KR20150037449A - A method of extracting hypericin from hypericum perforatum - Google Patents

Abstract

The present invention relates to a method for extracting hypericin from Hypericum perforatum, and more specifically, to a method for extracting hypericin from Hypericum perforatum comprising the following steps: extracting Hypericum perforatum crushed materials using a 75 percent ethanol solution; and preparing ethyl acetate fractions by adding ethyl acetate to the ethanol solution prepared by the extraction step and fractioning the same. The extraction method of the present invention can isolate the hypericin from the Hypericum perforatum with high purity and yield, thereby being widely used in various industries related to medicines and functional foods using the hypericin.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for extracting hyperlysine from a plant,

The present invention relates to a therapeutic agent for the treatment of female menopausal syndrome, particularly hyperlysine, in high purity and high efficiency.

In women, the function of the ovaries declines with age, and this leads to a significant decrease in estrogen secretion. Due to the reduced secretion of estrogen, the response to follicle-stimulating hormone and luteinizing hormone is reduced, the initial follicle shortening is shortened, the menstrual cycle is further shortened, the ovulation phase is further shortened, and progesterone production is reduced. Eventually, the follicle becomes unresponsive and does not produce estrogen.

It is known that climacteric is a transitional period in which physiological function and sexual function are reduced or eliminated due to an overall and gradual decrease of ovarian function, and ovulation is stopped after ovarian function is stopped as a part of menopausal period, A permanent stop is called menopause. On the other hand, the World Health Organization (WHO) defined menopause as a period when ovarian function is lost and estrogen secretion is no longer conceived, and that the transition from adulthood to old age is over.

As described above, women gradually start ovarian function gradually from the age of 40 after the menopausal period, and the average life span of women is gradually extended, the period of hormone deficiency is about 1/3 to half of the life span. Thus, there is an increasing need for special attention to the healthy life of women after menopause.

For example, in Korea, the proportion of elderly people aged 65 or older is estimated to be 9.1% in 2005, and it is expected to be more than 14% of the population in 2019. The average life expectancy of women is higher than that of men In 2002, the average life span of women was 80.4 years. Therefore, women in Korea should live for about 40 years after menopause. In this respect, interest in women's health after menopause is increasing .

The chronic chronological symptoms due to changes in the body during the menopausal period, including the menopause or previous menstrual changes due to changes in hormones, specifically estrogen production, follicle stimulating hormone and luteinizing hormone, are called menopausal symptoms or menopausal symptoms.

In particular, the decrease in estrogen affects not only the female urinary system, genitalia but also other parts of the body, so estrogen secretion decreases greatly throughout the body.

For example, most menopausal women are experiencing acute female hormone deficiency symptoms such as hot flush, tachycardia, sweating, anxiety or sleep disorder, and in some women, chronic female hormone deficiency causes cardiovascular disease, osteoporosis Of chronic illnesses.

Specifically, symptoms of menopausal symptoms include symptoms due to vascular changes such as hot flush, night sweat, tachycardia or headache, and symptoms due to musculoskeletal changes such as myalgia, joint pain and back pain , Memory loss, depression, loss of concentration, anxiety, and dizziness.

In addition, since the estrogen is reduced, the supply of blood is not smoothly performed and the elasticity and moisture of the skin and vaginal mucosa are lost. For example, when the mucous membranes of the eyes, nose, or mouth are dry, they may experience uncomfortable eyes or thirsty burns.

In addition, symptoms such as vaginal dryness, urinary tract infections, urinary frequency or urinary incontinence appearing in the years after menopause and epidermal symptoms, osteoporosis after years of menopause, cardiovascular disease such as atherosclerosis or cerebrovascular disease . In particular, heart disease is one of the major causes of death in postmenopausal women, and many women are suffering from osteoporosis because of the rapid bone loss caused by estrogen deprivation after menopause.

As described above, the necessity of treatment for postmenopausal women is becoming more and more important, and various methods have been studied for the treatment of the symptoms of postmenopausal women, namely, postmenopausal syndrome. For the treatment of postmenopausal syndromes, Hormone Replacement Therapy (HRT) is widely used to administer estrogen.

There are two types of estrogen: natural estrogen and synthetic estrogen. Oral administration is the most widely used method of administering the synthetic estrogen hormone preparation, and parenteral administration includes transdermal patches, transdermal cream, gel, and vaginal cream.

However, such an artificial synthetic estrogen is effective for the improvement of the menopausal symptoms, but it increases the activity of the cancer-inducing gene in the long-term use, thereby posing a risk of uterine cancer or breast cancer and possibly causing venous thrombosis or gallbladder disease. Is being heated.

Accordingly, studies have been made actively to find a estrogen derived from a natural product having a similar activity to a synthetic estrogen as a substitute for the synthetic estrogen used in the above-mentioned hormone replacement therapy, while having a less side effect.

Premarin, made from natural product-derived estrogens and from the animal's natural urine, has been shown to increase the levels of estradiol and estrone in clinical trials to a monthly level. However, there have been reports of serious side effects such as increased breast cancer (26%), heart attack (29%) and fainting (44%), and blood coagulation (2 times) It ended itself in five years, and the US Food and Drug Administration (FDA) decided in 2003 to put a warning on hormone medicines.

Although the use of conventional hormone replacement agents such as synthetic estrogen and animal estrogen products can cope with overall menopausal symptoms, there is a problem that their use is limited due to serious side effects.

Therefore, there is a trend toward research on natural estrogen derived from plant having a similar activity to synthetic estrogen as a substitute for synthetic estrogen used in the above-mentioned hormone replacement therapy and having less side effects.

In particular, plant-derived natural estrogens are often referred to as phytoestrogens.

The vegetable estrogen refers to a non-steroidal compound derived from a plant which is generally similar in function and structure to female hormone estrogen, and is found in the stem, root, flower or seed of a plant.

Examples of the above vegetable estrogens include isoflavone derived from soybean and bean, coumestan derived from clover, and lignan derived from flax seed. In addition, there are flavanones, flavones, dihydrocalchones, and the like.

For example, isoflavones exist as inactive glycosides of biochanin A and formononetin in plants, but are absorbed into the body and are similar to estrogen by glucosidase in intestinal bacteria (Genistein) and daidzein (daidzein), resulting in estrogen-like activity.

Genistein, which is a representative example of the vegetable estrogen, has been reported to inhibit the protein tyrosine kinase of cells and thus to inhibit the growth of cancer cells. However, it has also been reported that it may cause cancer in people who have no effect or who have a genetic predisposition for uterine cancer and breast cancer.

Therefore, it is required to develop new phytoestrogenic estrogens having excellent estrogenic activity without side effects such as the side effects of genistein. Due to this necessity, it is urgently required to objectively verify the natural resources that have been used for a long time as a food and have proved its safety, and to develop a natural substance having a therapeutic, improving or preventive effect on female postmenopausal syndrome.

As a result of research by the present inventors, hyperlysine, which is known to be effective for existing antidepressants, is expected to be effective for the treatment, improvement or prevention of female postmenopausal syndrome due to its excellent estrogenic activity. It was evaluated that the safety was recognized and the possibility of occurrence of side effects included in the drinking water was evaluated to be low.

Therefore, in order to utilize the hyperlysine, when the hyperlysine can be extracted in high yield and high purity using a highly safe solvent such as water or alcohol used for food, Is expected to be excellent.

KR 10-1067028 B KR 10-1075554 B KR 10-2006-0012316 A

In order to solve the problems of the prior art as described above, it is an object of the present invention to provide a method of extracting hyperlysine with high purity and high yield by using a solvent which can be used for various purposes.

In order to accomplish the above object, the present invention provides a method for extracting hyperlysine from a juice extract. More particularly, the present invention relates to a method for producing an extract, which comprises the steps of: extracting a supernatant of water with a 75% aqueous ethanol solution; and adding ethyl acetate to the ethanol aqueous solution produced by the extraction step to produce an ethyl acetate fraction The method comprising the steps of:

Hereinafter, the present invention will be described in more detail.

The present inventors confirmed that hyperlysine, which is an active ingredient contained in Jojoba, a plant that has been consumed in a long time difference form, exhibits estrogen-like activity and has therapeutic, ameliorative or preventive effects on menopausal diseases, In order to establish a method for extracting hyperlysine from the supernatant in a high purity and a high yield, the solvent extraction method is more preferable than the supercritical extraction method widely used for the extraction of active ingredients recently Further, in order to confirm the optimum conditions in relation to the solvent extraction method, further experiments on the object to be extracted, that is, the extraction site, the kind of solvent, the extraction condition and the fraction solvent were further conducted. As a result, As a result of using a water solvent and an aqueous solution of ethanol, the extracts of 75% ethanol It was confirmed that the high purity hyperlysine can be extracted at the highest yield when the fraction is extracted with an ethyl acetate fraction after the extraction at 60 ° C as an aqueous solution.

As used herein, the term " extract " means an extract obtained by treating an extractive solvent with a plant or a product prepared by the method of producing an extract, unless otherwise specified. The extract may be formulated ) Results.

As used herein, unless specified otherwise, menopausal syndrome or menopausal symptoms refers to symptoms resulting from a decrease in hormone production in postmenopausal women, and generally includes facial flushing, sweating, sweating during sleep, dry skin, vaginal dryness, vaginal dryness Is a concept that includes atrophy, atrophy of the lower urinary tract, intercourse, vaginitis, cystitis, dysuria, urinary incontinence, concentration disorders, short term memory impairment, anxiety, nervousness, memory loss, loss of libido, myalgia, arthralgia or osteoporosis.

Unless otherwise specified herein, treatment or amelioration refers to any action by which administration of a composition of the invention improves or alleviates symptoms of female postmenopausal syndrome.

As used herein, " prophylactic " means any action that inhibits the symptoms of female postmenopausal syndrome by administration of the composition of the present invention, unless otherwise specified herein.

In the present invention, a supercritical fluid is a material in which the two states of a liquid and a gas are in a critical state in which they can not be distinguished from each other, and when the vapor pressure is under a temperature and a pressure higher than its critical point Means a fluid in a state above a supercritical point, and supercritical extraction means extraction using a supercritical fluid.

The hyperlysine extracting method of the present invention comprises the steps of: preparing the extract of Needle Supernatant by a solvent extraction method; And a method for extracting hyperlysine from the jujitsu, which comprises the step of adding ethyl acetate to the thus-prepared jujuba extract and solvent fractionation to prepare an ethyl acetate fraction.

More specifically, the hyperlysine extraction method of the present invention comprises the steps of washing a required amount of water, drying and then pulverizing to produce a desired super-pulverized product; And a step of adding an aqueous ethanol solution to the supernatant and performing a solvent extraction method; And a method for extracting hyperlysine from the jujitsu, which comprises the step of adding ethyl acetate to the thus-prepared jujuba extract and solvent fractionation to prepare an ethyl acetate fraction.

The Hypericum perforatum , St. John? wort) is one of the hubs originating in Europe and West Asia. It grows up to about 1 m in height. The rootstock of this juvenile is thick and short, and there are many stems here, stems are hard at the bottom, The juvenile ginseng runs from June to August with a bright yellowish orange flower with an acute pedicel, with five petals in an oval shape. When the flower grows, the fruit is opened, and a small, round black seed is in it. It is easy to grow at home because it is strong enough to grow well in the sunlight as well as in the place where the sunlight shines well, and it breeds by the cutting and sowing. It has been known from the past that it has been eaten as a herbal tea for the purpose of treating depression, treating insomnia, relieving stress, etc., and has been known to have effects such as astringent action, antibacterial action or anti- .

The above-mentioned candle can be used both on the ground and underground, specifically flowers, leaves, stalks or roots, and the candle is preferably selected from the group consisting of flowers and leaf parts, It can be a mixture of flowers and leaves, such as more than species, for example, candles. Regarding the above-mentioned jujube root parts, estrogen-like activity was found in the extracts of the mixture of flowers and leaves of the above-mentioned Jojanga other than the extracts prepared from all parts of Jojangaproi extract, Is contained in higher purity and yield, and in this respect the essential candle may preferably be a blend of flowers and leaves of the candle.

The step of preparing the extract may include a step of preparing the supernatant and a step of performing a solvent extraction method.

The process for preparing the above-mentioned super-pulverized product may be carried out by washing the pulverized product, drying and then pulverizing the pulverized product to prepare a desired super-pulverized product.

The candle may be preferably a mixture of at least one candle selected from the group consisting of candle leaf and candle candle, for example, candle candle and candle candle.

In addition, the syringe may be prepared in powder form to maximize the contact area with the extraction solvent to increase the extraction yield. Thus, the step of preparing the desired extract may include a step of preparing the required superfine powder. The step of preparing the required ultrafine powder may be carried out by, for example, a method of hot-air-drying and pulverizing the ultrafine powder at 50 ° C to 70 ° C.

In this respect, the process for preparing the above-mentioned ultra-pulverized product may be carried out by washing the seedlings and drying the pulverized leaves, and then pulverizing the pulverized products to prepare pulverized products.

The solvent extraction method may be performed by adding an aqueous solution of ethanol to the supernatant and performing a solvent extraction method.

The aqueous ethanol solution may be 70% (w / w) ethanol aqueous solution to 80% (w / w) ethanol aqueous solution, preferably 75% (w / w) ethanol aqueous solution in terms of extraction yield and purity.

The solvent extraction method may be carried out at 55 to 65 ° C, preferably at 60 ° C in terms of extraction yield and purity. As a result, it was confirmed that extraction yield and purity were both low at 40 ° C and the yield was particularly low at 80 ° C, and it was confirmed that performing at 60 ° C was preferable in terms of yield and purity .

Regarding the extraction method, the extraction yield and purity of the supercritical extraction method used for the extraction of the active ingredient were found to be lower than those of the optimum extraction conditions using the aqueous ethanol solution, even when the extraction pressure was adjusted differently. It has been confirmed that a solvent extraction method using an aqueous ethanol solution is preferable.

The step of preparing the desired fraction may be carried out by fractionation using a conventional fraction solvent.

It was confirmed that ethyl acetate was preferable in terms of the extraction yield of the fraction solvent. The ethyl acetate fraction showed an extraction yield of 200% to 1,500% or more as compared with the other fraction solvents (FIG. 3).

The solvent fractionation may be performed by a conventional method. Specifically, ethyl acetate may be added to the crude extract to prepare an ethyl acetate fraction.

For example, the same amount of hexane is added to the extract prepared by the solvent extraction method using the 75% ethanol aqueous solution, left to shake from left to right, and then left to separate the layers. Then, a hexane layer and a water layer After separating the hexane fraction, the same amount of chloroform was added to the water layer. The mixture was shaken to the left and right so that the two solutions became well reacted. Then, the mixture was left to separate the layers, and then the chloroform layer ) And a water layer, the chloroform fraction was separated, and the same amount of ethyl acetate was added to the water layer. The mixture was shaken to the right and left to allow the two solutions to react well, When the ethyl acetate layer and the water layer are separated, they can be carried out by a method of obtaining an ethyl acetate fraction.

The present invention relates to a method for separating hyperlysine from an initial stage to a high yield and purity, which has been confirmed to have estrogenic activity and has been isolated from the essential extract, The present invention also provides a method for obtaining hyperlysine which is excellent in estrogen-like activity and can be used for various purposes in the future for the treatment, improvement or prevention of menopausal disease, It can be widely applied to a health functional food, medicine, or cosmetics related industry that can be applied to various diseases related to hyperlysine including diseases.

FIG. 1 is a view illustrating a process for preparing extract and fraction according to an embodiment of the present invention.
FIG. 2 is a graph showing hyperlysine content of an extract according to an extracting site according to an embodiment of the present invention. FIG.
FIG. 3 is a graph showing the hyperlysine content of a fraction of the aqueous extract of Yohan's ethanol aqueous solution according to the fraction solvent according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to production examples and examples. However, the following Preparation Examples and Examples are merely illustrative examples for the purpose of facilitating understanding of the present invention, and can be modified into various other forms, and the scope of the present invention is not limited to the following Examples.

Example  1: extract and Fraction  Manufacturing method

In the case of Johannesch, dried chrysanthemums imported from China were used. The chrysanthemums were washed, separated by site, dried in a hot air, and pulverized by a crusher. The leaves were separated from leaves and flowers (mixture of flowers and leaves), and stems were separated from the ground.

The extracts were prepared by solvent extraction and supercritical extraction. The fractions were also fractionated by solvent fractionation. A specific method of carrying out the present invention is shown in Fig.

First, in the solvent extraction method, 10 g of the pulverized product or the pulverized pulverized product of each of the above essential components and 200 ml of the solvent were put into an extraction container and extracted.

The solvents used were water, 25% ethanol aqueous solution, 50% ethanol aqueous solution, 75% ethanol aqueous solution and 100% ethanol. Extraction temperatures were 40 ° C., 60 ° C., 80 ° C. and 100 ° C.

The supercritical extraction was carried out by using supernatant extraction apparatus (SFE 5L, Natex, Austria) at 300 ° C. for 30 seconds at a temperature of 40 ° C. dhseh at 150 bar, 250 bar and 350 bar pressure condition. Carbon dioxide was used as supercritical extraction solvent.

Each of the extracted extracts was subjected to the extraction method, followed by filtration and concentration, and then dried at -20 ° C. to prepare an extract.

The extract prepared by the above-mentioned preparation method was fractionated while sequentially treating an organic solvent to prepare a fraction. Specifically, the same amount of hexane was added to the extract solution, and the mixture was shaken to the left and right, followed by allowing the layer to separate. After separation into a hexane layer and a water layer, the hexane fraction was transferred to water and an equal amount of chloroform was added to the remaining water layer. The solution was shaken to the left and right to allow the two solutions to react well, and then left to separate the layers. When the chloroform layer and the water layer were separated, the chloroform fraction was transferred to water and an equal amount of ethyl acetate was added to the remaining water layer. The solution was shaken to the left and right to allow the two solutions to react well, and then left to separate the layers. Ethyl acetate layer and water layer were separated. The ethyl acetate fraction was transferred to hand and the same amount of butanol was added to the remaining water layer. The solution was shaken to the left and right to allow the two solutions to react well, and then left to separate the layers. The Buthanol layer and the water layer were separated and the butanol fraction was transferred to hand and the remaining water layer was transferred to the hand. Fractions were prepared by evaporating fraction solvents such as hexane, chloroform, ethyl acetate, butanol and water from each of the fractions collected in the manual, followed by drying at -20 ° C.

The prepared extracts and fractions were stored under refrigeration conditions and dissolved in DMSO if necessary.

Example  2: Depending on extraction conditions Hyperlysine  Yield and purity

In order to measure the yield and purity of hyperlysine in the extracts and fractions according to each of the above extraction methods and conditions, the content of the extract and the content of hyperlysine in the extract were measured.

Hyperlysine in the extract and fraction was measured by the following method.

First, a hyperlysine standard solution was prepared to measure the hyperlysine content. Specifically, 10 mg of hyperlysine was precisely weighed and dissolved in methanol to prepare a solution having a final concentration of 1 mg / mL, and used as a standard stock solution. The standard stock solution was stored at 5 ° C to 15 ° C and used within 30 days. When quantifying, the standard stock solution was accurately taken and diluted with methanol to a concentration of 25 and 50? / ML, and used as a standard solution.

0.5 g of the extract or fraction was precisely weighed and placed in a stoppered flask equipped with a stopper. 25 mL of methanol was added, and the mixture was shaken occasionally and ultrasonically extracted for 15 minutes. After cooling the extract, collect the supernatant, and add 20 mL of methanol again to the residue, collect all the extracts, add methanol to make exactly 50 mL, and use this solution as the sample solution.

Each 20 μL of each of the prepared test solution and standard solution was tested according to the liquid chromatographic method under the following conditions, and the peak areas AT and AS of the test solution and the standard solution were measured and the hyperlysine content was calculated by the following calculation formula.

[formula]

The above operating conditions are as follows.

First, the detector was measured at 590 nm using an ultraviolet absorption spectrophotometer.

The column used in the liquid chromatography method was a stainless steel pipe having an inner diameter of about 4 mm to 6 mm and a length of 15 cm, and the column was filled with octadecylsilyl silica gel for a liquid chromatograph of 5 μm .

As the mobile phase, a mixed solution of methanol and ethyl acetate in a ratio of 4: 1 was used as the A mobile phase, and 10 mM phosphate buffer (pH 2.0) was used as the B mobile phase. The flow rate was maintained such that the flow rate was 1 mL / min and the retention time of the hyperlysine was adjusted to about 7 to 8 minutes.

The hyperlysine content determined according to the above assay was expressed in% using the weight of each extract and fraction.

First, when the supernatant was extracted with supercritical extraction method, the lower apicalin was detected to be 0.0001% or less regardless of the size of the supernatant, and it was confirmed that the extraction yield was not preferable.

With respect to the solvent extraction method, the extraction yield was found at 60 캜 using a 75% ethanol aqueous solution. As shown in Fig. 2, the extraction yield of the extracted portion was hardly extracted in the case of stem, It was found to be most preferable.

Further, as a result of checking the extraction solvent and extraction conditions, it was confirmed that almost no hyperlysine occurred in the water and the aqueous 40% ethanol solution, and it was hardly extracted even at 40 ° C. Therefore, as a result of performing at 60 ° C, 80 ° C and 100 ° C, it was confirmed that extraction with 75% ethanol at 60 ° C was most preferable as shown in Table 1 below.

In addition, in the case of the fraction solvent, as shown in Fig. 3, it was confirmed that the highest hyperlysine content was obtained when the fraction was fractionated by ethyl acetate fraction.

Claims (2)

A step of washing with water, drying and then pulverizing to prepare an ultra-pulverized product; And adding a 75% ethanol aqueous solution to the required superfine powder and conducting a solvent extraction method at a temperature of 60 ° C .; And
Adding ethyl acetate to the thus-prepared jujuba extract, followed by solvent fractionation to prepare a crude fraction for producing ethyl acetate fraction
A hyperlysine extraction method for extracting hyperlysine from the root of the plant. The method according to claim 1,
The process for preparing the above-mentioned super-pulverized product is characterized in that the process for preparing the pulverized product of the present invention is carried out by washing the seedlings and drying the pulverized product, Extraction of hyperlysine.

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