There is a need for an economic process suitable for the large-scale production of eucalyptus extracts having high arsenic content, low iron content and reduced coloration.

The present invention is based on the unexpected discovery that a combination of a particular alcohol and an acid can be used to obtain a low iron content and coloured eucalyptus extract from plants.

Additionally, a method of obtaining a rutin-rich extract from a plant is described herein. The method comprises dissolving a rutin-containing plant extract in methanol or ethanol to obtain a crude rutin extract solution, wherein the solid rutin-containing plant extract comprises 90% to 95% by weight of rutin and 100 to 300 Ppm of iron; reducing the volume of the crude rutin extract solution to obtain a concentrate; by using an acid (1-15% by weight of the rutin-containing plant extract), and selective C1-C4 An alcohol, water or both is added to the concentrate to obtain a mixture, wherein the musk-containing plant extract constitutes 8% to 30% (w/v) of the mixture and the acid is selected from ascorbic acid, citric acid, a group consisting of tartaric acid, gallic acid, and malic acid; allowing the mixture to stand to form a precipitate; and separating the precipitate, thereby obtaining an iron-rich eucalyptus extract containing less than 20 ppm.

The musk-containing plant extract can be obtained by extracting an elliptic leaf vine, eucalyptus or Fava d'anta with water, an alcohol or an aqueous alcohol solution, such as a coarse precipitate.

In certain embodiments, the acid is added in an amount of from 1 to 10% by weight of the musk-containing plant extract, for example, 1-5% or 2-4%. In the case of adding water and a C1-C4 alcohol, the ratio of the water to the C1-C4 alcohol can be 1:1. To 5:1, for example, 1:1 to 3:1. The water and the C1-C4 alcohol may be added separately, simultaneously or in the form of an aqueous solution of a C1-C4 alcohol.

Similarly, a feature of this article is a eucalyptus extract of Uncaria sylvestris, which contains 97% to 99% of rutin (as measured by HPLC) and less than 20 ppm of iron. The extract is at 550. The absorbance at the nano is less than 0.2. The extract may also contain from 5% to 10% by weight of water. It can be prepared by the above method.

The details of one or more specific embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and appended claims.

A method of obtaining a rutin-rich extract from a plant source using a novel combination of an alcohol and an acid is described herein. The eucalyptus extract obtained by the method has significantly reduced the coloration and iron content as compared to the extract prepared by the conventional method.

The solid gum-containing extract can be prepared from a variety of plant sources using conventional methods known in the art, for example, Eucalyptus (Asia), Fava d'anta (Brazil), and Ellipticus (Brazil). Such methods include, but are not limited to, soaking, heating, shaking, stirring, or injecting plant material (eg, stems and leaves) in a variety of solvents. The plant material used may be in the form of a whole dried plant, crushed plant or powder. The solvent includes water, an alcohol (for example, methanol) or an aqueous alcohol solution. After extraction, the extract solution can be separated from the solid by screening, filtration, pressurization or other suitable technique. The filtrate can be concentrated and cooled to allow A precipitate formed, thus obtaining a solid rutin-containing extract. For a description of various extraction methods, see, for example, Chinese Patent 1013579; US 2006/0099690; and Dighe et al, International Journal of Pharma and Bio Sciences, 2(1): 750-757 (2011).

Typically, the solid phase-containing extract prepared by these methods has a high iron content (for example, 100-300 ppm) and a dense coloring (for example, yellow or brown), particularly in extracts derived from Uncaria in. The rutin can constitute from 85% to 90% by weight of these solid rutin-containing extracts.

The thus prepared solid rutin-containing extract may be dissolved in an alcohol solvent (preferably methanol or ethanol) to obtain a crude rutin extract solution. For example, 0.05 to 0.13 grams of solid extract can be dissolved per 1 milliliter of solvent. The crude extract solution can be further concentrated, for example, at atmospheric pressure to reduce its volume by, for example, 4 to 10 times. The final volume of the crude extract solution can be predetermined according to the desired volume and composition of the mixture described below.

An acid and a selective C1-C4 alcohol (for example, methanol, ethanol, propanol or n-butanol) are added to the above crude rutin extract solution to obtain a mixture. Water can also be included in the mixture.

The acid used is selected from the group consisting of ascorbic acid, citric acid, tartaric acid, gallic acid and malic acid. It is added to the crude rutin by about 1-15% (for example, 1-10%, or 1-5%, based on the weight of the solid rutin-containing extract used to prepare the crude rutin extract solution) Extract solution.

The mixture must contain at least one C1-C4 alcohol. For example, as described above, the crude rutin extract solution can be prepared from the solid rutin-containing extract using methanol or ethanol (i.e., C1-C4 alcohol). No need to treat the crude rutin The extract solution was added with additional C1-C4 alcohol. In some cases, in order to obtain a mixture comprising 8% to 30% (w/v) of the solid rutin-containing extract, C1 may be added, for example, 2-12 times (v/w) of the solid rutin-containing extract. - C4 alcohol, aqueous solution of C1-C4 alcohol or C1-C4 alcohol and water. The additional C1-C4 alcohol added may be the same or different from the alcohol used to prepare the crude rutin extract solution.

The rutin is present in different hydrates such as monohydrate, dihydrate and trihydrate. Anhydrates and monohydrates are known to be unstable. Therefore, it is preferred to prepare a relatively stable dihydrate or trihydrate by hydration with water during the purification process. Further, water may be added to the crude rutin extract solution. It can be added separately. It can also be added in the form of a C1-4 aqueous alcohol solution. The ratio of the water to the C1-C4 alcohol may be from 1:1 to 5:1, for example, from 1:1 to 3:1. As mentioned above, the final mixture preferably comprises from 8% to 30% (w/v) of the solid phase-containing extract.

Then, the mixture containing the crude rutin extract solution, the C1-C4 alcohol, the acid, and the selective water is allowed to stand for precipitation, for example, to form crystals. For example, the mixture can be further concentrated, and the concentrated mixture is stirred and allowed to cool to form a precipitate. The precipitate can be collected by any suitable technique, for example, filtration, rinsing with alcohol and/or water, and drying.

The precipitate (i.e., the rutin-rich extract) may comprise 97% to 99% of rutin (as measured by HPLC) and less than 20 ppm of iron (i.e., less than 10 ppm). It also has a low coloration, for example, the extract has an absorbance below 0.25 at 550 nm. It may also contain from 5% to 10% by weight of water. The eucalyptus extract may have any particle size, for example, 5 microns or less. For example, it can have nano-sized particles.

The eucalyptus extract prepared by the above method is useful for a variety of applications. It can be formulated into a pharmaceutical composition, for example, a drug. It can also be used as a nutritional supplement or as a food additive.

The following specific examples are to be construed as illustrative only and in no way limiting the remainder of the disclosure. Without further elaboration, the skilled artisan can use the invention to its fullest extent in accordance with the description herein. All publications cited herein are hereby incorporated by reference in their entirety.

The iron content, purity and absorbance values described in the following Examples 1-4 were obtained by the following procedure:

(1) Iron content

Accurately obtained 1.0 gram of sample and sulfated. 2 ml of hydrochloric acid was added to the residue. The sample was then dried and heated in boiling water after the addition of 10 ml of water and a drop of hydrochloric acid. After cooling, the volume was accurately adjusted to 10.0 ml and then filtered through a 0.45 micron filter. The iron concentration in the sample solution was measured by ICP analysis.

(2) Visible light absorption

A 1.0 gram sample was dissolved in 15 ml of dimethylformamide (DMF). After the volume was adjusted to 20.0 ml with DMF, the solution was filtered through a 0.45 micron filter. At 550 nm, the absorbance of the filtered sample was measured using a path length of 1.0 cm.

(3) HPLC analysis

HPLC analysis was carried out according to liquid chromatography of the EP monograph rutin trihydrate and related substances. Briefly, the reagents and conditions set forth below were used: Test solution: 0.10 g of the substance to be tested was dissolved in 20 ml of methanol and diluted to 100.0 ml with phase B.

Reference solution (a): 10.0 mg of rutin trihydrate CRS was dissolved in 10.0 ml of methanol.

Reference solution (b): 1.0 ml of the reference solution (a) was diluted to 50.0 ml with phase B.

Column (LiChrospher 100 RP-8, C8, 4.0x250 mm, Agilent):

- Size: 1 = 0.25 m, i.d. = 4.0 mm,

- Static phase: octyl sulfonyl group gel for chromatography (5 micron)

- Temperature: 30 ° C

Phase:

- Phase A: Mix 5 volumes of tetrahydrofuran with 95 volumes of 15.6 g/L sodium dihydrogen phosphate solution and adjust to pH 3.0 with phosphoric acid.

- Phase B: Mix 40 volumes of tetrahydrofuran with 60 volumes of 15.6 g/L solution of sodium dihydrogen phosphate to adjust to pH 3.0 with phosphoric acid.

Gradient timetable

Flow rate: 1 ml/min.

Detection: The spectrophotometer is at 280 nm.

Injection: 20 microliters.

The acids and alcohols used in the following Examples 1-4 were obtained from Wako Pure Chemical Industries, Ltd. (Osaka): ascorbic acid (minimum 99.5% grade 1); citric acid (minimum 98.0% grade); malic acid (minimum) 99.0% premium); gallic acid (98.0-103.0% grade 1); tartaric acid (minimum 99.0% grade 1); methanol (minimum 99.5% grade 1); ethanol (minimum 99.5% grade); n-propanol (minimum 99.7) % extra grade); n-butanol (minimum 99.0% premium).

Example 1: Preparation of crude rutin extract solution

The stems and leaves of Uncaria serrata were extracted with water and/or alcohol using a conventional procedure to obtain a solid rutin extract. Three groups of solid rutin-containing extracts (50 g each) were dissolved in 600 ml of methanol and stirred at 30-35 ° C for two hours. Each solution was filtered and concentrated under atmospheric pressure to give 300 ml of a crude rutin extract solution.

The solid-containing musk extract thus prepared contains 85-90% (w/w) of rutin and 100-200 ppm of iron. It develops a dark brown color when dissolved in an organic solvent and an average absorbance of 0.8-1.3 at 550 nm in DMF. The data is summarized in Table 1 below.

Example 2: Effect of ascorbic acid in combination with various solvents (1) Methanol

Three sets of crude rutin extract solutions were concentrated under atmospheric pressure (each group was obtained by dissolving 50 g of the solid rutin-containing extract in 600 ml of methanol (MeOH)) to provide a volume of 300 ml. Each concentrate was allowed to receive 0, 1.0 or 5.0 grams of ascorbic acid and each was further concentrated to provide a volume of 150 milliliters. After crystallization by cooling, the purified rutin crystals were collected by filtration followed by washing with methanol. The purified rutin was dried at 60 ° C for 5 hours and analyzed as described above. The results are summarized in Table 2 below. The addition of ascorbic acid during the crystallization procedure reduces residual iron in a dose-dependent manner and reduces the absorbance at 550 nm.

(2) Ethanol

Two groups of 500 ml of the crude rutin extract solution (which was obtained by dissolving 25 g of the solid rutin extract in ethanol (EtOH)) were concentrated under atmospheric pressure to provide a volume of 100 ml. Each set of concentrate was allowed to receive 25 ml of water containing 0 or 0.5 grams of ascorbic acid. After crystallization by stirring and cooling, the purified rutin crystals were collected by filtration, followed by rinsing with 15 ml of 95% ethanol. The purified rutin was dried at 60 ° C for 5 hours and analyzed as described above. The results are summarized in Table 3 below. The effect of ascorbic acid is significantly less than when methanol is used during crystallization (see above).

(3) propanol

Two sets of crude rutin extract solutions were prepared by dissolving 25 grams of the solid rutin extract in 300 milliliters of MeOH. Each set of extract was concentrated under atmospheric pressure to provide a volume of 43 ml. Each set of concentrate was allowed to receive an aqueous solution of propanol containing 0 or 0.5 grams of ascorbic acid (n-propanol: water = 1:1; total 200 ml). After crystallization by stirring and cooling, the purified rutin crystals were collected by filtration. The rutin crystals were rinsed with 25 ml of n-propanol followed by water. The purified rutin was dried at 80 ° C for 6 hours and analyzed. The results of the analysis are summarized in Table 4 below. Ascorbic acid reduces residual iron and reduces the absorbance of the purified rutin at 550 nm.

(4) Butanol

Four crude rutin extract solutions were prepared by dissolving 25 grams of the solid rutin extract in 300 milliliters of MeOH. Each extract solution was concentrated under atmospheric pressure to provide a volume of 43 ml. Each concentrate was allowed to receive an aqueous solution of n-butanol (water: n-butanol = 133: 67 ml) containing 0, 0.25, 0.5 or 2.5 grams of ascorbic acid. After crystallization by stirring and cooling, the purified rutin crystals were collected by filtration, followed by rinsing with 25 ml of n-butanol followed by 100 ml of water. The purified rutin was dried at 80 ° C for 6 hours and analyzed as described above. The results of the analysis are summarized in Table 5. Ascorbic acid reduces residual iron and reduces the absorbance of the purified rutin at 550 nm.

Example 3: Effect of various acids in combination with aqueous butanol solution

Each of the seven crude rutin extract extract solutions was prepared by dissolving 25 grams of the solid rutin-containing extract obtained as described in Example 1 in 300 ml of MeOH. The extract was concentrated under atmospheric pressure to provide a volume of 43 ml each. Each concentrate was subjected to an aqueous butanol solution (water: n-butanol = 133: 67 ml) containing tartaric acid, citric acid, malic acid or gallic acid (the amounts are shown in Table 6 below).

After crystallization by stirring and cooling the concentrate, the purified rutin crystals were collected by filtration. The purified rutin was rinsed with 25 ml of n-butanol followed by 100 ml of water. The purified rutin was dried at 80 ° C for 6 hours and analyzed as described above. The results are shown in Table 6. With the exception of gallic acid, all other acids reduced residual iron and reduced the absorbency of the purified muscimol at 550 nm.

The results described in Examples 2 and 3 indicate butanol and ascorbic acid The combination is the most effective to reduce both the iron content and the yellow color that exhibits absorbance at 550 nm.

Example 4: Effect of solvent

Purified rutin was obtained using a variety of solvents without any acid. It has been observed that aqueous butanol and aqueous ethanol provide better yields of rutin (as compared to other solvents). See list 7 below.

Other specific examples

All of the features disclosed in this patent specification can be combined in any combination. Each feature disclosed in this patent specification can be replaced by another feature that provides the same, equivalent, or similar purpose. Therefore, unless expressly stated otherwise, each feature disclosed is merely an embodiment of a generic series of equivalent or similar features.

A person skilled in the art can readily ascertain the basic characteristics of the invention from the above, and various changes and modifications of the invention can be made without departing from the spirit and scope thereof. Therefore, other specific examples are also within the scope of the present patent application.

Claims (21)

A method for obtaining a rutin-rich extract from a plant, the method comprising: dissolving a rutin-containing plant extract in methanol or ethanol to obtain a crude rutin extract solution, the muscarin-containing plant extract comprising 90% Up to 95% by weight of rutin and 100 to 300 ppm of iron; reducing the volume of the crude rutin extract solution to obtain a concentrate; by adding a monoacid (1-15% to the rutin) to the concentrate A mixture of the weight of the plant extract and the selective C1-C4 alcohol, water or both, wherein the caroten-containing plant extract constitutes 8% to 30% (w/v) of the mixture and the acid ascorbic acid Or tartaric acid; allowing the mixture to stand to form a precipitate; and separating the precipitate, thereby obtaining a eucalyptus extract containing less than 20 ppm of iron. The method of claim 1, wherein the musk-containing plant extract is obtained by extraction with water, an alcohol or an aqueous alcohol solution from O. serrata, eucalyptus or Fava d'anta, such as a coarse precipitate. The method of claim 1, wherein a C1-C4 alcohol and water are added. The method of claim 1, wherein the acid is added in an amount of from 1 to 10% by weight of the rhizoma-containing plant extract. The method of claim 4, wherein the acid is added in an amount of from 1 to 5% by weight of the rhizoma-containing plant extract. The method of claim 5, wherein the acid is added in an amount of from 2 to 4% of the carotenoid-containing plant extract. The method of claim 3, wherein the C1-C4 alcohol and water are added as an aqueous solution of a C1-C4 alcohol comprising from 5 to 80% by volume of water. The method of claim 3, wherein the ratio of the water to the C1-C4 alcohol is 1:1 to 5:1 by volume. The method of claim 8, wherein the ratio of the water to the C1-C4 alcohol is 1:1 to 3:1 by volume. The method of claim 1, wherein the C1-C4 alcohol is methanol and the acid ascorbic acid. The method of claim 1, wherein the C1-C4 alcohol is n-butanol or propanol. The method of claim 11, wherein the acid is ascorbic acid. The method of claim 12, wherein the C1-C4 alcohol is n-butanol. The method of claim 13, wherein the acid is added in an amount of from 1 to 10% by weight of the rhizoma-containing plant extract. The method of claim 14, wherein the acid is added in an amount of from 1 to 5% by weight of the musk-containing plant extract. The method of claim 15, wherein the acid is added in an amount of from 2 to 4% by weight of the musk-containing plant extract. The method of claim 13, wherein the ratio of the water to the C1-C4 alcohol is 1:1 to 5:1 by volume. The method of claim 13, wherein the ratio of water to C1-C4 alcohol is 1:1 to 3:1 by volume. An eucalyptus extract of Uncaria serrata containing 97% to 99% of rutin (as measured by HPLC) and less than 20 ppm of iron, the absorbency of the extract at 550 nm Below 0.2, the extract is prepared by the method described in claim 1. The eucalyptus extract of claim 19, wherein the extract comprises at least 98% of rutin (as measured by HPLC) and less than 10 ppm of iron, which has a low absorbance at 550 nm. At 0.1. The eucalyptus extract of claim 19, wherein the extract comprises 5% to 10% by weight of water.