KR19980021297A - Immunostimulator Compositions Containing Mixed Water Extracts of Hwangbaekpi and Matari Plants - Google Patents

Abstract

The present invention relates to an immunoadjuvant composition containing a mixed water extract of Phellodendron amurense Rupr. Cortex and Matari (Pathnia scabiosaefolia FISCHl.) Plants as an active ingredient. The extract of the present invention has an excellent immunopotentiating effect to restore or enhance an impaired immune mechanism in patients undergoing chemotherapy or radiation therapy and to increase antibody production titers by using it as an adjuvant when using an immune-related vaccine. Effect. As a mixed water extract of baekbaekpi and matari used as an active ingredient in the present invention, an extract obtained by extracting a mixture of baekpipi and matari plants with water may be used, or an aqueous fraction obtained by further purification as needed. In particular, the cells are affected by the immunosuppressed state of cancer patients or by chemotherapy or radiation therapy administered to these cancer patients, and thus T cells, B cells, and macrophages (M) associated with immunity. In the case where the decrease in the number of cells and the like, the immune function is selectively suppressed according to the decrease, the mixed water extract of Hwangbaekpi and Matari according to the present invention can effectively inhibit the proliferation or metastasis of malignant tumors by enhancing biological immune function. In addition, the effect of increasing the titer of antibody formation by the vaccine is shown by the combination with an immune-related vaccine. Therefore, the extract of the present invention is administered in combination with an anticancer chemotherapeutic agent, or used in combination with radiotherapy to improve the immunosuppression of cancer patients or selective immunosuppression according to anticancer chemotherapy or radiotherapy to inhibit cancer proliferation or cancer cells. It can inhibit metastasis and can be used as an excellent anticancer adjuvant, and can also be used together with an immune-related vaccine as an adjuvant.

Description

Immunostimulator Compositions Containing Mixed Water Extracts of Hwangbaekpi and Matari Plants

FIG. 1 is a graph showing the effect of the mixed water extract of Hwangbaekpi and Matari of the present invention on the T cell dependent antibody production reaction in vivo.

2 is a graph showing the effect of the mixed water extracts of Hwangbaekpi and Matari plants of the present invention on the proliferation of T cells.

3 is a graph showing the effect of the mixed water extracts of Hwangbaekpi and Matari of the present invention on the proliferation of immune cells.

4 is a graph showing the effect of the mixed water extracts of Hwangbaekpi and Matari plants of the present invention on the normal immune cell line

5 is a photograph showing the effect of the mixed water extract of Hwangbaekpi and Matari plants of the present invention on the liver, kidney and heart of the mouse (15 g / kg administration, HE staining, 5A Figure: liver x400, 5B Figure: kidney x200, 5C degrees: heart, x200).

The present invention relates to an immunoadjuvant composition containing a mixed water extract of Hwangbaekpi (黃 柏皮) and Matari plants (packing) as an active ingredient. More specifically, the present invention contains a mixed water extract obtained by extracting a mixture of Pel1odendron amurense RUPRCHT codex and Matari (Patrinia scabiosaefolia FISCH.) With water as an active ingredient, the inducer of activation of a potential immune response to the antigen. It relates to a pharmaceutical composition that acts as an effect to enhance biological immune function. The present invention also relates to a process for the preparation of the mixed water extracts of such baekbaekpi and matari plants.

Immunodeficiency disease is deeply involved in clinical immunological refractory diseases, cancer, diabetes, and male infertility. In addition, AIDS, various pathogenic viral diseases, opportunistic infections, parasitic infections, etc. All can be said to be diseases related to immunodeficiency.

The bioimmune system is regulated by the jaul nervous system and the endocrine system, and has the property of maintaining homeostasis by excluding non-self invading from the outside or internally through humoral and cellular immunity. However, if the system is abnormally disturbed for some reason, immunodeficiency or immunodeficiency may be caused. As such, immune enhancing agents and immune function activators are generally used to correct and restore an abnormally disturbed immune system and to restore damaged immune function.

Immune function enhancers are drugs mainly used for congenital and acquired immunodeficiency syndrome, and are urgently needed as part of pharmacotherapy for tumors and AIDS. Representative immune enhancers currently in use include immunoglobulin, inteferon and the like. Among them, an agent that concentrates and purifies IgG, one of human immunoglobulins, is used for the prophylactic treatment of measles, chicken pox, hepatitis B, mumps, etc., but anaphylaxis due to pain at the injection site and lowering blood pressure Anaphylaxis A positive reaction has the disadvantage of high doses. On the other hand, interferon (INF) was found as an antiviral factor, but since it has been confirmed to inhibit cell proliferation and immune function, it is used as an antiviral agent and an antitumor agent, but β type is a fever, boredom and anorexia. , Side effects such as injection communication or alopecia are observed, and α type shows side effects such as leukocyte reduction and elevated GOT due to temporary suppression of bone marrow function.

In addition, immunopotentiators improve the immune response of the living body and improve the secondary immunodeficiency state caused by excessive therapy with radiotherapy and antitumor agents or corticosteroids. Bacillus Calmette-Guerin, an attenuated tuberculosis bacterium, is attenuated. CWS (Cell Wal1 Skelton) and Water Soluble Adjuvant (WSA), which have improved side effects, are used. Lentinan extracted and purified from protein polysaccharides obtained from picibanil or Ganoderma lucidum obtained from purulent Streptococcus Group A type 3 Su strains, and Lentinus edodes. lentinan) is recognized as an immune resiliency function, but antitumor activity and anti-AIDS effect is difficult to expect.

In general, immunodeficiency decreases the resistance to infection, and patients with antibody-producing insufficiency cannot prevent infection by bacteria, and the so-called immune opsonin effect is attenuated, thus reducing the phagocytosis of neutrophils. do. In addition, in this case, the complement system (complement system) activation response is also lowered, so the leukocyte flow factor is not generated, the incidence of inflammation is increased, and viremia occurs and spreads to the central nerve or elsewhere.

In case of T cell dysfunction, varicella infection becomes severe, and infant's vaccinia is accompanied by fatal side effects such as pancreatic head. Tuberculosis, typhus, candidiasis, etc., because cellular immune is the main defense system is aggravated by the failure of T cells. In primary or secondary immunodeficiency, the ability of neutrophils to deteriorate the bactericidal ability of Pseudomonas aeruginosa, and Pseudomonas aeruginosa or Klebsiella, which do not usually cause pneumoconiosis, may also be a cause of purulent growth.

Humoral immunodeficiency is manifested by a lack of immunoglobulin in herpes. B cell disorders are thought to occur during the differentiation of B cells from bone marrow cells or during the differentiation of B cells into antibody-producing cells.

However, drugs that can augment or repair immunodeficiency or immunodeficiency due to the destruction of the immune system are rare, and even though there are problems that are difficult to actually use due to side effects.

Therefore, the present inventors conducted an intensive study to find a safe pharmaceutical ingredient that shows excellent immune function enhancing function and immune function revival effect and has little side effects, and in particular, studied various natural medicinal plants. That is, the inventors of the present invention not only can restore immune function of cancer patients in immunodeficiency state or minimize side effects due to immunosuppression by conventional anticancer chemotherapy and radiotherapy, as well as immune-related vaccines and toxoids. The natural plant extract was intended to be used as a means to further enhance immune activity when used in combination with biological agents such as (toxoid). As a result, it was confirmed that the mixed water extract extracted with water by mixing Hwangbaekpi and Matari plants among various natural plants can achieve the object as described above and completed the present invention.

Therefore, the first object of the present invention relates to an immunoadjuvant composition containing a mixed water extract of the baekbaekpi and matari plants.

Another object of the present invention is to provide an adjuvant composition for reviving immune function inhibited by anticancer chemotherapy and radiotherapy by containing a mixed water extract of Hwangbaekpi and Matari plants as an active ingredient.

It is still another object of the present invention to provide an immunoadjuvant for increasing antibody formation ability by using a mixed water extract of baekbaekpi and matari as an active ingredient in combination with an immune-related vaccine.

It is a further object of the present invention to provide a method for preparing a mixed water extract which is used as an active ingredient with immunopotentiation according to the present invention from a mixture of baekbaekpi and matari.

Hereinafter, each object of the present invention will be described in detail.

The present invention relates to an immunoadjuvant composition containing a mixed water extract of Hwangbaekpi and Matari as an active ingredient, in particular, an immunosuppressive composition for chemotherapy and radiotherapy or an immunoadjuvant for an immune-related vaccine. .

The mixed water extract of Hwangbaekpi and Matari, which is used as an active ingredient according to the present invention, is harmless to normal cells as demonstrated in the following experimental examples, and shows an excellent immunostimulating effect, in particular, immunosuppression by drugs or radiation. In addition to reviving the condition, in combination with immune-related vaccines and toxoids it is an excellent pharmacological benefit of increasing the titer of antibodies by increasing immune function.

Hwangbaekpi used in the preparation of the extract of the present invention is a bark of a yellow wall or a yellow tree stem native to Korea, Japan, China, etc. A representative of the yellow-back tree is Phellodendron amurense RUPRECHT. His variants include Latifolia Nagai Kawamodo, Zapponicombe Cucumber, Pelodendron Insulin Nagai, Pelodendron Mullet Nagai, and Pelodendron Ricegent. Yellow and white skin contains 1.5 to 4.5% of yellow or tan pigment and several alkaloids. The main component of alkaloids is berberine. Others include palmatine, magnoflohne, guanidine, jateorrizine, phellodendrine, candicine and menisperine It is known that high-density compounds include obakunone, obakulactone, and β-sitosterol. These ingredients have strong antibacterial action, blood pressure lowering action, central nervous system suppression effect, acetylcholine enhancement action and anti-inflammatory action. It has been used for bone disease and jaundice in herbal wood or pharmacy. In addition, it is effective for typhoid fever and cholera, and it is effective for stomach and stomach, and their bark has been used for gastroenteritis, abdominal pain, jaundice, etc. However, there have been no reports of immune-related effects such as Hwangbaekpi skin enhancement or reactivation.

Matari, another plant used in the manufacture of the extract of the present invention, is also known as a so-called patch (Patrinia scabiosaefolia FISCH.), Dettali (白花 敗 醬; Patrinia villosa JUSS.), Dolmatari (岩 敗 醬) ; Patrinia sibirica JUSS.). Matari is a perennial herbaceous plant native to Korea and in the temperate regions of the world. The herb is used as an anti-inflammatory agent in ophthalmology, Streptococcus pyogens, edema, and fevers. . It has also been reported that hydrothermal extracts of matari have antitumor effects (eg, uterine cancer, esophageal cancer, gastric cancer, intestinal cancer, lung cancer, etc.). However, there is no report on the mechanism of the anticancer effect of these matari plants. In addition, Matari strongly inhibits the development of Staphylococcus aureus, Streptococcus, etc. in the in vitro test, prevents the degeneration due to the regenerative ability of damaged liver cells, and improves the circulation of the portal vein, promoting the regeneration of liver cells. In addition, it acts on the central nervous system to stabilize the nerves, has a strong analgesic effect, has been known to have a blood pressure lowering effect and antidiuretlc (antidiuretlc) effect.

Matari's root contains trace alkaloids such as essential oils, various saponin components, carbohydrates and cumarin. The dermal layer of the root includes acetic acid, formic acid, valeric acid, and the like, and contains alkaloids such as catinine and valerianine. Valeric acid is known to have a very strong analgesic effect. In addition, triterpine-based ursolic acid (ursolic acid) and oleanilic acid (oleanilic acid) is also contained. Ursolic acid has been reported to inhibit carcinogenesis by TPA (12-O-tetradecanoylphorball-13-acetate) and to inhibit metastasis by reducing the expression of matrix metalloproteinase-9 (MMP-9). (Cha, HJ et al., Cancer Res. 56: 228l-2284, 1996 and Huang MT et al., Cancer Res. 54: 701-708, 1994). Dried seeds contain 19.4-l9.9% protein and 30-34.4% fat. However, no specific studies have yet been conducted on which active ingredients exhibit their respective pharmacological properties.

In the present invention, the mixed water extract obtained by mixing Hwangbaekpi and Matari and extracting with water shows a strong immune function enhancing activity including immune function revival and immune supplementation, which is not known to Hwangpipi and Matari so far, and the mixed water extract is also Experimentally confirmed that showing the synergistic effect is significantly superior to the case of using the respective extracts of Hwangbaekpi and Matari alone and completed the present invention.

The mixed water extract of Hwangbaekpi and Matari used as an active ingredient in the present invention is obtained by the following method.

That is, the mixture of Hwangbaekpi and Matari plants was extracted with water, the extract was filtered, and the filtrate was saturated under high pressure to remove the precipitate of coagulated protein. The precipitate was removed by centrifugation, and the organic solvent was added to the filtrate to remove the organic solvent-soluble substance. By separating the aqueous layer in lyophilized, a mixed water extract (RI-P) of sulfur white skin and matari desired in the present invention can be obtained in powder form.

At this time, Hwangbaekpi and Matari are used in a weight ratio of 1: 0.1-5, preferably 1: 1-2 by weight. In particular, in the present invention, it is preferable to combine the yellow and white skin and matari in a weight ratio of 1: 1.

In addition, the mixed water extract obtained above may be further purified as necessary to separate only the water-soluble fraction thereof and used as an active ingredient in the present invention. That is, by dissolving the mixed water extract (RI-P) in water, purifying by physicochemical method, filtration and lyophilization of the separated filtrate to obtain the water-soluble fraction (RI-W) of the mixed water extract according to the present invention. It can also be used as an active ingredient of the composition.

The present invention also includes a method for producing a mixed water extract of baekbaekpi and matari as described above.

In the following, the mixed water extract should be understood to mean both the extract obtained by extracting a mixture of Hwangbaekpi and Matari with water or its water-soluble fraction obtained by purifying it, unless otherwise specified.

Hereinafter, the method for preparing the mixed water extract of the present invention will be described in more detail.

According to the method of the present invention, the mixed water extract is heated under saturated steam pressure (121 ° C., 15 pounds / in a ² ) by grinding a mixture of sulfur white skin and matari dried in the first step, using water such as constant or distilled water as a solvent. Extract.

The water used to extract the mixture of sulfur and skin in the first step is used in a ratio of 5-10 parts by weight, particularly preferably 7 parts by weight, based on 1 part by weight of the mixture.

Next, the extract was centrifuged in the second step to remove the precipitate and the extract was saturated again under high pressure, for example, boiled under vapor pressure (121 ° C., 15 pounds / in a ² ) in an autoclave to remove residual protein. After coagulation, the solution is filtered off by centrifugation or the like.

In the third step, the separated filtrate is extracted with an organic solvent such as chloroform, hexane, dichloromethane, cyclohexane, preferably chloroform or hexane to remove impurities such as resin and fiber, and the aqueous layer is again subjected to talc or the like. After purification by lyophilization to give the desired mixed water extract (RI-P).

The mixed water extract (RI-P) thus obtained may be separated only by its water-soluble fraction through subsequent purification as follows, if necessary. That is, the mixed water extract (RI-P) is again dissolved in water such as constant or distilled water and left at a low temperature of about 1 to 5 ° C. for 18 to 24 hours, and then the precipitate is filtered off to remove the filtrate by a physicochemical method. For example, it refine | purifies using a talc at low temperature of 1-5 degreeC. The water-soluble fraction (RI-W) of the mixed water extract according to the present invention is obtained by lyophilizing the filtrate separated by filtration of the purified aqueous solution again at 1-5 ° C. in a membrane filter apparatus.

The mixed water extract of Hwangbaekpi and Matari obtained by the method of the present invention as described above exhibits a pharmacologically useful immune function reactivation and augmentation effect as mentioned above.

The mixed water extract of Hwangbaekpi and Matari or the water-soluble fraction thereof according to the present invention obtained by the method as described above can be used in the pharmaceutical field either alone or in combination with a pharmaceutically acceptable carrier, in a therapeutically effective amount. It may be formulated in the form of a suitable pharmaceutical composition by conventional methods and administered in a manner customary in the pharmaceutical art, preferably orally. In general, the compositions of the present invention may be formulated in the form of tablets, capsules, solutions, suspensions, syrups or edible beverages suitable for oral administration.

In addition, the dosage of the mixed water extract according to the present invention may vary depending on whether the extract is a mixed water extract or a water-soluble fraction thereof, or depending on the sex, age, weight, and desired effect of the patient to be administered. In the case of oral administration to an adult, a dose of 5 to 50 mg, preferably 10 to 30 mg per day per kg of body weight can be administered. In addition, the extract according to the present invention may be administered or used in combination with drugs such as anticancer agents, anti-inflammatory agents, antiviral agents and the like as necessary.

Anticancer agents that can be used in combination or in combination with the composition according to the present invention include adriamycin, aerophosphamide, 5-FU, amsacrine and the like. In addition, the extract according to the present invention can be used with all kinds of vaccines, including hepatitis vaccine or pandemic hemorrhagic fever vaccine.

The present invention is explained in more detail by the following examples and experimental examples, but these are provided merely to illustrate the present invention, and the present invention is not limited in any way by them.

Example 1 Preparation of Mixed Water Extract (RI-P)

Mix 100 g of dried powdered Phellodendron amurense RUPRECHT and Matari (Patrinia scabiosaefolia FISCH.) 1: 1 by weight, and grind 100 g of pulverized powder, and add 3000 ml of distilled water and steam pressure at 121 ℃ and 15 lbs. After extraction for 40-60 minutes under / in a ² , the extract was aliquoted and the residue was removed. The extract was centrifuged to remove the precipitate, and the filtrate was concentrated and heated to 1500 ml in total and filtered. The filtrate was again saturated with steam at 121 ° C. under 15 pounds / in a ^{2 for} 15 minutes, and the precipitate containing the coagulated protein was removed by centrifugation and filtered. The filtrate was added to a separatory funnel, and 400 ml of chloroform was added to elute the resin and fiber, and the chloroform layer was separated therefrom. After the same operation was repeated twice, 200 ml of n-hexane was added to the aqueous layer again to elute remaining resins, fibers, and n-hexane soluble substances. The aqueous layer was separated, warmed to 60-80 ° C., and then stirred by adding 500 g of talc, followed by filtration under reduced pressure to remove talc, and the filtrate was gradually filtered again, and the filtrate was lyophilized to be powdered. By this method, about 15000 mg of mixed water extract (RI-P) was obtained from the mixture of Hwangbaekpi and Matari in a yield of about 15% (based on dry weight).

Example 2 Preparation of Water-Soluble Fraction (RI-W)

1000 mg of the mixed water extract RI-P obtained in Example 1 was aliquoted, dissolved in 500 ml of distilled water, and the resulting solution was left at a low temperature of about 1 ° C. for one day, followed by stirring with 300 g of talc, followed by filtration under reduced pressure. Purification was carried out by removing talc. The purified filtrate was slowly passed through a membrane filter (Nucleopore) apparatus at a temperature of about 5 ° C to lyophilize the aqueous fraction of the filtrate passed through and powdered. This method yielded about 800 mg of an aqueous fraction (RI-W) according to the invention in a yield of about 80% (dry weight basis).

[Composition]

[Composition 1] Tablet

250 mg of the lyophilized powdered mixed water extract according to the present invention prepared in Example 1, 260 mg of lactose for excipients and 35 mg of Avicel (microcrystalline cellulose), 15 mg of sodium starch glyconate as a dissolving aid, 80 mg of -HPC (Low-hydroxypropylcellulose) was mixed into a U-type mixer and mixed for about 20 minutes. After the mixing was completed, 100 mg of magnesium stearate was further added as a lubricant and mixed for about 3 minutes, and then tableted and film-coated by a conventional method to prepare a tablet containing 250 mg of mixed water extract per one party.

[Composition 2] Syrup

4.5 g of paraoxy methyl benzoate and 16 mg of paraoxypropyl benzoate were added as a preservative to 4.5 g of lyophilized powdered mixed water extract according to the present invention prepared in Example 1 The solution was maintained at 60 ° C, completely dissolved, cooled, and distilled water was added so that the total amount was 150 ml. A syrup containing 30 mg of mixed water extract was prepared per 1 ml.

[Composition 3] Capsule

300 mg of the lyophilized powdered mixed water extract according to the present invention prepared in Example 1 was mixed with 200 mg of lactose as a carrier to fill a hard gelatin capsule to prepare a capsule containing 300 mg of the mixed water extract per capsule.

Composition 4 Beverage Composition

After dissolving 500 mg of the freeze-dried powdered mixed water extract according to the present invention prepared in Example 1 in an appropriate amount of water, vitamin C as an auxiliary component, citric acid, sodium citrate, and high fructose as co-agents were added, and as a preservative, After adding an appropriate amount of sodium benzoate, water was added to make 100 ml of the total amount to prepare a beverage composition.

Experimental Example 1 Effect of Mixed Water Extract on T Cell-Dependent Antibody Production in Vivo

Twenty four Balb / c mice weighing 17-20 g were divided into six groups of four, and the first group was an untreated control group, and the second group was intraperitoneally transplanted with SRBC (Sheep Red Blood Cell) 2.4x108 cells. Groups 3 to 6 were treated with SRBC as in group 2, and then the mixed water extract RI-P of yellow skin and matari obtained in Example 1 according to the present invention was treated for 3 days (0 days, 1 day, Day 2) Orally administered in the amount of 3, 10, 30, 100mg per kg of body weight. Four days after transplantation, the animals were sacrificed and the spleens were removed aseptically. The splenocytes were isolated, and the cells were liberated using a syringe plunger in a petri dish containing 3 ml of EBSS (Earle's Balanced Salt Solution). . The obtained cell suspension was transferred to 15 ml conical tube, left for 5 minutes, and then 2 ml of the supernatant was taken and centrifuged at 1200 rpm for 10 minutes. The supernatant was discarded and the residue was again suspended in 2 ml of EBSS, diluted 30-fold with EBSS, and the splenocyte dilutions were used in 100 μl portions for one plaque forming cell assay. 100 μl of the splenocyte dilution thus obtained was washed with EBSS SRBC (24 μl, target cells), guinea pig complement (25 μl) diluted twice with EBSS, and agarose (0.85% in EBSS, 350 μl), then poured into Petri dishes to solidify and left in a CO ₂ incubator at 37 ° C. for about 1 hour. The modified number of plaques and cells were then counted using a modified Jerne Plaque Assay (Hwan M. Kim, et al., J. Toxicological Sciences 21: 41-45, 1996), and the results were obtained per 106 cells. The number of antibody producing cells is shown. The average value of the measured results is shown in Table 1 and FIG.

TABLE 1 Effect on T-cell dependent antibody production response in vivo

As can be seen from these results, when the mixed water extract according to the present invention was administered, the number of antibody-forming cells increased proportionally with the concentration of the drug, so that the 100 mg / kg administration group produced antibody-producing cells as compared to the SRBC treatment group. An increase of 307% was shown. Therefore, it can be seen that the immuno-enhancing effect of the mixed water extract of Hwangbaekpi and Matari according to the present invention is very excellent.

Experimental Example 2 Synergistic Effects of Mixed Water Extracts on T Cell-Dependent Antibody Production In Vivo

In each group, 65 Balb / c mice weighing about 17 to 20 g were divided into 13 groups, and ⁸ groups of SRBC (Sheep Red Blood Cell) 2.4xl0 were injected intraperitoneally. After SRBC transplantation, the first group was treated with physiological saline without further treatment as a control group, and the other groups were sulfur white skin extracts obtained by the same method as Example 1 alone. Group 5) and Matari water extracts (Groups 6-9), and the mixed water extracts (RI-P) of the present invention obtained in Example 1 (Groups 10-13), respectively, for 3 days from the first day of transplantation (0 (1, 2 days) were administered orally in amounts of 3 mg, 10 mg, 30 mg and 100 mg per kg body weight. After 4 days, the animals were sacrificed to remove the spleen aseptically, the tissue was then chopped, the splenocytes were separated using a mesh, and the cells were liberated using a syringe plunger in a petri dish containing 3 ml of EBSS. The cell suspension was transferred to a 15 ml conical tube, left for 5 minutes, 2 ml of the supernatant was centrifuged at 1200 rpm for 10 minutes. The supernatant was discarded and resuspended in 2 ml of EBSS, diluted 30-fold with EBSS, and used in 100 μl portions of one plaque forming cell assay. 100 μl of the splenocyte dilution thus obtained was washed with EBSS using SRBC (25 μl, target cells), guinea pig complement (25 μl) diluted twice with EBSS, and agarose (0.85%, 350 μl in EBSS). ), Poured into Petri dishes, solidified, and left in a CO ₂ incubator at 37 ° C. for about 1 hour. The number of aqntibody forming cells was then determined using a modified Journey plaque assay. The number of plaques and the number of cells were counted and expressed as the number of antibody producing cells per 10 ⁶ cells. The measured results are shown in Table 2 below.

TABLE 2 Synergistic Effects of Mixed Water Extracts on T-Cell-Dependent Antibody Production In Vivo

As can be seen from the results shown in Table 2, the mixed water extract of the present invention significantly increased the number of antibody-producing cells compared to the case where the respective plants, namely, extracts of Hwangbaekpi and Matari, were administered alone. . Therefore, it can be seen that the mixed water extract of the present invention exhibits significantly superior synergistic effect compared to the extract of each plant.

Experimental Example 3 Effect of Mixed Water Extract on T Cell Activation Reaction (Mixed Immune Cell Reaction)

The effect on the T cell activation reaction of the mixed water extracts of Hwangbaekpi and Matari according to the present invention was measured by the mixed immune cell reaction method.

Major histocompatibility complex (MHC) class [2] Mice with different antigens from B6C3F1 (H-2k) and BDFl (H-2d) mice with different spleens are aseptically removed and finely chopped using a mesh. To release the splenocytes. After the free splenocytes were adjusted to 25xl06 cells / ml, 100 μl of B6C3Fl (H-2k) and BDF1 (H-2d) splenocytes were put into 96 well plates, respectively, to finally 200 μl.

Here, the mixed water extract RI-P obtained in Example 1 according to the present invention was treated at a concentration of 0.01 to 100 µl / ml, and then incubated in 37 ° C. in a CO ₂ incubator for 3 days. 18 h before the end of the incubation, 1 μCi [ ³ H] thymidine was added per well. After the cells were collected using an automatic cell harvester, the proliferation of immune cells was measured by measuring the degree of absorption of [ ³ H] thymidine. The measured results are shown in Table 3 and FIG. 2.

TABLE 3 Effect of the mixed water extract of the present invention on the proliferation of T cells

From the results described in Table 3 and FIG. 2, it can be seen that when the mixed water extract of the present invention is applied, the proliferation of T cells increases with the concentration thereof.

Experimental Example 4 Effect of Mixed Water Extract on Immune Cell Proliferation

After cervical distal bone, the abdomen was disinfected with 70% alcohol. In the sterile state, the abdomen was dissected, the spleen was removed, placed in a Petri dish containing buffer (EBSS), and the splenocytes were released using the plunge of the syringe. The suspension containing the splenocytes thus obtained was transferred to a tube, left for about 5-10 minutes, and the supernatant was collected and centrifuged (1200 rpm, 10 minutes). The supernatant was discarded and the precipitate was resuspended in RPMI 1640 culture [10% FCS (fetal calf serum), 2-mercaptoethanol (2-ME)], and cell number and cell viability were measured. The isolated splenocytes were treated with mixed water extract (RI-P) obtained in Example 1 according to the present invention for 3 days at a concentration of 0.01 to 100 μl / ml, followed by 5 psi of mixed gas (7%, O ₂ , 10% CO ₂ , 83% N ₂ ) was incubated using a rocking plate (rocking plate) in an incubator filled with. After incubation, [ ³ H] thymidine was added at a concentration of 5 μCi / ml to measure the degree of absorption, thereby measuring the proliferation of immune cells. The measured results are shown in Table 4 and FIG. 3.

TABLE 4 Influence on immune cell proliferation

As shown in Table 4 and the accompanying FIG. 3, it can be seen that when the mixed water extract of the present invention is applied, the proliferation of immune cells increases in proportion to its concentration.

Experimental Example 5 Changes in Immune Cell Composition of Spleen, Thymus, and Bone Marrow by In Vivo Treatment of Mixed Water Extracts

After the oral administration of the mixed water extract RI-P of Hwangbaekpi and Matari prepared in Example 1 to mice at concentrations of 100 and 300 mg / kg for 3 days, the mice were sacrificed and the spleen, thymus and bone marrow were separated. . Immune cells were separated from these organs by the same method as that of separating splenocytes in Experimental Example 2, followed by centrifugation.

After the supernatant was discarded, the cell precipitate was treated with red blood cell hemolysis buffer (ACK buffer, 0.15M NH ₄ C1, 0.0lM KHCO ₃ , 0.1 mM Na ₂ EDTA, pH 7.2) for 1 to 2 minutes for 2 to 3 minutes. . After washing the immune cells with medium, the cell concentration was adjusted to 10 ⁷ cells / ml, and then 100 μl per tube was dispensed. In order to measure B cells, phycoerythrin (PE) is bound to rat monoclonal antibody B220 against mouse B cell antigen, and Thy-1-PE (Caltag Laboratories) is used for T cells. For CD4 T cells, CD4-FITC (Caltag Laboratories) and for CD8 T cells, Ly2-FlTC (Caltag Laboratories) were used. These antibodies were added to each tube and allowed to react on ice for about 40 minutes. After the reaction, the cells were washed with phosphate buffered saline (PBS), and the composition ratio of each immune cell was measured using FACScan (Becton Dickenson, San Jose, Calif.). As a result of measuring 10,000 cells, the composition ratio of B cells, T cells, and CD4 T cells in the spleen was increased as shown in Table 4 below, and in the thymus, the composition ratio of T cells with Thy-1 antibody was increased. CD4 T cells with CD4 antibody were also increased. In the bone marrow, the proportion of B cells with B220 antibody was increased.

[Table 5] Change in the percentage of immune cell composition by the mixed water extract of the present invention (unit%)

Experimental Example 6 Toxicity to Normal Cells of Mixed Water Extracts

In order to find out how the mixed water extract of Hwangbaekpi and Matari according to the present invention affects normal immune cells, in particular, it shows toxicity.

In this experimental example, the NIH-3T3 cell line, which was distributed from the National Cancer Institute, USA, was used. All experiments were performed using cells passaged up to 10 times after receiving the culture. Cells were cultured in a medium (R10) to which 10% FCS was added to the RPMI1640 culture medium and subjected to SRB analysis, using RPMI1640 culture solution (R5) containing 5% FCS under conditions of 5% CO ₂ and 37 ° C. Incubated. Cell cultures containing 4 × 10 4 cells / mL were dispensed into 96-well microplates and preincubated for about 24 hours under conditions of 5% CO ₂ , 37 ° C. to stabilize prior to drug administration.

Cytotoxicity was measured by administering the mixed water extract RI-P of Hwangbaekpi and Matari obtained in Example 1 at various concentrations between 0.03 µg / ml and 3 µg / ml (final concentration). After adding the mixed water extract, the cells were incubated for 48 hours under conditions of 5% CO ₂ , 37 ° C. and 100% relative humidity. Cytotoxicity was measured using SRB (Sulforhodamine B) assay. Monks, A et al., J. Natl. Cancer Inst. 83: 757-766, 1991]. The measured results are shown in FIG. In FIG. 4, the X-axis (concentration) is represented by converting the concentration value into a log value.

As shown in Figure 4, the mixed water extract according to the present invention had no effect on the normal cell line NiH-3T3 cells, and no increase in cytotoxicity was observed even with increasing concentration.

Experimental Example 7 Toxicity Test

LD ₅₀ (amount capable of killing 50% of experimental animals) is an important value that is an indicator of acute toxicity of drugs in order to confirm the safety of the mixed water extracts of Hwangbaekpi and Matari according to the present invention. Obtained by the same method.

Thirty normal ICR mice (♀, 19 ± 1 g) were divided into five groups of A to E, each six to one group, and the mixed number of Hwangbaekpi and Matari prepared in Example 1 according to the present invention per 1 kg of body weight in group A. The amount of the extract was gradually increased from the administration of 3 g of the extract, and 6 g in the B group, 9 g in the C group, 12 g in the D group and 15 g in the E group, respectively, and the Berens-Karber method [Reference] : LD ₅₀ value by oral (po) administration of the mixed water extract of the present invention was measured by Kogyo Co., Ltd., Namsan, Japan, p131, 1960]. The results are shown in Table 6 below.

Table 6 Lethal dose by oral administration of the mixed water extract of the present invention (LD ₅₀ )

* Z: ½ of the number of dead animals in two consecutive doses

** d: difference between two consecutive doses

As shown in the results shown in Table 6, even in the group that orally administered the mixed water extract of Hwangbaekpi and Matari according to the present invention in a high dose amount of 15 g per 1 kg of body weight, no animals died and oral administration of the mixed water extract LD ₅₀ value was 15 g / kg or more, and thus it was found to be very safe for living bodies. In other words, it can be clearly seen that the mixed water extract of Hwangbaekpi and Matari according to the present invention can be safely administered with little toxicity.

In addition, autopsies and histopathological examinations were performed on the test animals used to measure the LD ₅₀ value in the following manner. At the end of the LD ₅₀ test, all living animals were bled to death after ether anesthesia, and the organs were extracted. After autopsy, the whole organs were fixed in 10% neutral formalin solution for 10 days, and then dehydrated and embedded in paraffin-embedded (Fisher, Histomatic Tissue Processor, 166A, Shadon, UK). 5 micrometer sections were made with a microtome (AO Rotary Microtome, LEICA, Germany) and observed with hematoxylin and eosin staining.

Microscopic examination of all animals in each group was shown in the attached FIG. That is, when administration of the mixed water extract of baekbaekpi and matari according to the present invention up to 15g per 1kg of mouse body weight, the ideal findings by drug administration in the liver tissue was not observed (Fig. 5A). In addition, no abnormal findings by drug administration were observed in kidneys (Fig. 5B), and no abnormal findings by drug administration were observed in cardiomyocytes of the heart (Fig. 5C). Abnormalities in drug administration were not observed in other major organs, including the gastrointestinal tract, pancreas, lung, spleen, adrenal gland, brain, testes, ovaries, and bone marrow.

Therefore, the mixed water extract of Hwangbaekpi and Matari according to the present invention had no side effects due to acute toxicity to all organs even when 15g per 1kg of body weight, the maximum dose that can be administered to mice, and also toxins such as any organ damage. It was determined to be a safe drug that does not cause it.

Claims (15)

An immunoadjuvant composition containing a mixed water extract of baekbaekpi and matari plants. The immunopotentiator composition according to claim 1, which is used for reviving the immune function inhibited by an anticancer chemotherapeutic agent and radiotherapy. 2. The immunoadjuvant composition according to claim 1, which is used as an adjuvant for increasing antibody formation ability in the use of an immune-related vaccine. The immunopotentiator composition according to any one of claims 1 to 3, comprising a mixed water extract obtained by mixing the baekbaekpi and matari plants in a ratio of 1: 0.1 to 1: 5 by weight. 5. The immunopotentiator composition according to claim 4, comprising a mixed water extract obtained by mixing the baekbaekpi and matari plants in a ratio of 1: 1 to 1: 2 by weight. 6. The immunopotentiator composition according to claim 5, comprising a mixed water extract extracted by mixing Hwangbaekpi and Matari plants in a ratio of 1: 1 by weight. The immunopotentiator composition according to claim 1, wherein the mixed water extract of baekbaekpi and matari is a purified water soluble fraction thereof. 2. The immunopotentiator composition of claim 1, further formulated in pharmaceutical unit dosage form using a pharmaceutically acceptable carrier. 9. The immunopotentiator composition according to claim 8, wherein the pharmaceutical unit dosage form is in the form of a tablet, capsule, solution, suspension, syrup or food beverage. 1) The mixture of Hwangbaekpi and Matari plants was extracted with water and the extract was filtered. 2) The precipitate of coagulated protein produced by saturation of the filtrate under high pressure was removed by centrifugation. 3) The organic solvent was added to the filtrate by adding an organic solvent. Method for producing a mixed water extract of baekbaekpi and matari plants, characterized in that the material is removed and then separated by a water layer to lyophilize. The method according to claim 10, wherein the extracting water in step 1) is used in a ratio of 5 to 10 parts by weight based on 1 part by weight of the mixture of sulfur white skin and matari plant. 11. The method according to claim 10, wherein the extracting water is used in the step 1) in a ratio of 7 parts by weight based on 1 part by weight of the mixture of baekbaekpi and matari plants. The process according to claim 10, wherein chloroform or hexane is used as the organic solvent in step 3). The method according to claim 10, wherein the obtained lyophilisate is subsequently dissolved again in water, purified by physicochemical method, and then the purified aqueous solution is filtered to lyophilize the filtrate to obtain an aqueous fraction of the mixed water extract. 15. The method of claim 14, wherein talc is used as a physicochemical purification method.