KR100834966B1 - Manufacturing method of fraction from stem of Lonicera japonica Thunb. and injection drug comprising the fraction - Google Patents

Abstract

The present invention relates to a method for producing a phosphorescent lamp fraction containing an active active ingredient from a phosphorescent lamp (phosphorus stem removed from the copper leaf, hereinafter phosphorus lamp) and an injection composition containing the phosphorescent lamp fraction, more specifically, It removes unnecessary tannins, poorly soluble flavonoids, saponins, etc., and is effective as an injectable ingredient, and has excellent efficacy, safety, and stability. Iridoids such as sweroside and loganin The present invention relates to a process for producing a phosphorescent fraction containing an active ingredient of a) -based substance and to an anti-inflammatory and analgesic injection composition containing the above-mentioned phosphorous fraction.

Honeycomb, sweroside, loganin, injection

Description

A method for preparing a phosphorous lamp fraction containing an active ingredient from a phosphorescent lamp, and an anti-inflammatory and analgesic injection composition containing the phosphorescent lamp fraction.Manufacturing method of fraction from stem of Lonicera japonica Thunb. and injection drug comprising the fraction}

1 shows a graph of the efficacy according to the amount of the active ingredient of the final tablet.

The present invention removes unnecessary tannins, poorly soluble flavonoids, saponins, and the like from the phosphorus, and has excellent drug efficacy when used as a raw material for injection, and has excellent efficacy, safety, and stability, such as sweroside, loganin, and The present invention relates to a method for preparing a phosphorescent light fraction containing an active ingredient of the same iridoid-based substance and to an anti-inflammatory and analgesic injection composition containing the phosphorous light fraction.

Indong ( Lonicera japonica Thunb.) Is a semi-herbaceous vine shrub that grows in the foothills and near the authorized banks of 50-600 m above sea level in mountain areas in Japan, China and Korea. Gold, silver, stem and branches are called diuresis, detoxification, boil, edema, cold, hemostasis, haemostasis, pain, vomiting It is used as a herbal medicine [Korea Plant Book, Chang Bok Lee, 709, 1989, Anal History, Seoul; Korean Medicinal Herbs (Medicinal Herbs) Standard Collection, Ko, Ji-Joon, Sang-In Lee, 87, 305, 1988, Korea Medical Index, Seoul; Plant Resources in Korea, Tae-Jung Kim, vol. 4, 148-149, 1996, Seoul National University Press, Seoul]. In addition, there are records in oriental medicine books such as Sasang Medicine and Minjeoljeung, which are useful for Onggi, a variety of inflammatory diseases both inside and outside the human body. [Korea Chosun National Medicine, Yanbian Institute of Korean Medicine, 276, 1991, Yeogang Publishing Co., Seoul ; Light-spray, Lee Kyung-hwa, 349-351, 1991, Yeogang Publishing Co., Seoul], privately known anti-inflammatory and analgesic activity has long been used in the treatment of upper respiratory tract infections, tonsillitis and neuralgia such as cold. Recently, the anti-inflammatory and analgesic activity of human sinus has been scientifically proven through several experimental animal models, and effective physiologically active substances have been isolated and reported to the academic community. [Development of vegetable anti-inflammatory agents: comparison of anti-inflammatory and analgesic effects on human extracts, Song, Jin-Jin et al., Korean Journal of Pharmacognosy, 363-367, 25, 1994; Flavonoids from the aerial parts of Lonicera japonica, Son et al., Arch. Pharm. Res., 365-370, 15, 1992; Antiinflammatory activity of Lonicera japonica, Lee et al., Phytother. Res., 445-447, 12, 1998; Triterpenoid saponins from the aerial parts of Lonicera japonica, Son et al., Phytochem., 1005-1008, 35, 1994; Anti-inflammatory activity of the major constituents of Lonicera japonica, Lee et al., Arch. Pharm. Res., 133-135, 18, 1995].

Active active ingredients contained in phosphorus, etc. known to date, such as caffeoylquinic acid, methyl caffeate, chlorogenic acid, iso-chlorogenic acid Degradable tannins and iridoid glycosides such as loganin, sweroside, vogeloside and epi-vogeloside Etc. are known. In the existing literature, most studies on the composition of gold buds and copper leaves, which are mostly buds, are conducted. However, it is important to note that the distribution of the components of the phosphorus lamp, the phosphorus leaf, and the gold and silver coin are different. In other words, unlike honeysuckle, such as honeydew leaves or gold coins, flavonoids such as lonicerin, rhoifolin, orcnaflavon, flavonoids, hederagenin, and oleanolic acid Triterpene-based saponins and various hydrolyzable tannins, which are present as non-sugars, are the main components.

However, there are some problems in developing injections from phosphorus having components of this class. In other words, if a large amount of polymer tannins are contained in the injection material, there is a risk of co-precipitation by combining with other components as time passes, and when the body is administered, it forms a poorly soluble precipitate by combining with blood plasma proteins. It may also cause obstruction. In addition, since flavonoids contained in phosphorus are generally very poorly soluble in water, it is difficult to dissolve more than the effective concentration without using a certain amount of organic solvents or other dissolution aids, and fractions having a high flavonoid content are generally main. Use Insoluble in physiological saline and long-term storage in alkaline buffers, its stability may be a problem. Finally, human saponins, especially monodesmosides, are known to have strong hemolytic action and are therefore not possible to be injected intravenously without purification [Studies on the saponins of Lonicera japonica Thunb., Kawai et al. , Chem. Pharm. Bull., 4769-4775, 36 (12), 1988]. In particular, the leaves and gold and silver leaves of tannin and poorly soluble flavonoids were significantly higher than those of phosphorus and not only caused toxicity at low doses compared to phosphorus in the acute toxicity test when used as an injection.

Therefore, the present inventors have conducted research to prepare injections by purifying fractions having excellent anti-inflammatory and analgesic effects and excellent safety and stability. As a result, the present inventors removed unnecessary tannins, poorly soluble flavonoids, saponins, etc. Very high content of ingredients and excellent safety and stability, extracts and purifies active ingredients containing iridoid-based substances such as sweroside and loganin, which are suitable as raw materials for injection. This material has completed the present invention by revealing that the anti-inflammatory, analgesic effect is significantly superior to the existing extract.

Accordingly, an object of the present invention is to provide a method for producing a fraction of phosphorous, etc., containing an active ingredient having excellent anti-inflammatory and analgesic activity from phosphorous and the like, and having excellent safety and stability.

In addition, the present invention is another object to provide an anti-inflammatory, analgesic injection composition containing the above-mentioned fractions such as excellent anti-inflammatory, analgesic activity obtained from phosphorus and the like, excellent safety and stability.

The present invention is extracted by refluxing the phosphorus and the like with water and filtered, the same amount of saturated lower alcohol is added to the filtrate to separate the layer, and the saturated lower alcohol layer is concentrated under reduced pressure to obtain a powder extract, the polyamide resin or poly It is characterized by the method of producing fractions of phosphorus such as phosphorus, which has excellent anti-inflammatory, analgesic effect and excellent safety and stability from phosphorus, etc. after purification with vinylpyrrolidone resin and octadecylsilane resin. .

Also included are anti-inflammatory, analgesic injection composition containing fractions such as phosphorus.

Referring to the present invention in more detail as follows.

The present invention removes unnecessary tannins, poorly soluble flavonoids, saponins, and the like from the phosphorus, and has excellent drug efficacy when used as a raw material for injection, iridoids such as sweroside and loganin, which are excellent in safety and stability. The present invention relates to a method for producing a phosphorescent light fraction containing an active ingredient containing an (iridoid) -based substance as a main component, and to an anti-inflammatory and analgesic injection composition containing the phosphorous light fraction.

First, the previously used phosphorus outpost (Indong lamp and the like leaf mixed out state, the following copper outpost), phosphorus leaves and phosphorus used in the present invention is extracted and purified as follows.

Each sample, such as phosphorus outpost, honeysuckle leaf, phosphorus, etc., was extracted under reflux for 2 hours to 3 hours with distilled water of about 7 to 10 times the weight of the crude drug, filtered, and the filtrate was collected, and again for 2 hours to 3 hours with 5 to 7 times of distilled water. After extracting reflux for a period of time, the collected liquid is filtered, combined with the previous filtrate and concentrated under reduced pressure so that the volume is about 1 to 3 times the weight of the original herbal medicine (v / w) and then filtered again. When the amount of water is too small to extract with distilled water, it becomes difficult to stir, and the solubility of the extract becomes low, so that the extraction efficiency is lowered. When the amount of distilled water is too large, the amount of distilled water to be concentrated compared to the extraction efficiency increases, resulting in time and economic irrationality. Since it occurs, proceed to the above process. Thereafter, the same amount of saturated lower alcohol is added, stirred for about 10 to 20 minutes at 30 to 50 rpm, and allowed to stand, and the layers are separated. The saturated lower alcohol layer is filtered and concentrated under reduced pressure to obtain a primary active purified fraction. The saturated lower alcohol used in this case is a saturated aqueous solution of lower alcohol, which is diluted with distilled water by adding distilled water to the lower alcohol, stirring the mixture, and taking a lower alcohol layer saturated with distilled water. Separation is performed 2-3 times. If a small amount of lower alcohol is used to obtain the lower alcohol solvent fraction, the purification efficiency is lowered, and the yield and the content of the active ingredient are lowered. When the lower alcohol is used, the economical efficiency is increased by using excessive alcohol compared to the purification efficiency. Dropped. Therefore, it is advisable to use 1 to 3 times the lower alcohol (v / w) of the weight of the crude drug, the amount described above being consistent with such purification efficiency and economics.

The activity fractions of the phosphorus lamps were the most excellent in analgesic and anti-inflammatory effects. In addition, the acute toxicity test and blood safety test for each fraction also concluded that the active fraction, such as phosphorus, was the safest. In particular, the leaves of the leaves were inadequate for the development of injectables in both blood safety, solubility, and acute toxicity tests, which resulted in poorly soluble flavonoids, tannins, and saponins in the leaves. It was due to the field. Therefore, in order to remove such unnecessary substances and to search for medicinal substances, first, column chromatography on activated outpost, phosphorous lamp, and copper leaf active fraction is performed using polyamide resin or polyvinylpyrrolidol resin. The amount of the filler is used 1 to 10 times (w / w) of the saturated lower alcohol layer, the solvent is distilled water, 50% (v / v) aqueous methanol, eluting 2-3 times the volume of the filler in the order of methanol A step-gradient method of eluting the next solvent is used. Among the fractions thus obtained, the fractions obtained when eluted with distilled water not only significantly removed aromatic organic acids, tannins and flavonoids, but also showed excellent effects such as increased drug efficacy, markedly reduced acute toxicity, increased solubility, and increased blood safety. This fraction was subjected to column chromatography again using ODS (Octadecylsilane) resin to increase the amount of methanol by 10% (v / v) from 10% (v / v) aqueous methanol solution, and then to 2 ~ 3 times the volume of the resin. The solvent is eluted in a step-gradient manner, and the amount of resin is 10 to 20 times the weight of the distilled water elution fraction weight purified using the polyamide resin or polyvinylpyrrolidol resin. At this time, the fraction eluted with 20 to 30% (v / v) aqueous methanol solution was the most effective, it was determined that it is safe and suitable for development in the form of an injection.

As a result of tracking the anti-inflammatory and analgesic active ingredients of the fractions extracted from phosphorus and the like, in particular, the most active 20 to 30% (v / v) methanol solution elution fraction by the method according to the present invention as described above Iridoid-based substances such as sweroside and loganin are the main active substances, wherein the content of sweroside is 15.1-72.1 wt% and the content of loganin is 13.9-41.4 It was confirmed by weight percent. ED ₅₀ in animal experiments was 0.03 mg / Kg in acetic acid-induced riding test for analgesic effect, and ED ₃₀ was less than 0.3 mg / Kg in croton oil-induced ear edema measurement for anti-inflammatory effect. In addition, LD ₂₀ was measured at 2.0 g / Kg in the acute toxicity test. All of the drugs were injected intravenously in these efficacy and acute toxicity studies. The topical toxicity test does not develop toxicity at 150 mg / Kg and is therefore safe for use above normal normal doses.

Compared to the prior art, the anti-inflammatory and analgesic effect is very excellent, and it can be used as an injection using a pharmaceutically acceptable carrier or dissolving aid, etc., in addition to the active ingredient composition obtained from the above phosphorus, which is most suitable for solubility, acute toxicity, and blood safety. have.

The effective dosage of the active ingredient may vary depending on the age, physical condition, weight, etc. of the patient, but is generally administered within the range of 5 to 100 mg / kg (weight) / day. In addition, administration is carried out once a day or divided several times a day within the effective dosage range per day.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Example 1 Drug Effects by Phosphorus Use Site

In July 1999, samples of Indong Outpost, Indong Leaf, Indong, etc. were collected from Yeongcheon, Gyeongbuk, and the mixture was dried and refluxed for 2.5 hours with distilled water of 7 times the weight of herbal medicine, filtered and collected. The filtrate was collected again with 7 times of distilled water. After extracting reflux for 2.5 hours, the collected solution was filtered, combined with the previous filtrate and concentrated under reduced pressure so that the volume is about 2 times the weight of the original herbal medicine (v / w) and filtered again. Then, an equal amount of saturated normal-butyl alcohol was added thereto, stirred at about 30 rpm for 15 minutes, and the layers were separated twice. The alcohol layer was filtered and concentrated under reduced pressure to obtain a primary active fraction. Column chromatography was performed on the purified fractions with polyamide resin (CAS NO. 63428-83-1). The amount of the resin was used five times the amount of the sample and the second active fraction obtained by eluting twice the volume of the filler in the order of distilled water, 50% (v / v) aqueous methanol solution, methanol in order to use the next test. Croton oil induced ear edema test was performed through the tail vein of 6-week-old ICR mice (body weight: 20-30 g, n = 6, SLC, Japan) fasted 4 hours before the experiment. After 15 minutes, the second active fraction of copper outpost, honeysuckle, and honeysuckle leaves were infested with 2.5% croton oil, and after 4 hours, the thickness of the left and right ears was measured using a dial thickness gauge. Measured by the following equation 1 to calculate the rate of fire, the following Table 1 shows the efficacy.

As shown in Table 1, the active light fractions were the best anti-inflammatory and analgesic activity during intravenous injection.

Example 2 Comparison of Active Fraction Drugs Obtained from Phosphor

Croton oil induced ear edema was measured in the same manner as in Example 1.

In addition, the arachidonic acid induced ear edema test was performed on 6-week-old ICR mice (body weight: 20-30 g, n = 6, SLC, Japan) fasted 4 hours before the experiment. 15 minutes after the drug (Mariebiene, Indong, etc., the first active fraction, second active fraction), and after 15 minutes to spread with 0.05% arachidonic acid, and after 1 hour using a measuring instrument to measure the thickness of the left and right ears, respectively After the calculation by the formula (1) is shown in Table 2 the inhibition rate.                     

As shown in Table 2, the secondary active fractions obtained from phosphorus, etc., were not only removed from aromatic organic acids, tannins, flavonoids compounds, but also increased the content of the active ingredient, which is an excellent improvement effect compared to the primary purified products. It was confirmed to have.

Example 3 Comparison of Active Fraction Drugs Obtained from Phosphor

In Example 1, polyamide resin was replaced with polyvinylpyrrolidone resin (CAS NO. 25249-54-1), and the procedure and test method were the same as in Example 1.

Example 4: Preparation of Final Tablets and Drug Tests

The powder obtained after concentration of the second active fraction of phosphorescent lamp in Example 1 under reduced pressure was again ODS resin (YMC * GEL ODS-A 12nm, S-150m or ODS-AM 12nm, S-50m or ODS-AQ 12 nm, S-50 m) was used to perform column chromatography, and the final purified product eluting three times the amount of 20% (v / v) methanol aqueous solution of resin was used in the next experiment.

Croton oil induced ear edema was measured in the same manner as in Example 1.

In addition, the acetic acid induced writhing test was carried out through the tail vein of ICR mice (body weight: 20-30 g, n = 8, SLC, Japan) fasted the day before the experiment. 20 minutes after intraperitoneal injection of 0.7% acetic acid 20 minutes after the administration, and measured the number of riding for 10 minutes after 15 minutes, the inflammation inhibition rate is shown in Table 4 below.

Example 5 Content and Drug Test of Active Active Ingredients

As a result of performing high performance liquid chromatography (HPLC) on the final purified products of the various samples prepared by the methods of Examples 1 and 4, the active ingredient sweroside (sweroside) is 15.1-72.1 wt%, and loganine ( loganin) was found to contain 13.9 ~ 41.4% by weight. The contents of sweroside and loganin are shown in Table 5 according to the place and time of sample, and the effects of the final tablets of each sample are shown in Table 6 below.

Example 6 Hemolysis Test on Final Purified Product

20 ml of blood was collected from the rabbit heart with a syringe treated with Heparin and centrifuged for about 10 minutes. Discard the upper layer sufficiently, dilute the residue 10 times with physiological saline for injection, and shake carefully to mix the drug of each concentration of Example 1 (final purified product such as incandescent lamp and primary purified product of honeycomb lamp, honeysuckle leaf, and outpost) And 0.5 ml of the final solution of the final tablet of Example 4 and 0.5 ml of diluted blood were prepared in a test tube (a saline control group and a distilled water (100% hemolysis) control group were also prepared). Thereafter, the mixture was left to stand for 15 minutes in a constant temperature bath at 37 ℃, and again left at room temperature for 45 minutes. Finally, after centrifugation at 2500 rpm for 2 minutes, the supernatant was measured at 540 nm.

Example 7: Toxicity Test

SD rats (body weight: 120-170 g, 5 females per unit dose, SLC, Japan) fasted 4 hours before the experiment were subjected to 1.0 g / Kg, 1.5 of the final purified product in Example 3 via the tail vein. g / Kg and 2.0 g / Kg were administered and visually observed for 30 minutes and then visually observed at 30 minute intervals. In addition, two weeks after the drug administration, mortality, general symptoms, and body weight were measured, and autopsy confirmed abnormalities of each organ.

LD ₅₀ of the final tablets was higher than 2.0 g / Kg (LD ₂₀ = 2.0 g / Kg) and increased breathing and decreased activity for about 10 minutes after administration at a dose of 2.0 g / Kg, but recovered soon. There was no change in body weight and no increase or decrease by administration of test substance. Autopsy results were also unchanged from the control group.

At the doses of 50, 100 and 150 mg / Kg, no differences were observed between the physiological saline-administered groups and no toxicity, such as tissue necrosis or inflammatory reactions.

Preparation Example 1 Preparation of Injection

An injection was prepared in the following composition using the final tablet obtained in Example 4.

Injectable ampoule: 20 mg of final tablet

Mannitol 60 mg

Corresponding solvent sample: 2000 mg of saline for injection

2080 mg total

Preparation Example 2 Preparation of Injection

An injection was prepared in the following composition using the final tablet obtained in Example 4.

Injectable ampoule: 50 mg of final tablet

KH ₂ (PO ₄ ) 8.5 mg

                    Physiological saline for injection 3000 mg

Total 3058.5 mg

Preparation Example 3 Preparation of Injection

An injection was prepared in the following composition using the final tablet obtained in Example 4.

Injectable ampoule: 100 mg of final tablet

Mannitol 300 mg

KH ₂ (PO ₄ ) 17 mg

                   Physiological saline for injection 3000 mg

3417 mg total

As described above, the active fractions obtained from phosphorus and the like by the extraction and purification methods have significantly increased solubility and blood safety compared to general extraction and purification, and analgesic and anti-inflammatory drugs are also excellent, resulting in excellent safety and stability. It is suitable as a raw material.

Claims (5)

After extracting the phosphorus lamp with water under reflux, the mixture was filtered, and the filtrate was added with a saturated aqueous solution of 1 to 4 carbon atoms, and the layers were separated. The alcohol layer was concentrated under reduced pressure to obtain a powder extract, and the extract was extracted with polyamide or polyvinylpyrroly. Swae, characterized in that the purified from the resin resin in the order of water, a C 1-4 alcohol solution and a C 1-4 alcohol as a developing solvent, and then octadecyl silane resin with a developing solvent of 1 to 4 carbon atoms aqueous solution Process for preparing fractions of phosphorous, etc., containing roroside and loganin. The method of claim 1, wherein the saturated aqueous solution of 1 to 4 carbon atoms is a saturated aqueous solution of butyl alcohol or propyl alcohol. delete An analgesic, anti-inflammatory injection composition comprising the fraction of swineside (sweroside) and loganin prepared in claim 1 or 2 as an active ingredient. 5. The analgesic and anti-inflammatory injection composition according to claim 4, wherein the sweroside is 15.1-72.1 wt%, and the loganin is 13.9-41.4 wt%.

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