WO2010013987A2 - Extraction method for increasing liquiritigenin content in glycyrrhizae radix et rhizoma or glycyrrhizae radix extract - Google Patents

Extraction method for increasing liquiritigenin content in glycyrrhizae radix et rhizoma or glycyrrhizae radix extract Download PDF

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WO2010013987A2
WO2010013987A2 PCT/KR2009/004331 KR2009004331W WO2010013987A2 WO 2010013987 A2 WO2010013987 A2 WO 2010013987A2 KR 2009004331 W KR2009004331 W KR 2009004331W WO 2010013987 A2 WO2010013987 A2 WO 2010013987A2
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Prior art keywords
liquiritigenin
glycyrrhizae radix
content
organic solvent
extract
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PCT/KR2009/004331
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French (fr)
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WO2010013987A4 (en
WO2010013987A3 (en
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Chankyu Jeong
Seil Sohn
Hongki Kim
Hyungson Kim
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Daewon Pharm., Co., Ltd
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Priority claimed from KR1020090064903A external-priority patent/KR101158535B1/en
Application filed by Daewon Pharm., Co., Ltd filed Critical Daewon Pharm., Co., Ltd
Priority to CN2009801308750A priority Critical patent/CN102112142A/en
Publication of WO2010013987A2 publication Critical patent/WO2010013987A2/en
Publication of WO2010013987A3 publication Critical patent/WO2010013987A3/en
Publication of WO2010013987A4 publication Critical patent/WO2010013987A4/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A61K36/484—Glycyrrhiza (licorice)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/02—Antidotes

Definitions

  • the present disclosure relates to an extraction method for increasing liquiritigenin content in Glycyrrhizae Radix et Rhizoma or Glycyrrhizae radix extract.
  • Glycyrrhizae Radix et Rhizoma is a perennial plant which belongs to the Rosales order and the Leguminosae family, and its root is reddish brown and grows deep in soil and its angular stem grows vertically to about 1 m to about 1.5 m. Its leaves are obliquely oval-shaped with pointed tips. The leaves are about 2 cm to about 5 cm long and about 1 cm to about 3 cm wide without any lacinia. Its flowers are similar to those of soybean, bloom in an indigo-purple color in summer, and are a kind of raceme.
  • Glycyrrhizae Radix et Rhizoma in an oriental pharmacy the plant is a herbal medicine widely used in the oriental pharmaceutical practice. According to the pharmacological action of Glycyrrhizae Radix et Rhizoma, it binds to toxic materials in the liver and detoxifies the liver, and is useful in treatment for drug addiction, hepatitis, urticaria, dermatitis, eczema, etc. In addition, it has not only cough and sputum removal action, but also antihistaminergic and antiacetylcholinergic actions.
  • the plant usually controls han-yeol (cold and heat) and four-qi (warm hot cool heat), normalizes the physiology of ears, eyes, mouth, nose, and urine and feces, facilitates flow in all the blood vessels, strengthens muscles and bones, and improves the systemic nutritional state.”
  • Glycyrrhizae Radix et Rhizoma has been used as an antidote to all kinds of poisonings, and it has been widely used as a very important galenical such as an antitussive and expectorant, a flavor, and a palliative, in not only Oriental medicine but also new medicine and folk medicine.
  • Glycyrrhizae radix extract contains high contents of liquiritin, liquiritigenin, liquiritin apioside, isoliquiritin, and flavonoids containing isoliquiritin apioside and aglycones, and methanol fractions containing mostly liquiritin and liquiritin apioside (Kamei, J., et al., Eur. J. Pharmacol., 469, pp 159-63, 2003).
  • liquiritigenin has been known to show various physiological activities such as anti-dementia, anti-bacterial action, anti-oxidant, anti-angiogenesis, anti-skin cancer, detoxification of poisoning caused by cadmium, etc.
  • KR Patent No. 5353872 disclosed a composition containing liquritigenin for treatment and prevention of disease caused by heavy metal addiction
  • KR Patent No. 697056 disclosed a composition containing liquiritigenin as an active ingredient for prevention and treatment of liver cancer.
  • Liquiritigenin having these various psychological activities is generally extracted by a conventional method using water or lower alcohol.
  • a small amount of liquiritigenin exists in Glycyrrhizae radix extract many studies have been conducted to increase the content of liquiritigenin in Glycyrrhizae radix extract.
  • KR Patent No. 592482 disclosed a method for increasing liquiritigenin extract content in Glycyrrhizae Radix et Rhizoma, including:
  • the method has problems in that each step is complicated and costs are increasing because germs or enzymatic liquid of the germs are used.
  • KR Patent No. 37789 disclosed a method of increasing the content of liquritigenin, an active ingredient, in Glycyrrhizae Radix et Rhizoma or Glycyrrhizae radix extract, including:
  • dissolving liquiritin in DMSO dissolving liquiritin in DMSO; adding 1 M HCl to the solution; performing a bath heating of the mixture to perform an acid hydrolysis of the liquiritin; adding an equivalent amount of 1 M HCl to neutralize the acidic part; and performing silica gel chromatography using a solvent combination comprising chloroform and acetone to purify impurities such as sugar ingredients and salts generated during the acid hydrolysis.
  • the present inventors have studied a simple and highly efficient method of increasing the liquirtigenin content from Glycyrrhizae radix extract, understood that when water and strong acid or base was added at about 80°C to about 100°C into an extract extracted by introducing Glycyrrhizae Radix et Rhizoma into water, organic solvent, or a mixed solvent of organic solvents and using ultrasonic waves, a product containing liquiritigenin was generated as a precipitate, which may be easy to isolate later, and the extraction content of liquiritigenin may be increased from the Glycyrrhizae radix extract by a simple method, and finally made the present invention.
  • One object of the present invention is to provide an extraction method for increasing liquiritigenin content in Glycyrrhizae Radix et Rhizoma or Glycyrrhizae radix extract.
  • the present invention provides an extraction method for increasing liquiritigenin content in Glycyrrhizae Radix et Rhizoma or Glycyrrhizae radix extract, the method including:
  • Step 1 Using water, an organic solvent, or a mixed solvent of water and the organic solvent to Glycyrrhizae Radix et Rhizoma to extract a product containing liquiritin (Step 1); and introducing water, an organic solvent, or a mixed solvent of water and the organic solvent into the product containing liquiritin extracted in Step 1, adding acid or base into the solution, and hydrolyzing the mixture to prepare a product containing liquiritigenin (Step 2).
  • a column chromatography process may be omitted as to simplify the steps, and an extract containing a higher liquiritigenin content (about 8 to 23 %) than that obtained by a conventional method may be obtained using the method of the present invention, the method is useful during an extraction of liquiritigenin.
  • the present invention provides an extraction method for increasing liquiritigenin content in Glycyrrhizae Radix et Rhizoma or Glycyrrhizae radix extract, the method including:
  • Step 1 Using water, an organic solvent, or a mixed solvent of water and the organic solvent to Glycyrrhizae Radix et Rhizoma to extract a product containing liquiritin (Step 1); and putting water, an organic solvent, or a mixed solvent of water and the organic solvent in the product containing liquiritin extracted in Step 1, adding acid or base into the solution, and hydrolyzing the mixture to prepare a product containing liquiritigenin (Step 2).
  • Step 1 is a step in which water, an organic solvent, or a mixed solvent of water and the organic solvent is used to Glycyrrhizae Radix et Rhizoma to extract a product containing liquiritin.
  • liquiritin may be first extracted from Glycyrrhizae Radix et Rhizoma and then hydrolyzed to obtain liquiritigenin.
  • Glycyrrhiza uralensis Fisch. Glycyrrhiza glabra Linne.
  • Glycyrrhiza uralensis Fisch. Glycyrrhiza glabra Linne.
  • Glycyrrhiza inflata Bat. Glycyrrhiza uralensis Fisch.
  • Glycyrrhiza glabra Linne. Glycyrrhiza glabra Linne., etc.
  • Glycyrrhizae Radix et Rhizoma Glycyrrhizae Radix et Rhizoma.
  • Water, an organic solvent, or a solvent of water and the organic solvent may be preferably used as a solvent during the extraction in the step, and the organic solvent may preferably include a lower alcohol such as methanol, ethanol, n-propanol, isopropanol, n-butanol, t-butanol, and etc., acetone, acetonitrile, ethyl acetate, tetrahydrofuran, dimethyl ether, diethyl ether and dimethyl formamide, benzene, toluene, xylene, etc.
  • a lower alcohol such as methanol, ethanol, n-propanol, isopropanol, n-butanol, t-butanol, and etc.
  • acetone acetonitrile
  • ethyl acetate tetrahydrofuran
  • dimethyl ether diethyl ether and dimethyl formamide
  • the solvent may be added preferably at a 2-fold to 10-fold larger amount than the weight of Glycyrrhizae Radix et Rhizoma for extraction, more preferably at a 4-fold to 8-fold larger amount, but it is not limited thereto.
  • the extraction temperature may be preferably about 30 °C to about 100 °C, more preferably about 80 °C to about 100 °C, but it is not limited thereto.
  • the extraction time may be preferably about 1 hour to about 10 hours, more preferably about 4 hours to about 8 hours, but it is not limited thereto.
  • the extraction method may include, but is not limited to, heating and stirring extraction, reflux extraction, ultrasonic extraction, and maceration.
  • the number of extraction may be preferably, but is not limited to, once to five times.
  • Glycyrrhizae radix extract containing liquiritin may be extracted and the obtained extract may be filtered and concentrated under reduced pressure to yield a product containing liquiritin.
  • Step 2 is a step in which water, an organic solvent, or a mixed solvent of the organic solvent is introduced into the product containing liquiritin extracted in Step 1, acid or base is added into the product, and liquiritin contained in the product is hydrolyzed to prepare a product containing liquiritigenin.
  • the organic solvent then used may preferably include a lower alcohol such as methanol, ethanol, n-propanol, isopropanol, n-butanol, t-butanol, and etc., acetone, acetonitrile, ethyl acetate, tetrahydrofuran, dimethyl ether, diethyl ether and dimethyl formamide, benzene, toluene, xylene, etc.
  • the concentration of methanol or ethanol can be 70% to 100%.
  • the acid may be preferably a strong acid, for example, hydrochloric acid, sulfuric acid, nitric acid, or any mixed solvent thereof.
  • sulfuric acid may be used. More preferably, about 1 M to about 5 M sulfuric acid may be used.
  • sodium hydroxide, potassium hydroxide, calcium hydroxide, or any mixture thereof may be used and preferably, calcium hydroxide may be used. More preferably, about 1 M to about 5 M calcium hydroxide may be used.
  • the reaction temperature may be preferably about 50 °C to about 100 °C, and more preferably about 80 °C to about 100 °C.
  • the mixture may be cooled down to room temperature to obtain a precipitate due to the insolubility of liquritigenin in water, and the liquiritigenin content amounts to about 5% to about 15%.
  • the precipitate may be dissolved in an organic solvent, washed several times with water, and then the organic layer may be concentrated and driedunder reduced pressure to increase liquiritigenin content to about 8% to about 23%.
  • the organic solvent then used may include a lower alcohol such as n-butanol, t-butanol, and etc., ethyl acetate, dimethyl ether, diethyl ether and dimethyl formamide, benzene, toluene, xylene, etc.
  • a lower alcohol such as n-butanol, t-butanol, and etc.
  • ethyl acetate dimethyl ether, diethyl ether and dimethyl formamide
  • benzene toluene
  • xylene etc.
  • drying under reduced pressure may be preferably used rather than a hot air drying, and after the organic solvent of the precipitate is dried under reduced pressure, the precipitate may be dissolved again in ethanol and a drying under reduced pressure may be additionally performed on the precipitate to reduce the amount of the residual solvent more.
  • a column chromatography process may be omitted as to simplify the steps, and an extract containing a higher liquiritigenin content than that obtained by a conventional method may be obtained using the method of the present invention, the method is useful during an extraction of liquiritigenin.
  • Glycyrrhizae Radix et Rhizoma was purchased from an oriental medicine trader and exactly differentiated, it was segmented into small pieces (Glycyrrhizae Radix et Rhizoma segments) and used.
  • the content of liquiritin included in the Glycyrrhizae Radix et Rhizoma was within 1%.
  • About 800 ml of about 80% to about 90% methanol was added into about 100 g of the Glycyrrhizae Radix et Rhizoma segments and a heat stirring was performed at about 85 °C for about 8 hours. Subsequently, cooling and filtering was performed, followed by concentration under reduced pressure to obtain an extract containing about 25.8 g of liquiritin.
  • the liquiritin content was measured according to the following method. Specifically, about 0.5 g of the extract was precisely weighed and an ultrasonic extraction was performed using about 50 ml of about 80 % methanol, followed by a filtration to prepare a test solution. Separately, about 10 mg of a standard liquiritin product was precisely used to prepare a 50-ml solution as a standard solution, and then each 20 ⁇ l of the test and standard solutions was used for test according to liquid chromatography under the following conditions to measure the content, considering dilution factors.
  • UV spectrophotometer (measurement wavelength: about 254 nm)
  • a measurement according to the method showed that the liquiritin content obtained from the Glycyrrhizae radix extract was about 2.3 %.
  • the liquiritigenin content was measured according to the following method. Specifically, about 0.1 g of the product was precisely weighed and an ultrasonic extraction was performed using about 50 ml of about 80 % methanol, followed by a filtration to prepare a test solution. Separately, about 10 mg of a standard liquiritin product was precisely used to prepare a 50-ml solution as a standard solution, and then each 20 ⁇ l of the test and standard solutions was used for test according to liquid chromatography under the following conditions to measure the content, considering dilution factors.
  • UV spectrophotometer (measurement wavelength: about 254 nm)
  • Example 2 The same method as in Example 1 was performed to yield about 23 g of a dark brown material (liquiritigenin content: about 11.2 %), except that about 80 % ethanol was added into the Glycyrrhizae Radix et Rhizoma to prepare an extract.
  • Example 2 The same method as in Example 1 was performed to yield about 23 g of a dark brown material (liquiritigenin content: about 11.8 %), except that about 100 % methanol was secondarily added into the extract obtained from addition of about 80 % methanol into the Glycyrrhizae Radix et Rhizoma to yield an extract containing liquiritin (about 2.9 %).
  • Example 2 The same method as in Example 1 was performed to yield about 25 g of a dark brown material (liquiritigenin content: about 12.1 %), except that an extraction process was performed twice on the Glycyrrhizae Radix et Rhizoma by using about 80 % ethanol.
  • the mixture was cooled down to room temperature, an equivalent amount of 1 N NaOH was added to neutralize the acidic part, and then a freeze-drying was performed for about 48 hours to obtain about 28 g of an extract (liquiritigenin content: about 0.8 %).
  • Example 2 The same method as in Example 1 was performed to yield about 29 g of an extract (liquiritigenin content: about 0.7 %) containing liquiritigenin, except that ethanol was used as a solvent instead of methanol.
  • liquiritin content in Glycyrrhizae radix extract was about 1 % to about 2 % in methanol, ethanol, isopropanol, n-propanol, n-butanol, t-butanol, acetone, ethyl acetate, tetrahydrofuran, dimethyl ether, diethyl ether, dimethyl formamide, and acetonitrile as an extraction solvent, and it is confirmed that liquiritin extraction was poorly achieved in n-hexane, benzene, toluene, xylene, dichloromethane, and chloroform.
  • Glycyrrhizae radix extract containing liquiritin was introduced and dispersed into water, an acid was added into the solution, and then reaction temperature was increased to increase the liquiritigenin content.
  • Liquiritigenin contents in the product were measured using a method for measuring liquiritigenin content in Example 1, and the results were recorded in Table 9.
  • the extract containing liquiritigenin in ⁇ Example 1> was dissolved in ethyl acetate and dried using the following method during the process of drying the ethyl acetate, and then the amount of the residual solvent, in which the Glycyrrhiza radix extract containing liquiritigenin was dissolved, was measured. The results were recorded in Table 11.
  • the extract was dried at about 50 °C for about 48 hours using a hot air drying method.
  • the extract was entangled each other as to tend to become a highly viscous material.
  • the extract was dried at about 50 °C or higher for about 5 hours using a method of drying under reduced pressure, and then about 30 g of a dry matter was dissolved in about 300 ml of ethanol and the solution was dried at about 50 °C for about 24 hours using a method of drying under reduced pressure.
  • the extract was dried at about 50 °C or higher for about 5 hours using a method of drying under reduced pressure, and then about 30 g of a dry matter was dissolved in about 300 ml of ethanol and the solution was dried at about 60 °C for about 24 hours using a method of drying under reduced pressure.
  • a precipitate containing liquiritigenin is dissolved in an organic solvent, which may be dried using a drying method under reduced pressure, and then the resulting precipitate may be again dissolved in ethanol, which may be additionally dried using the same drying method under reduced pressure.
  • a column chromatography process may be omitted as to simplify the steps, and an extract containing a higher liquiritigenin content (about 8 to 23 %) than that obtained by a conventional method may be obtained using the method of the present invention, the method is useful during an extraction of liquiritigenin.

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Abstract

Disclosed herein is an extraction method for increasing liquiritigenin content in Glycyrrhizae Radix et Rhizoma or Glycyrrhizae radix extract, the method comprising: using water, an organic solvent, or a mixed solvent of water and the organic solvent to Glycyrrhizae Radix et Rhizoma to extract a product containing liquiritin (Step 1); and introducing water, an organic solvent, or a mixed solvent of water and the organic solvent into the product containing liquiritin extracted in Step 1, adding acid or base into the solution, and hydrolyzing the mixture to prepare a product containing liquiritigenin (Step 2).

Description

EXTRACTION METHOD FOR INCREASING LIQUIRITIGENIN CONTENT IN GLYCYRRHIZAE RADIX ET RHIZOMA OR GLYCYRRHIZAE RADIX EXTRACT
The present disclosure relates to an extraction method for increasing liquiritigenin content in Glycyrrhizae Radix et Rhizoma or Glycyrrhizae radix extract.
In general, Glycyrrhizae Radix et Rhizoma is a perennial plant which belongs to the Rosales order and the Leguminosae family, and its root is reddish brown and grows deep in soil and its angular stem grows vertically to about 1 m to about 1.5 m. Its leaves are obliquely oval-shaped with pointed tips. The leaves are about 2 cm to about 5 cm long and about 1 cm to about 3 cm wide without any lacinia. Its flowers are similar to those of soybean, bloom in an indigo-purple color in summer, and are a kind of raceme.
In keeping with an age-old phrase, “Glycyrrhizae Radix et Rhizoma in an oriental pharmacy”, the plant is a herbal medicine widely used in the oriental pharmaceutical practice. According to the pharmacological action of Glycyrrhizae Radix et Rhizoma, it binds to toxic materials in the liver and detoxifies the liver, and is useful in treatment for drug addiction, hepatitis, urticaria, dermatitis, eczema, etc. In addition, it has not only cough and sputum removal action, but also antihistaminergic and antiacetylcholinergic actions. It has alleviatory action against pain caused by abrupt tension of muscle or tissue, body weight increase, and increase in the number of leukocytes, diuretic action, anti-inflammatory action, etc. and specifically, ingredients thereof such as liquiritin, liquiritigenin, etc. inhibit the generation of peptic ulcers.
According to <Donguibogam>, a time-honored scientific compendium for oriental medicine produced by Huh Jun during the Chosun Dynasty (1392-1910), it is the documented that “Glycyrrhizae Radix et Rhizoma removes toxicities of all medicines, has an action that brings 72 different kinds of stone medicines (mineral medicines) and about 1200 different kinds of herbal medicines in harmony to demonstrate the efficacy of the medicines, and so has been called ‘national elder’ meaning an elder of a country. The plant usually controls han-yeol (cold and heat) and four-qi (warm hot cool heat), normalizes the physiology of ears, eyes, mouth, nose, and urine and feces, facilitates flow in all the blood vessels, strengthens muscles and bones, and improves the systemic nutritional state.”
It is known that in folk remedies for poisoning from bamboo shoots or mushrooms, people have decocted the plant into medical juice for drinking, and it has been known to have effects on tobacco intoxication, drug addition, and other addictions by boiling down the plant and drinking the boiled plant juice. Therefore, Glycyrrhizae Radix et Rhizoma has been used as an antidote to all kinds of poisonings, and it has been widely used as a very important galenical such as an antitussive and expectorant, a flavor, and a palliative, in not only Oriental medicine but also new medicine and folk medicine.
Glycyrrhizae radix extract contains high contents of liquiritin, liquiritigenin, liquiritin apioside, isoliquiritin, and flavonoids containing isoliquiritin apioside and aglycones, and methanol fractions containing mostly liquiritin and liquiritin apioside (Kamei, J., et al., Eur. J. Pharmacol., 469, pp 159-63, 2003). Among these ingredients, liquiritigenin has been known to show various physiological activities such as anti-dementia, anti-bacterial action, anti-oxidant, anti-angiogenesis, anti-skin cancer, detoxification of poisoning caused by cadmium, etc.
As conventional technologies associated with liquiritigenin, KR Patent No. 5353872 disclosed a composition containing liquritigenin for treatment and prevention of disease caused by heavy metal addiction, and KR Patent No. 697056 disclosed a composition containing liquiritigenin as an active ingredient for prevention and treatment of liver cancer.
Liquiritigenin having these various psychological activities is generally extracted by a conventional method using water or lower alcohol. However, because a small amount of liquiritigenin exists in Glycyrrhizae radix extract, many studies have been conducted to increase the content of liquiritigenin in Glycyrrhizae radix extract.
For example, KR Patent No. 592482 disclosed a method for increasing liquiritigenin extract content in Glycyrrhizae Radix et Rhizoma, including:
Segmenting Glycyrrhizae Radix et Rhizoma into small pieces, performing extraction on the pieces under reflux using water at 100℃, and filtering the extract, followed by freeze-drying the filtrate; treating the dry matter freeze-dried by the above step with germs having sugar hydrolysis activity, or enzymatic liquid of the germs, followed by leaving still at about 20℃ to about 50℃ for about 10 minutes to about 36 hours; adding a 2-fold to 10-fold amount of ethanol to the culture incubated by the above step to divide it into an ethanol soluble part and an ethanol insoluble part, followed by concentrating the ethanol soluble part under reduced pressure to obtain an extract; dispersing the extract obtained by the above step in distilled water and performing a three-phase partitioning extraction, followed by concentrating the soluble fraction; and performing a re-crystallization on the seventh fraction in the above step in a mixed solvent of dichloromethane and methanol to obtain pure liquiritigenin.
However, the method has problems in that each step is complicated and costs are increasing because germs or enzymatic liquid of the germs are used.
KR Patent No. 37789 disclosed a method of increasing the content of liquritigenin, an active ingredient, in Glycyrrhizae Radix et Rhizoma or Glycyrrhizae radix extract, including:
dissolving liquiritin in DMSO; adding 1 M HCl to the solution; performing a bath heating of the mixture to perform an acid hydrolysis of the liquiritin; adding an equivalent amount of 1 M HCl to neutralize the acidic part; and performing silica gel chromatography using a solvent combination comprising chloroform and acetone to purify impurities such as sugar ingredients and salts generated during the acid hydrolysis.
However, because DMSO in the method is difficult to remove due to its high melting point, a lot of efforts such as checking the residual solvent should be made, and when an acid hydrolysis is performed using HCl, problems arose that a time-consuming acid hydrolysis is inevitable due to its slow hydrolysis reaction, and the purity of the liquritigenin yield is not good.
Thus, the present inventors have studied a simple and highly efficient method of increasing the liquirtigenin content from Glycyrrhizae radix extract, understood that when water and strong acid or base was added at about 80℃ to about 100℃ into an extract extracted by introducing Glycyrrhizae Radix et Rhizoma into water, organic solvent, or a mixed solvent of organic solvents and using ultrasonic waves, a product containing liquiritigenin was generated as a precipitate, which may be easy to isolate later, and the extraction content of liquiritigenin may be increased from the Glycyrrhizae radix extract by a simple method, and finally made the present invention.
One object of the present invention is to provide an extraction method for increasing liquiritigenin content in Glycyrrhizae Radix et Rhizoma or Glycyrrhizae radix extract.
In order to achieve the objects, the present invention provides an extraction method for increasing liquiritigenin content in Glycyrrhizae Radix et Rhizoma or Glycyrrhizae radix extract, the method including:
Using water, an organic solvent, or a mixed solvent of water and the organic solvent to Glycyrrhizae Radix et Rhizoma to extract a product containing liquiritin (Step 1); and introducing water, an organic solvent, or a mixed solvent of water and the organic solvent into the product containing liquiritin extracted in Step 1, adding acid or base into the solution, and hydrolyzing the mixture to prepare a product containing liquiritigenin (Step 2).
Because a high-priced solvent is not needed, a column chromatography process may be omitted as to simplify the steps, and an extract containing a higher liquiritigenin content (about 8 to 23 %) than that obtained by a conventional method may be obtained using the method of the present invention, the method is useful during an extraction of liquiritigenin.
Features and advantages of the present invention will be more clearly understood by the following detailed description of the present preferred embodiments by reference to the accompanying drawings. It is first noted that terms or words used herein should be construed as meanings or concepts corresponding with the technical sprit of the present invention, based on the principle that the inventor can appropriately define the concepts of the terms to best describe his own invention. Also, it should be understood that detailed descriptions of well-known functions and structures related to the present invention will be omitted so as not to unnecessarily obscure the important point of the present invention.
The present invention provides an extraction method for increasing liquiritigenin content in Glycyrrhizae Radix et Rhizoma or Glycyrrhizae radix extract, the method including:
Using water, an organic solvent, or a mixed solvent of water and the organic solvent to Glycyrrhizae Radix et Rhizoma to extract a product containing liquiritin (Step 1); and putting water, an organic solvent, or a mixed solvent of water and the organic solvent in the product containing liquiritin extracted in Step 1, adding acid or base into the solution, and hydrolyzing the mixture to prepare a product containing liquiritigenin (Step 2).
Hereinafter, the present invention will be described in detail.
First, Step 1 is a step in which water, an organic solvent, or a mixed solvent of water and the organic solvent is used to Glycyrrhizae Radix et Rhizoma to extract a product containing liquiritin.
Because liquiritigenin content is nearly 0% in Glycyrrhizae Radix et Rhizoma, liquiritin may be first extracted from Glycyrrhizae Radix et Rhizoma and then hydrolyzed to obtain liquiritigenin.
In this step, Glycyrrhiza uralensis Fisch., Glycyrrhiza glabra Linne., Glycyrrhiza uralensis Fisch., Glycyrrhiza glabra Linne., Glycyrrhiza inflata Bat., Glycyrrhiza uralensis Fisch., and Glycyrrhiza glabra Linne., etc. can be used as Glycyrrhizae Radix et Rhizoma.
Water, an organic solvent, or a solvent of water and the organic solvent may be preferably used as a solvent during the extraction in the step, and the organic solvent may preferably include a lower alcohol such as methanol, ethanol, n-propanol, isopropanol, n-butanol, t-butanol, and etc., acetone, acetonitrile, ethyl acetate, tetrahydrofuran, dimethyl ether, diethyl ether and dimethyl formamide, benzene, toluene, xylene, etc.
In a method according to the present invention, the solvent may be added preferably at a 2-fold to 10-fold larger amount than the weight of Glycyrrhizae Radix et Rhizoma for extraction, more preferably at a 4-fold to 8-fold larger amount, but it is not limited thereto.
In a method according to the present invention, the extraction temperature may be preferably about 30 ℃ to about 100 ℃, more preferably about 80 ℃ to about 100 ℃, but it is not limited thereto.
In a method according to the present invention, the extraction time may be preferably about 1 hour to about 10 hours, more preferably about 4 hours to about 8 hours, but it is not limited thereto.
In a method according to the present invention, the extraction method may include, but is not limited to, heating and stirring extraction, reflux extraction, ultrasonic extraction, and maceration.
In a method according to the present invention, the number of extraction may be preferably, but is not limited to, once to five times.
According to the method, Glycyrrhizae radix extract containing liquiritin may be extracted and the obtained extract may be filtered and concentrated under reduced pressure to yield a product containing liquiritin.
Next, Step 2 is a step in which water, an organic solvent, or a mixed solvent of the organic solvent is introduced into the product containing liquiritin extracted in Step 1, acid or base is added into the product, and liquiritin contained in the product is hydrolyzed to prepare a product containing liquiritigenin.
The organic solvent then used may preferably include a lower alcohol such as methanol, ethanol, n-propanol, isopropanol, n-butanol, t-butanol, and etc., acetone, acetonitrile, ethyl acetate, tetrahydrofuran, dimethyl ether, diethyl ether and dimethyl formamide, benzene, toluene, xylene, etc. In the organic solvent, the concentration of methanol or ethanol can be 70% to 100%.
The acid may be preferably a strong acid, for example, hydrochloric acid, sulfuric acid, nitric acid, or any mixed solvent thereof. Preferably, sulfuric acid may be used. More preferably, about 1 M to about 5 M sulfuric acid may be used. As a base, sodium hydroxide, potassium hydroxide, calcium hydroxide, or any mixture thereof may be used and preferably, calcium hydroxide may be used. More preferably, about 1 M to about 5 M calcium hydroxide may be used.
In a method according to the present invention, the reaction temperature may be preferably about 50 ℃ to about 100 ℃, and more preferably about 80 ℃ to about 100 ℃.
After a hydrolysis, the mixture may be cooled down to room temperature to obtain a precipitate due to the insolubility of liquritigenin in water, and the liquiritigenin content amounts to about 5% to about 15%. Subsequently, the precipitate may be dissolved in an organic solvent, washed several times with water, and then the organic layer may be concentrated and driedunder reduced pressure to increase liquiritigenin content to about 8% to about 23%. The organic solvent then used may include a lower alcohol such as n-butanol, t-butanol, and etc., ethyl acetate, dimethyl ether, diethyl ether and dimethyl formamide, benzene, toluene, xylene, etc.
When the organic solvent is dried, drying under reduced pressure may be preferably used rather than a hot air drying, and after the organic solvent of the precipitate is dried under reduced pressure, the precipitate may be dissolved again in ethanol and a drying under reduced pressure may be additionally performed on the precipitate to reduce the amount of the residual solvent more.
Because a high-priced solvent is not needed, a column chromatography process may be omitted as to simplify the steps, and an extract containing a higher liquiritigenin content than that obtained by a conventional method may be obtained using the method of the present invention, the method is useful during an extraction of liquiritigenin.
Hereinafter, preferred Examples are given for a better understanding of the present invention. However, the following Examples are only for the understanding of the present invention, and the present invention is not limited to the following Examples.
<Example 1> Preparation of liquiritigenin 1
(1) Preparation of Glycyrrhizae radix extract containing liquiritin
After Glycyrrhizae Radix et Rhizoma was purchased from an oriental medicine trader and exactly differentiated, it was segmented into small pieces (Glycyrrhizae Radix et Rhizoma segments) and used. The content of liquiritin included in the Glycyrrhizae Radix et Rhizoma was within 1%. About 800 ml of about 80% to about 90% methanol was added into about 100 g of the Glycyrrhizae Radix et Rhizoma segments and a heat stirring was performed at about 85 ℃ for about 8 hours. Subsequently, cooling and filtering was performed, followed by concentration under reduced pressure to obtain an extract containing about 25.8 g of liquiritin.
1H NMR (CD3OD, 300 MHz) δ 7.61 (1H, d, 8.6 Hz) 7.08 (2H, d, 8.4 Hz) 6.81 (2H, d, 8.4 Hz) 6.49 (1H, dd, 8.6 Hz), 6.37 (1H, d, 2.4 Hz), 5.41 (1H, dd, 13.2, 3.1) 5.37 (2H, dd, 13.0, 2.9 Hz), 3.49 (3H, t, 2.4 Hz), 3.05 (1H, dd, 17.0, 13.0), 2.68 (1H, dd, 17.0, 2.9)
In the obtained Glycyrrhizae radix extract, the liquiritin content was measured according to the following method. Specifically, about 0.5 g of the extract was precisely weighed and an ultrasonic extraction was performed using about 50 ml of about 80 % methanol, followed by a filtration to prepare a test solution. Separately, about 10 mg of a standard liquiritin product was precisely used to prepare a 50-ml solution as a standard solution, and then each 20 ㎕ of the test and standard solutions was used for test according to liquid chromatography under the following conditions to measure the content, considering dilution factors.
* Detector: UV spectrophotometer (measurement wavelength: about 254 nm)
* Column: Octadecyl silica gel, about 5 ㎛, about 4.6 mm × about 15 cm
* Mobile phase: about 1 % acetic acid mixed solution:ACN = 9:1
* Flow rate: about 1 ml/min
A measurement according to the method showed that the liquiritin content obtained from the Glycyrrhizae radix extract was about 2.3 %.
(2) Preparation of liquiritigenin
About 100 g of the extract obtained in (1) was dispersed in about 500 ml of water, into which 250 ml of 3M sulfuric acid was introduced, and a heat stirring was performed at about 90 ℃ to about 100 ℃ for about 2 hours. Subsequently, the mixture was cooled down to room temperature and a precipitate thus produced was filtered and dried to yield about 34 g of a dark-brown material (liquiritigenin content: about 8.5 %).
Subsequently, about 30 g of the material was dissolved in about 300 ml of ethyl acetate, washed twice with about 300 ml of water, and concentrated and dried under reduced pressure for removal of the solvent to obtain about 22 g of a dark brown crystal (liquiritigenin content: about 11 %).
1H NMR (CD3OD, 300 MHz) δ 7.73 (1H, d, 8.6 Hz, H-5) 7.33 (2H, d, 8.4 Hz) 6.81 (2H, d, 8.4 Hz) 6.49 (1H, dd, 8.6 Hz), 6.35 (1H, d, 2.4 Hz), 5.37 (2H, dd, 13.0, 2.9 Hz), 3.05 (1H, dd, 17.0, 13.0), 2.68 (1H, dd, 17.0, 2.9)
Then, the liquiritigenin content was measured according to the following method. Specifically, about 0.1 g of the product was precisely weighed and an ultrasonic extraction was performed using about 50 ml of about 80 % methanol, followed by a filtration to prepare a test solution. Separately, about 10 mg of a standard liquiritin product was precisely used to prepare a 50-ml solution as a standard solution, and then each 20 ㎕ of the test and standard solutions was used for test according to liquid chromatography under the following conditions to measure the content, considering dilution factors.
* Detector: UV spectrophotometer (measurement wavelength: about 254 nm)
* Column: Octadecyl silica gel, about 5 ㎛, about 4.6 mm × about 15 cm
* Mobile phase: about 0.01M phosphoric acid:ACN = 67:33
* Flow rate: about 1 ml/min
<Example 2> Preparation of liquiritigenin 2
The same method as in Example 1 was performed to yield about 23 g of a dark brown material (liquiritigenin content: about 11.2 %), except that about 80 % ethanol was added into the Glycyrrhizae Radix et Rhizoma to prepare an extract.
<Example 3> Preparation of liquiritigenin 3
The same method as in Example 1 was performed to yield about 23 g of a dark brown material (liquiritigenin content: about 11.8 %), except that about 100 % methanol was secondarily added into the extract obtained from addition of about 80 % methanol into the Glycyrrhizae Radix et Rhizoma to yield an extract containing liquiritin (about 2.9 %).
<Example 4> Preparation of liquiritigenin 4
The same method as in Example 1 was performed to yield about 25 g of a dark brown material (liquiritigenin content: about 12.1 %), except that an extraction process was performed twice on the Glycyrrhizae Radix et Rhizoma by using about 80 % ethanol.
<Comparative Example 1> Conventional preparation method of liquiritigenin 1
About 800 ml of about 80 to 100 % methanol was added into about 100 g of Glycyrrhizae Radix et Rhizoma segments and left still at room temperature for 48 hours. Subsequently, the mixture was filtered and concentrated under reduced pressure to obtain about 18.4 g to about 21.2 g of an extract (liquiritigenin content: about 1.3 to about 1.6). The obtained extract was dissolved in 200 ml of DMSO, 100 ml of 1 N HCl was introduced into the solution, and a bath heating was performed on the mixture at 100 ℃ for 6 hours. The mixture was cooled down to room temperature, an equivalent amount of 1 N NaOH was added to neutralize the acidic part, and then a freeze-drying was performed for about 48 hours to obtain about 28 g of an extract (liquiritigenin content: about 0.8 %).
<Comparative Example 2> Conventional preparation method of liquiritigenin 2
The same method as in Example 1 was performed to yield about 29 g of an extract (liquiritigenin content: about 0.7 %) containing liquiritigenin, except that ethanol was used as a solvent instead of methanol.
<Experimental Example 1> Highly efficient extraction experiment of liquiritin
(1) Liquiritin content according to an extraction solvent
In order to measure liquiritin content in Glycyrrhizae radix extract according to an extraction solvent, a heat stirring was performed on about 100 g of Glycyrrhizae Radix et Rhizoma segments under conditions of extraction solvents and extraction temperatures in the following Table 1 for 8 hours, a liquiritin content in the extract was measured using a method for measuring liquiritin content in Example 1, and the results were recorded in Table 1.
Table 1
Extraction solvent Extraction temperature (℃) Liquiritin content (%)
Methanol 85 2.2
Ethanol 85 1.9
Isopropanol 85 1.9
n-propanol 85 1.9
n-butanol 85 1.7
t-butanol 85 1.6
Dimethyl formamide 85 1.8
Acetone 80 1.7
Acetonitrile 80 1.7
Ethyl acetate 80 1.8
Tetrahydrofuran 80 1.8
Dimethyl ether 50 1.1
Diethyl ether 55 1.2
n-hexane 70 0.2
Benzene 85 0.7
Toluene 85 0.8
Xylene 85 0.8
Dichloro methane 40 0.3
Chloroform 40 0.4
As indicated in Table 1, it is determined that liquiritin content in Glycyrrhizae radix extract was about 1 % to about 2 % in methanol, ethanol, isopropanol, n-propanol, n-butanol, t-butanol, acetone, ethyl acetate, tetrahydrofuran, dimethyl ether, diethyl ether, dimethyl formamide, and acetonitrile as an extraction solvent, and it is confirmed that liquiritin extraction was poorly achieved in n-hexane, benzene, toluene, xylene, dichloromethane, and chloroform.
(2) Liquiritin content according to methanol or ethanol content in extraction solvent
In order to measure liquiritin content in Glycyrrhizae radix extract according to methanol or ethanol content in an extraction solvent, a heat stirring was performed on about 100 g of Glycyrrhizae Radix et Rhizoma segments at about 85 ℃ for about 8 hours by changing the concentration of methanol or ethanol as an extraction solvent to yield a Glycyrrhizae radix extract, liquiritin content in the extract was measured using a method for measuring liquiritin content in Example 1, and the results were recorded in Table 2.
Table 2
Extraction solvent Liquiritin content (%)
50% methanol 1.7
60% methanol 1.9
70% methanol 2.0
80% methanol 2.3
90% methanol 2.3
100% methanol 2.2
50% ethanol 1.6
60% ethanol 1.5
70% ethanol 2.1
80% ethanol 2.3
90% ethanol 2.0
100% ethanol 1.9
As indicated in Table 2, it is determined that liquiritin content in Glycyrrhizae radix extract was high when methanol or ethanol was about 80 % to about 100%.
(3) Liquiritin content according to extraction temperature
To measure liquiritin content in Glycyrrhizae radix extract according to extraction temperatures, experiments were performed as follows.
About 800 ml of about 80% to about 100% methanol or ethanol was introduced into about 100 g of Glycyrrhizae Radix et Rhizoma segments and a heat stirring was performed at about 85 ℃ for about 8 hours for extraction. As a comparative example, an extraction was performed for 24 hours using the same method. A liquirtin content in an extract subsequently obtained was measured using a method for measuring liquiritin content in Example 1, and the results were recorded in Table 3.
Table 3
Extraction solvent Liquiritin content(%)
Room temperature (25 ℃) 85 ℃
80% methanol 1.4 2.3
90% methanol 1.3 2.3
100% methanol 1.6 2.2
80% ethanol 1.7 2.3
90% ethanol 1.5 2.0
100% ethanol 1.6 1.9
As indicated in Table 3, it is determined that the amount of liquiritin was extracted at 80℃ by about 1.5-fold more than that of the extract at room temperature.
Therefore, to extract liquiritin highly efficiently from Glycyrrhizae Radix et Rhizoma, it is determined that about 80% to about 100% methanol or ethanol is used as an extraction solvent and preferably the extraction is performed at temperatures of about 80℃ or higher.
(4) Liquiritin content according to ultrasonic extraction
In order to measure liquiritin content in Glycyrrhizae radix extract according to extraction temperatures and supersonic extraction, experiments were performed as follows.
About 800 ml of about 80% to about 100% methanol or ethanol was introduced into about 100 g of Glycyrrhizae Radix et Rhizoma segments and an ultrasonic wave was used at about 60 ℃ for about 8 hours for extraction. As a comparative example, an extraction was performed for 8 hours using the same method. A liquirtin content in an extract subsequently obtained was measured using a method for measuring liquiritin content in Example 1, and the results were recorded in Table 4.
Table 4
Extraction solvent Liquiritin content(%)
Room temperature (25 ℃) 60 ℃
80% methanol 1.8 2.4
90% methanol 1.6 2.3
100% methanol 1.5 2.3
80% ethanol 1.8 2.5
90% ethanol 1.7 2.4
100% ethanol 1.6 2.1
As indicated in Table 4, it is determined that the amount of liquiritin was extracted at 60 ℃ by about 1.4-fold more than that of the extract at room temperature.
Therefore, to extract liquiritin highly efficiently from Glycyrrhizae Radix et Rhizoma, it is determined that about 80% to about 100% methanol or ethanol is used as an extraction solvent and preferably the extraction is performed at temperatures of 60 ℃ or higher.
<Experimental Example 2> Experiment of hydrolyzing liquiritin into liquiritigenin
(1) Liquiritigenin content according to hydrolysis temperatures
In a reaction of hydrolyzing the liquiritin into liquiritigenin by adding acid into Glycyrrhizae radix extract containing liquiritin according to the present invention, experiments were performed as follows to measure a content of liquiritigenin produced after a hydrolysis was performed according to hydrolysis temperatures.
About 100 g of the Glycyrrhizae radix extract containing liquiritin obtained in <Experimental Example 1> was dispersed in about 500 ml of water, and about 250 ml of about 3M sulfuric acid was introduced into the solution to proceed a hydrolysis reaction. Subsequently, the reaction was performed for about 2 hours by changing the hydrolysis reaction temperature from about 50 ℃ to about 100 ℃. Subsequently, the reaction mixture was cooled down to room temperature to form a precipitate, which was filtered and dried to yield a product containing liquiritigenin. A liquiritigenin content in the product was measured using a method for measuring liquiritigenin content in Example 1 and the results were recorded in Table 5.
Table 5
Reaction temperature (℃) Liquiritigenin content(%)
50 2.4
60 3.7
70 5.2
80 7.2
90 8.5
100 8.5
As indicated in Table 5, it is determined that as the reaction temperature increased, the amount of liquiritigenin produced from a hydrolysis increased, and it is confirmed that when the hydrolysis reaction was performed at about 90 ℃ to about 100 ℃, a liquiritigenin content increased by about 8.5%.
(2) Liquiritigenin content according to a kind of acid
In a reaction of hydrolyzing the liquiritin into liquiritigenin by introducing and dispersing Glycyrrhizae radix extract containing liquiritin according to the present invention into water and then adding acid into the solution, experiments were performed as follows to measure liquiritigenin content according to a kind of acid.
About 100 g of Glycyrrhizae radix extract containing liquiritin obtained in <Experimental Example 1> was introduced and dispersed into about 500 ml of water, and about 250 ml of about 1M to about 5M hydrochloric acid, sulfuric acid, nitric acid, and a mixed solution thereof were introduced into the mixture to perform a reaction at about 100℃ for about 2 hours. Subsequently, the reaction mixture was cooled down to room temperature to form a precipitate, which was filtered and dried to yield a product containing liquiritigenin. A liquiritigenin content in the product was measured using a method for measuring liquiritigenin content in Example 1, and the results were recorded in Table 6.
Table 6
Acid Liquiritigenin content (%)
1M hydrochloric acid 2.2
3M hydrochloric acid 4.3
5M hydrochloric acid 5.1
1M sulfuric acid 7.3
3M sulfuric acid 8.5
5M sulfuric acid 7.9
1M nitric acid 7.2
3M nitric acid 8.1
5M nitric acid 7.2
3M hydrochloric acid+3M sulfuric acid 7.4
3M hydrochloric acid+3M nitric acid 7.3
3M nitric acid+3M sulfuric acid 8.1
As indicated in Table 6, it is confirmed that when sulfuric acid or nitric acid was used as an acid, an extract containing a high liquiritigenin content could be obtained, and when about 3 M sulfuric acid was used, the liquiritigenin content could increase by about 8.5%.
(3) Liquiritigenin content according to a kind of base
In a reaction of hydrolyzing the liquiritin into liquiritigenin by putting and dispersing Glycyrrhizae radix extract containing liquiritin according to the present invention in water and then adding base into the solution, experiments were performed as follows to measure a liquiritigenin content according to a kind of base.
About 100 g of Glycyrrhizae radix extract containing liquiritin obtained in <Experimental Example 1> was introduced and dispersed into about 500 ml of water, and about 250 ml of about 1M to about 5M sodium hydroxide, potassium hydroxide, calcium hydroxide, and a mixed solution thereof were introduced into the mixture to perform a reaction at about 100 ℃ for about 2 hours. Subsequently, the reaction mixture was cooled down to room temperature to form a precipitate, which was filtered and dried to yield a product containing liquiritigenin. A liquiritigenin content in the product was measured using a method for measuring liquiritigenin content in Example 1, and the results were recorded in Table 7.
Table 7
Base Liquiritigenin content (%)
1M sodium hydroxide 1.5
3M sodium hydroxide 3.2
5M sodium hydroxide 5.7
1M potassium hydroxide 2.1
3M potassium hydroxide 4.3
5M potassium hydroxide 6.2
1M calcium hydroxide 2.2
3M calcium hydroxide 4.4
5M calcium hydroxide 6.5
3M sodium hydroxide+3M potassium hydroxide 6.1
3M sodium hydroxide+3M calcium hydroxide 6.3
3M potassium hydroxide+3M calcium hydroxide 6.4
As indicated in Table 7, it is confirmed that when sodium hydroxide, potassium hydroxide, and calcium hydroxide were used as a base, an extract containing liquiritigenin could be obtained, and when about 5 M calcium hydroxide was specifically used, the liquiritigenin content could increase by about 6.5%. It is thought that due to an increased solubility of liquiritigenin in a basic solution, all of the extract was not precipitated and some of the extract was dissolved and lost in the solution, leading to a less liquiritigenin content compared to those in Table 6.
(4) Liquiritigenin content according to a kind of solvent
In a reaction of hydrolyzing liquiritin into liquiritigenin by introducing and dispersing Glycyrrhizae radix extract containing liquiritin according to the present invention into water and then adding acid into the solution, experiments were performed as follows to measure a change of liquiritigenin content in an organic solvent when an organic solvent other than water was added. As a control group, only water was used without any addition of an organic solvent.
About 500 ml of an organic solvent, about 500 ml of water, and about 250 ml of about 3 M sulfuric acid were introduced into about 100 g of the Glycyrrhizae radix extract containing liquiritin obtained in <Experimental Example 1>, and then a heat stirring was performed at about 85℃ for about 2 hours, or about 500 ml of an organic solvent and sulfuric acid corresponding to about 250 ml of about 3 M sulfuric acid were only added into about 100 g of a Glycyrrhizae radix extract containing liquiritin, and then a heat stirring was performed at about 85 ℃ for about 2 hours. Subsequently, the solution was cooled down to room temperature, an organic layer was separated and washed twice with water, and concentrated and dried at reduced pressure to yield a product containing liquiritigenin. Liquiritigenin contents in the product were measured using a method for measuring liquiritigenin content in Example 1, and the results were recorded in Table 8.
Table 8
Hydrolytic solvent Liquiritin content (%)(Organic solvent + water) Liquiritin content (%)(Organic solvent)
Water 8.5
Methanol 6.9 5.4
Ethanol 6.3 4.5
Isopropanol 6.4 3.1
n-propanol 5.3 3.4
n-butanol 7.2 3.2
t-butanol 6.2 2.8
Dimethyl formamide 7.3 5.4
Acetone 6.1 4.7
Ethyl acetate 7.5 6.4
Tetrahydrofuran 6.9 5.8
Dimethyl ether 5.2 2.8
Diethyl ether 5.3 2.3
n-hexane 6.9 0.9
Benzene 5.7 3.2
Toluene 5.9 3.8
Xylene 5.9 3.7
Dichloromethane 4.7 1.4
Chloroform 4.8 1.8
As indicated in Table 8, it is determined that when water and an organic solvent were added during the hydrolysis, liquiritigenin content was slightly lower than the content when only water was used, and higher than the content when an organic solvent was only used.
Therefore, in a method according to the present invention, Glycyrrhizae radix extract containing liquiritin was introduced and dispersed into water, an acid was added into the solution, and then reaction temperature was increased to increase the liquiritigenin content.
<Experimental Example 3> Method of increasing liquiritigenin precipitate content
(1) Liquiritigenin content according to a kind of solvent
An acid was added into a Glycyrrhizae radix extract containing liquiritin according to the present invention to hydrolyze the liquiritin into liquiritigenin, and then a resulting precipitate was dissolved in an organic solvent, washed with purified water, and experiments were performed as follows to know how much impurities were removed and liquiritigenin content was increased.
About 10 g of the Glycyrrhizae radix extract containing liquiritigenin obtained in <Experimental Example 2> was introduced into about 100 ml of an organic solvent, the solution was washed twice with about 100 ml of water, and the organic solvent was distilled and dried under reduced pressure to obtain a product containing liquiritigenin.
Liquiritigenin contents in the product were measured using a method for measuring liquiritigenin content in Example 1, and the results were recorded in Table 9.
Table 9
Hydrolytic solvent Liquiritigenin content (%) Weight of Glycyrrhizae radix extract (g)
n-butanol 10.3 8.6
t-butanol 10.7 8.1
Ethyl acetate 11.5 7.5
Dimethyl ether 8.9 9.3
Diethyl ether 9.2 9.6
Dimethyl formamide 10.4 8.2
n-hexane Insoluble
Benzene 8.6 9.1
Toluene 9.0 9.4
Dichloromethane 8.7 9.8
Chloroform 8.5 9.9
As indicated in Table 9, it is determined that butanol, ethyl acetate, and dimethyl formamide as a polar solvent showed more than about 10 % liquiritigenin content while Glycyrrhizae Radix et Rhizoma was not well dissolved or an increase in liquiritigenin content was infinitesimal in non-polar solvents. It is confirmed that during the process in which the extract was dissolved in the organic solvent and washed with water, impurities other than liquiritigenin were removed.
(2) Liquiritigenin content according to liquiritin content in Glycyrrhizae Radix et Rhizoma
In order to measure liquiritigenin content according to the amount of liquiritin in Glycyrrhizae Radix et Rhizoma, experiments were performed on each Glycyrrhizae Radix et Rhizoma having different liquiritin contents in the same way as in <Example 4> to yield a product containing liquiritigenin, liquiritigenin contents in the product were measured using a method for measuring liquiritigenin content in Example 1, and then the results were recorded in Table 10.
Table 10
Liquiritin content(%) Liquiritigenin content (%) Weight of Glycyrrhizae radix extract (g)
0.52 5.7 6.1
0.78 8.4 6.2
0.98 11.5 6.3
1.35 14.2 6.3
1.81 18.3 6.8
2.54 23.1 7.1
As indicated in Table 10, it is determined that as the liquiritin content in Glycyrrhizae Radix et Rhizoma increased, the liquiritigenin content and the yield of Glycyrrhizae Radix et Rhizoma or Glycyrrhizae radix extract increased. To increase the yield, it is preferred to select a Glycyrrhizae Radix et Rhizoma having a high liquiritin content and perform an extraction on it.
<Experimental Example 4> Method for drying a Glycyrrizae radix extract containing liquiritigenin
According to the present invention, the extract containing liquiritigenin in <Example 1> was dissolved in ethyl acetate and dried using the following method during the process of drying the ethyl acetate, and then the amount of the residual solvent, in which the Glycyrrhiza radix extract containing liquiritigenin was dissolved, was measured. The results were recorded in Table 11.
1) The extract was dried at about 40 ℃ for about 48 hours using a hot air drying method.
2) The extract was dried at about 50 ℃ for about 48 hours using a hot air drying method. The extract was entangled each other as to tend to become a highly viscous material.
3) The extract was dried at about 50 ℃ for about 24 hours using a method of drying under reduced pressure.
4) The extract was dried at about 60 ℃ for about 24 hours using a method of drying under reduced pressure.
5) The extract was dried at about 70 ℃ for about 24 hours using a method of drying under reduced pressure.
6) The extract was dried at about 50 ℃ or higher for about 5 hours using a method of drying under reduced pressure, and then about 30 g of a dry matter was dissolved in about 300 ml of ethanol and the solution was dried at about 50 ℃ for about 24 hours using a method of drying under reduced pressure.
7) The extract was dried at about 50 ℃ or higher for about 5 hours using a method of drying under reduced pressure, and then about 30 g of a dry matter was dissolved in about 300 ml of ethanol and the solution was dried at about 60 ℃ for about 24 hours using a method of drying under reduced pressure.
8) The extract was dried at about 50 ℃ or higher for about 5 hours using a method of drying under reduced pressure, and then about 30 g of a dry matter was dissolved in about 300 ml of ethanol and the solution was dried at about 70 ℃ for about 24 hours using a method of drying under reduced pressure.
Table 11
Drying method Residual solvent (%)
Ethyl acetate ethanol Total
1) 7.2 0 7.2
2) 6.4 0 6.4
3) 2.7 0 2.7
4) 2.5 0 2.5
5) 2.2 0 2.2
6) 0 1.4 1.4
7) 0 1.1 1.1
8) 0 0.7 0.7
As indicated in Table 11, it is preferred to dissolve the precipitate containing liquiritigenin in <Example 1> in ethyl acetate and dry the ethyl acetate using a drying under reduced pressure rather than a hot air drying, in terms of reduction in the residual solvent. It is determined that as the temperature increased during the drying under reduced pressures, the amount of the residual solvent decreased.
In addition, it is confirmed that when a drying was performed under reduced pressure, for example, the precipitate was dissolved in ethyl acetate, which was then dried under reduced pressure, and then the resulting precipitate was dissolved in ethanol, which was then dried under reduced pressure, the amount of the residual solvent significantly decreased.
Therefore, a precipitate containing liquiritigenin is dissolved in an organic solvent, which may be dried using a drying method under reduced pressure, and then the resulting precipitate may be again dissolved in ethanol, which may be additionally dried using the same drying method under reduced pressure.
Because a high-priced solvent is not needed, a column chromatography process may be omitted as to simplify the steps, and an extract containing a higher liquiritigenin content (about 8 to 23 %) than that obtained by a conventional method may be obtained using the method of the present invention, the method is useful during an extraction of liquiritigenin.
Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Claims (12)

  1. An extraction method for increasing liquiritigenin content in Glycyrrhizae Radix et Rhizoma or Glycyrrhizae radix extract, the method comprising:
    using water, an organic solvent, or a mixed solvent of water and the organic solvent to Glycyrrhizae Radix et Rhizoma to extract a product containing liquiritin (Step 1); and
    introducing water, an organic solvent, or a mixed solvent of water and the organic solvent into the product containing liquiritin extracted in Step 1, adding acid or base into the solution, and hydrolyzing the mixture to prepare a product containing liquiritigenin (Step 2).
  2. The method as set forth in claim 1, wherein the organic solvent in Step 1 and Step 2 is selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, n-butanol, t-butanol, acetone, acetonitrile, ethyl acetate, tetrahydrofuran, dimethyl ether, diethyl ether and dimethyl formamide, benzene, toluene, and xylene.
  3. The method as set forth in claim 2, wherein the concentration of the methanol or ethanol is 70% to 100%.
  4. The method as set forth in claim 1, wherein the extraction in Step 1 is performed at 30 ℃ to 100 ℃.
  5. The method as set forth in claim 1, wherein the extraction method in Step 1 is heating and stirring extraction, reflux extraction, or ultrasonic extraction.
  6. The method as set forth in claim 1, wherein the acid in Step 2 is sulfuric acid, hydrochloric acid, nitric acid, or a mixed solution thereof and the base in Step 2 is sodium hydroxide, potassium hydroxide, calcium hydroxide, or any mixture thereof.
  7. The method as set forth in claim 1, wherein the acid in Step 2 is 1 M to 5 M sulfuric acid, and the base in Step 2 is 1 M to 5 M calcium hydroxide.
  8. The method as set forth in claim 1, wherein the hydrolysis reaction in Step 2 is performed at 50 ℃ to 100 ℃.
  9. The method as set forth in claim 1, further comprising:
    dissolving the product containing liquiritigenin obtained in Step 2 in an organic solvent, washing the solution several times with water, and concentrating and drying an organic layer under reduced pressure to increase liquiritigenin content.
  10. The method as set forth in claim 9, wherein the organic solvent is selected from the group consisting of n-butanol, t-butanol, ethyl acetate, dimethyl ether, diethyl ether, diethyl ether and dimethyl formamide, benzene, toluene, and xylene.
  11. The method as set forth in claim 9, wherein the drying is a drying under reduced pressure.
  12. The method as set forth in claim 9, further comprising:
    dissolving the product containing liquiritigenin obtained in Step 2 in an organic solvent, washing the solution several times with water, concentrating and drying an organic layer under reduced pressure, and then dissolving the product containing liquiritigenin in ethanol and drying the solution under reduced pressure to decrease the amount of the residual solvent.
PCT/KR2009/004331 2008-08-01 2009-08-03 Extraction method for increasing liquiritigenin content in glycyrrhizae radix et rhizoma or glycyrrhizae radix extract WO2010013987A2 (en)

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KR100637358B1 (en) * 2004-01-13 2006-10-20 재단법인서울대학교산학협력재단 Composition comprising the extract of Glycyrrhizae radix for preventing the toxicity of Endocrine Disruptors
KR100592482B1 (en) * 2004-07-19 2006-06-26 경북대학교 산학협력단 Methods for increasing liquiritigenin content in a licorice and its extracts and the method for the isolation and extraction of liquiritigenin from them
KR100535872B1 (en) * 2005-07-28 2005-12-09 재단법인서울대학교산학협력재단 Composition comprising extract of Glycyrrhizae radix or liquiritigenin for the treatment and prevention of diseases caused by heavy metal poisoning
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