KR0163813B1 - Extraction and Purification of Active Active Ingredients from Phosphorus and Herbal Composition Containing the Extracts - Google Patents

Abstract

The present invention relates to a method for extracting an active ingredient from phosphorus and a herbal composition containing the extract, and more particularly, to adjust the mixing ratio of the leaves and stems of phosphorus used as a herbal medicine and to extract the active ingredient therefrom. The present invention relates to a method for efficiently extracting and purifying an effective ingredient having excellent anti-inflammatory analgesic activity and to a herbal composition containing the extracted and purified extract.

Description

A method for extracting and purifying an active ingredient from phosphorus and a herbal composition containing the extract

The present invention relates to a method for extracting an active ingredient from phosphorus and a herbal composition containing the extract, and more particularly, to adjust the mixing ratio of the leaves and stems of phosphorus used as a herbal medicine and to extract the active ingredient therefrom. The present invention relates to a method for efficiently extracting and purifying an effective ingredient having excellent anti-inflammatory analgesic activity and to a herbal composition containing the extracted and purified extract.

Indong is a semi-evergreen vine shrub of the Capriofoliaceae family that grows under the forests of the country's wild field, and the bud is called 'Gold Silver Coin' and the leaves and stems are called 'Indongdeung'. This is a plant that has been widely used in herbal medicine.

The active ingredients contained in phosphorus and the like so far known are Lonicerin (luteolin-7-rhamnoglucoside), caffeoylquinicacid, methyl caffeate, chlorogenic acid, iso-chlorogenic acid (iso flavonoids such as -chlorogenic acid; Biflavonides, such as ochnaflavon; A kind of saponin is fulvotomentoside A, α-hederin, sapindoside B, and new material Loniceroside A identified by the present inventors, B (Korean Patent Publication No. 94-2267); Contains iridoid glycosides such as Loganin, Vogeloside, Epi-vogeloside, etc. Turned out to be. These sinus has been widely used in arthritis, bronchitis, asthma, etc., since the prosthesis is effective for all scabs (inflammation of the inside and the outside), and in recent years, pandemic flu, various purulent infections, moist fever It is known to be effective in restoring joint redness, swelling, hepatotoxicity, and recovery of decreased cellular immunity.

However, most of the traditional Chinese medicine and related literatures mention only herbal medicines for humans themselves. It has never been.

In particular, there are no studies on the active ingredients contained in the stems and leaves of the phosphorus, the changes in the contents of the active ingredients according to the harvesting time, and the composition of the active ingredients representing the optimal condition for the anti-inflammatory analgesic effect. Since the use of phosphorus extract was only the situation to use by the traditional method of herbal medicine.

Therefore, the present inventors have tried to maximize the extraction efficiency of the herbal ingredients and use the phosphorus more scientifically, and as a result, the stem and leaves of the phosphorus are collected at the growth period with the highest content of the active substance, and the addition ratio of the stem and the leaf is optimized. The present invention was completed by finding a method of extracting an active ingredient, and developing a herbal composition from which the extract having excellent anti-inflammatory analgesic activity was scientifically formulated and standardized.

The present invention is to provide a method for extracting the active ingredient with anti-inflammatory analgesic physiological activity to the most active composition by adjusting the timing of harvesting, mixing ratio of leaves and stems, and a herbal composition containing the extract active ingredient There is this.

Hereinafter, the present invention will be described in detail.

In the present invention, in extracting and purifying an active ingredient from phosphorus, the dried and ground phosphorus leaves are collected after harvesting the dried phosphorus flower and the dried and ground phosphorus leaves are collected in the flowering period of the phosphorus. The mixture is mixed at a weight ratio of 5 to reflux with water, and then filtered and purified.

In addition, the present invention is 0.5-3.0% by weight of Loniceroside A extracted from phosphorus, 0.5-2.0% by weight of Lonieroside B, 0.5-3.0% by weight of Lonicerin, Loganin Herbal composition containing 2.0 to 5.0% by weight).

Referring to the present invention in more detail as follows.

The present invention is a method for extracting and purifying an active ingredient having excellent anti-inflammatory analgesic physiological activity from the stems and leaves of phosphorus, and the stems and leaves of the phosphorus used in the present invention can be maximized by using the ones collected at specific times. Can be.

According to the present invention, it is most preferable to collect the honeysuckle leaves immediately after the flowers of the honeysuckle are in full bloom, and the seasons in Korea usually correspond to the end of June to the beginning of July.

In addition, it is most preferable to collect the stem of the stem after the flower of Indong has completely disappeared, which is usually between October and December.

In this way, the leaves and stems collected at the most appropriate time are well dried and ground to a suitable size and mixed. The mixing ratio is such that the weight ratio of the stem and the leaves is 7: 3 to 5: 5.

According to the present invention, there is a big difference in the content of the active ingredient contained therein according to the harvesting time of the stem and leaves of the honeysuckle, in the case of the honeysuckle stem, which corresponds to the winter season, that is, after the flower has been completely lost Compared to that, the active ingredient was increased about 2 times, and in case of artificial leaves, the active ingredient was increased about 3 to 5 times immediately after the flower was in full bloom.

In addition, in the present invention, the mixing ratio of the phosphorus stem and leaves with the harvesting time of the phosphorus also has an important meaning. Because of the ratio of stem and leaves collected at the highest active ingredient content, the anti-inflammatory analgesic changes greatly, probably due to the synergistic effect at the most suitable ratio. Therefore, the case where the ratio of use of the stem and the leaf should be selected should be selected. According to the present invention, it was found that the case where the stem was mixed at a slightly higher weight than the leaf showed the most desirable anti-inflammatory and analgesic activity.

Therefore, in the present invention, it was found that it is most preferable that the use ratio of the stem and leaves of the phosphorus collected at a suitable time is set to 7: 3 to 5: 5 by weight. Here, when the harvesting time of the copper leaf and the stem is different or the added weight ratio is out of the ratio, there is a problem that the drug efficacy is lowered by causing the content change of the anti-inflammatory analgesic physiologically active ingredient.

In the present invention, the mixed herbal leaves and stems of the herbal medicines extracted with water, 8 to 12 times the weight of the herbal raw material is added by reflux extraction for about 5 hours and then filtered to collect the filtrate, and the residue 3 again the weight of the herbal raw material weight The water extraction efficiency is increased by adding ˜7 times of water, heating and filtration and mixing the filtrate with the previous filtrate.

If the amount of water is too small in the extraction with water, it is difficult to stir and the solubility of the extract is lowered, the extraction efficiency is reduced, if too much, the amount of the lower alcohol solvent used in the next purification step becomes more economically disadvantageous. Defects occur in handling, and the extraction again is to improve the extraction efficiency. Even though the filtration is effective in mass production, the loss of the herbal medicine is high because the water content of the herbal medicine is high. This is to prevent this.

As such, the active ingredient extract extracted from phosphorus contains impurities such as chlorophyll and unnecessary fatty acids, and thus purification is necessary. In the present invention, the same fraction of the saturated filtrate as the filtrate is added to the filtrate obtained by extraction and solvent fractionation is performed by layer separation. Get

At this time, the saturated alcohol used as a saturated alcohol is a saturated aqueous solution of a lower alcohol. The lower alcohol is stirred by adding distilled water to the lower alcohol, stirring, and stagnating. As the lower alcohol, for example, propyl alcohol, butyl alcohol and the like can be used, and the layer separation is performed 2-3 times.

If a lower alcohol is used to obtain a fraction of the lower alcohol, the lower the extraction efficiency, the lower the extraction yield, and the higher the lower alcohol, the more effective the Loniceroside A, B, etc. The bioactive component content is lowered and the activity is lowered, which makes it inefficient. Therefore, in order to maintain an effective physiologically active ingredient content at a certain level and to exhibit an excellent physiological activity effect, it is most preferable to suffice twice with a filtrate and the same amount of a lower alcohol solvent.

In addition, in order to remove unnecessary fatty acids such as fatty acids and chlorophyll in the extract and to limit the remaining amount of lower alcohol solvent, the lower alcohol solvent fraction was concentrated under reduced pressure at 60 to 70 ° C., and water of 20 to 30 times the weight of the extract was obtained. After adding azeotropically, add an equal amount of water to remove insoluble matter by membrane filtration and freeze-drying to obtain a powdery extract.

In this case, if the azeotropic concentration of the purified lower alcohol fraction is not concentrated with water, the residual lower alcohol content becomes too high, which causes problems in use as a pharmaceutical raw material, and does not undergo the removal process by membrane filtration of fatty acids and nonpolar substances. Membrane filters are performed to maintain the purity of the extract and to remove foreign substances, as the concentration of the extract may cause a delay in the concentration time and a poor drying condition.

The extract obtained by this simple and economic method is not only excellent in anti-inflammatory action but also analgesic action compared to the anti-inflammatory medicines in the traditional Chinese medicine, such as Sasang Medicine, Dong-Bo-gam. Can be.

As a result of analyzing the anti-inflammatory analgesic effective physiologically active substances of the standardized extract extracted from the leaves and stems of the phosphorus by the method according to the present invention as described above, the new substance Loniceroside A, B was first identified by the inventors. In addition, known ingredients such as Loganin and Loginerin (Lonicerin) are contained.

However, according to the present invention, the anti-inflammatory analgesic effect was found to be the most preferable in the case that 0.5-3.0% by weight of Ronicereroside A, 0.5-3.0% by weight of Ronnieseroside B.

In other words, it was found that the active ingredient composition extracted from phosphorus showed the best activity in a certain composition range of the compounding ratio.

In addition, the extract extracted and purified by the above method contains 0.5-2.0% by weight of lonicerin and 2.0-5.0% by weight of loganine together with the above-mentioned compositions of Loneroside A and B. appear.

This fact was surprisingly found to be consistent with the case of extracting and mixing the stem and leaves of the phosphorus according to the extract purification method of the present invention.

Therefore, when preparing a herbal composition using the powder extract obtained by the extraction and purification method according to the present invention can be usefully used as an anti-inflammatory analgesic.

The herbal composition prepared from the powder extract according to the present invention may be formulated in a conventional manner, and then administered in the form of tablets, soft capsules, injections, etc., and bases such as lactose, fine When using a base ingredient such as crystalline cellulose (Microcrystalline Cellulose), magnesium stearate (Magnesium Stearate) and phosphorus powder extract in a ratio of 1.5: 1, it exhibits superior anti-inflammatory analgesic activity compared to the conventional one.

Particularly, when the herbal composition of the present invention is administered to the human body, there is little concern about side effects compared to other synthetic drugs because it is a natural extract, and as an example of such side effects reduction, a non-steroidal anti-inflammatory drug (NSAID) -based drug is represented. It has been demonstrated in animal model experiments for gastrointestinal disorders and ulcerative side effects.

That is, as can be seen from the results of the ulcer inhibition test induced by indomethacin, ethyl alcohol (EtOH) and hydrochloric acid (HCI), the effect of inhibiting the ulcer induction caused by the ulcer inducing factor in the human extract prepared in the present invention. Was excellent (see Experimental Examples), and acute toxicity studies in mice and rats proved completely non-toxic. (LD ₅₀ = 5 g ext's / Kg or more, Journal of Pharmacognosy Vol. 25, No. 4, 1994).

As described above, according to the present invention, in extracting and purifying the active physiologically active ingredient from phosphorus, unlike the conventionally known extraction method, the stem and leaves of the phosphorus are extracted at a specific time and extracted using a raw material mixed at a predetermined ratio. · Purification is possible to obtain an extract with a noticeable difference in effect compared to the extract obtained by the conventional extraction method according to the conventional method, it is possible to extract scientific natural products. In addition, there is an effect that can be administered in the form of a convenient ointment, tablets, injections and the like, which is conventionally used only in the form of medicinal herbs such as herbal medicine.

Hereinafter, the present invention will be described in more detail with reference to the following Examples, which are not intended to limit the present invention.

Reference Example 1

To determine the proper timing of harvesting of stems and leaves, Ronicerose A, B content of leaf and stem of Rosincerside A, Roniceroseoside B, Roniserine, and Roganin among anti-inflammatory and analgesic bioactive substances The monthly change in the content of garnin was analyzed by HPLC and the results are shown in Table 1 below.

In addition, the results of the analysis of the active ingredients for the leaves in June and July and the results of the analysis of the active ingredients for the stems in November and December are as follows.

Reference Example 2

The content of the active drug extracted from indong was confirmed in the extracts from June to July immediately after the flowers bloomed in the case of leaves, and from November to December after the flowers had fallen from the stems. And leaves were collected and the results of experiments on the anti-inflammatory, analgesic bioactive changes according to the mixing ratio is shown in Table 2 below.

Example 1

Dry the dried stems collected in Andong, Gyeongbuk in the end of November, and cut them into 1.0 ~ 1.5cm size, and dry the dried leaves collected at the end of June in the same area and crush them to 3-5mm, and then stalk 700g and leaves 300g. 8 liters of water was added to the mixture, and the mixture was extracted with reflux for 5 hours while stirring well, the residue was collected and added with water of 7, and then extracted by heating for 2 hours and mixed with the filtrate of the leaves to increase water extraction efficiency. After adding the extract filtrate with the same amount of saturated n-butyl alcohol and saturating twice, the n-butyl alcohol layer was collected and concentrated under reduced pressure at 60-70 ° C., followed by azeotropic concentration with 0.8 distilled water, followed by addition of the same amount of distilled water. Dissolved and membrane filtered to remove insoluble components and then lyophilized to obtain 41g of extract. The amount of butyl alcohol remaining in the extract was 159 ppm, and the active ingredient composition was 1.34% of Loniceroside A, 1.52% of Loniceroside B, 1.1% of Lonicerin, and Loganin. 4.96%.

Example 2

Dry the dried stems collected at the end of November in Andong, Gyeongbuk, and cut them into 1.0 ~ 1.5cm size, and dry the dried leaves collected at the end of June at the end of June to crush them to 3-5mm, and then stalk 500g and leaves 500g. 12 liters of water was added, and the mixture was extracted with reflux for 5 hours while stirring well, and the residue was collected and 3 liters of water was added. Then, the mixture was heated and extracted for 2 hours to increase water extraction efficiency. After extracting the filtrate with the same amount of saturated n-butyl alcohol and dividing the layer twice, the n-butyl alcohol layer was collected and concentrated under reduced pressure at 60-70 ° C., and then azeotropically concentrated with 1 L of distilled water. The resultant was dissolved, membrane filtered to remove insoluble components, and lyophilized to obtain 52 g of extract. The amount of butyl alcohol remaining in the extract was 178 ppm, and the active ingredient composition was 1.89% of rosineroside, 2.08% of rosineroside, 1.56% of rosinerine, and 3.34% of roganine.

Example 3

Dry the dried stems collected in Andong, Gyeongbuk at the end of November and cut them into 1.0 ~ 1.5cm size, and dry the dried leaves collected at the end of June at the end of June. Take 10 liters of water, add stirring, and stir well for 5 hours while extracting the reflux for 5 hours, collect the residue, add 5 liters of water, and then extract the mixture for 2 hours with warming to increase water extraction efficiency. After extracting the filtrate with the same amount of saturated n-butyl alcohol and separating the layer twice, the n-butyl alcohol layer was collected and concentrated under reduced pressure at 60-70 ° C., followed by azeotropic concentration with 1.2 L of distilled water. It was added to dissolve, membrane filtration to remove the insoluble components and then lyophilized to obtain 44g of extract. The amount of butyl alcohol remaining in the extract was 198 ppm, and the effective composition was 1.47% of roroniseroside A, 1.78% of roroniseroside B, 1.32% of roniserine, and 4.28% of roganine.

Comparative Example 1

The extract obtained by filtration and freeze-drying the filtrate after extracting water from the commercially available phosphorus etc. by the standard liquid solution method was made into Comparative Example 1. At this time, the effective cohesiveness was Roniseroside A 0.03%, Ronisseroside B 0.05%, Roniserin 0.07%, Loganin 0.92%.

Comparative Example 2

After extracting the filtrate twice with the same amount of n-butyl alcohol and extracting the filtrate twice according to the extraction method of Comparative Example 1, the n-butyl alcohol layer was concentrated under reduced pressure at 60-70 ° C., azeotropically concentrated with water, and dissolved in water to make membrane insoluble. After removing the substance, the extract obtained by lyophilization of the filtrate was set to Comparative Example 2. At this time, the effective cohesiveness was 0.16% of rosineroside A, 0.21% of rosineroside, 0.31% of rosinerine B, and 3.78% of roganin.

Experimental Example 1

As a comparison of the acute anti-inflammatory activity of the extract prepared in Example 3 and Comparative Examples 1 and 2, the mouse ear edema model test (Mice ear edema model test) was carried out as shown in the following experimental methods and the results are shown in the following Tables 3 and 4 It was.

Experimental Method: After 1 hour oral administration of each phosphorus extract to mice, 2.5% croton oil or 2% arachidonic acid was applied to both ears and 5 hours after croton oil, and for arachidonic acid. After 1 hour each ear thickness was measured with a dial thickness gauge. The control group was orally administered sterile distilled water instead of phosphorus extract.

Experimental Example 2

Chronic inflammation (Adjuvant induced Arthritis Model Test) as a comparison of the chronic anti-inflammatory activity of the extract prepared in Example 3 and Comparative Examples 1 and 2 shown in Table 5 below.

Experimental Method: Inject chronically edible mycobacteriun buthylicum suspended in mineral mineral oil at 0.6mg / Rat in rat's right foot to induce chronic edema. Edema was measured using water-displacement method. Andong extract was orally administered daily for 18 days.

Experimental Example 3

As a comparison of the pain inhibitory effect of the extract prepared in Example 3 and Comparative Examples 1 and 2, the acetic acid induced writhing test was carried out as in the following experimental method, and the results are shown in Table 6 below.

Experimental Method: After 1 hour oral administration of phosphorus extract to ICR mice, intraperitoneal injection of 0.6 acetic acid at a dose of 0.1 10 body weight, and pain response of each mouse 10 minutes after injection for 10 minutes. The number of writhing was observed.

Experimental Example 4

In order to compare the ulcer inhibition effect induced by the extract of Example 3 and the extract prepared in Comparative Examples 1, 2, Indomethacin, ethyl alcohol (EtOH) and hydrochloric acid (HCI) as in the following experimental method The results are shown in the following Table 7,8.

Experimental method: Rats fasted for 24 hours were orally administered with a comparative drug, and after 1 hour to induce ulcer by oral administration of indomethacin or ethyl alcohol and hydrochloric acid solution, and after 1 hour, the stomach was extracted After leaving it in a 3% formalin solution for 30 minutes, the ulcer area was calculated using an image analyzer, and then the ulcer area reduction was shown as a percentage.

[Production Example 1]

Using the powder extract obtained in Example 3 to prepare a tablet for oral administration (standardized Ext's 100mg) with the following composition.

[Production Example 2]

Using the powder extract obtained in Example 3, an ointment was prepared in the following composition.

[Production Example 3]

An injection was prepared in the following composition using the powder extract obtained in Example 3.

Claims (4)

In extracting and refining the active ingredient from phosphorus, the dried and ground phosphorus leaves are collected after the flowers of the phosphorus have been dried, and the dried and ground phosphorus leaves are collected at the flowering stage of the phosphorus at a weight ratio of 7: 3 to 5: 5. A method for extracting and purifying an active ingredient from phosphorus, characterized in that the mixture is purified by mixing under reflux with water. The method of claim 1, wherein the purification is performed by reflux extraction, filtration, layer separation by adding the same amount of saturated lower alcohol to the filtrate, and concentration of the lower alcohol layer under reduced pressure to obtain a powder extract. Method for extracting and purifying the active ingredient from The method for extracting and purifying the active ingredient from phosphorus according to claim 2, wherein the saturated lower alcohol is a saturated aqueous solution of butyl alcohol or propyl alcohol. According to any one of claims 1 to 3, wherein the effective physiologically active substance extracted from the phosphorus is 0.5 to 3.0% by weight of Loniceroside A, 0.5 to 3.0% by weight Lonieroside B, Lonnie A method for extracting and purifying an active ingredient from phosphorus, characterized by containing 0.5 to 2.0% by weight of serine and 2.0 to 5.0% by weight of loganin.