KR100207293B1 - Immunostimulating composition containing mixed aqueous extract of phellodendri cortex and patrinia scabiosaefolia - Google Patents

Abstract

The present invention relates to an immunoadjuvant composition containing a mixed water extract of Phellodendron amurense Rupr. Cortex and Matari (Patrinia scabiosaefolia FISCH.) Plants as an active ingredient. The extract of the present invention has an excellent immunopotentiating effect to restore or increase damaged immune mechanisms in patients undergoing chemotherapy or radiation therapy, and to increase antibody production titers by using 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 immunocompromised state of cancer patients or by chemotherapy or radiation therapy for these cancer patients, thereby reducing T cells, B cells, and macrophages (MΦ) related to immunity. In the case where the immune function is selectively inhibited accordingly, the mixed water extract of Hwangbaekpi and Matari according to the present invention may not only effectively inhibit the proliferation or metastasis of malignant tumors by enhancing biological immune function, but also use in combination with immune-related vaccines. This also shows the effect of increasing antibody titer by the vaccine. Therefore, the extract of the present invention is administered in combination with port chemotherapy, or used in combination with radiation therapy to improve the immunosuppression of cancer patients or selective immunosuppression according to 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 extracts of Hwangbaekpi and Matari of the present invention on T cell dependent antibody production 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 extracts of Hwangbaekpi and Matari plants of the present invention on the liver, kidney and heart of mice (15g / Dosing, HE staining, 5a: hepatic x400, 5b: kidney x200, 5c: heart, x200).

The present invention relates to an immunoadjuvant composition containing a mixed water extract of Hwangbaekpi (黄 타) and Matari plants (packing) as an active ingredient. More specifically, the present invention contains a mixed water extract obtained by extracting a mixture of Phellodendron amurense RUPRCHT cortex and Matari (Patrinia scabiosaefolia FISCH.) With water as an active ingredient to induce 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 disease, cancer, diabetes, and male infertility. In addition, AIDS, as well as opportunistic infections of various pathogenic viral diseases, parasitic infections, etc. It can be called a disease associated with immunodeficiency.

The bioimmune system is controlled by the autonomic nervous system and the endocrine system, and has the property of maintaining homeostasis by excluding non-self from 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 function enhancers, immune function activators, etc. are generally used to correct and restore an abnormally disturbed immune system and to restore damaged immune functions.

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, a preparation of concentrated and purified IgG of human immunoglobulin is used for the prophylaxis 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 (interferon, INF) was found as an antiviral factor, after which cell proliferation inhibitory activity and immune function control action was confirmed, and is used as antiviral agent and antitumor agent, but β type is fever, boredom, appetite Side effects such as sluggishness, injection, or alopecia are observed, and type α exhibits side effects such as leukocyte reduction and elevated GOT due to temporary inhibition of bone marrow function.

In addition, immunopotentiators improve the immune response of the living body to improve secondary immunodeficiency caused by radiotherapy and excessive use of antitumor agents or corticosteroids, and the attenuated tuberculosis bacterium BCG (Bacillus Calmette-Gurin) Cell Wall Skelton (CWS) 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., is a major defense system of cellular immunity is aggravated by the failure of the T cell system. 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 the lack of immunoglobulins in serum. 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 enhance or repair immunodeficiency or immunodeficiency due to 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. In other words, 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 chemotherapeutic agents and radiotherapy, as well as immune-related vaccines and toxoids. The natural plant extract was used as a means to further enhance immunity 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 yellow blood 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 producing a mixed water extract which is used as an active ingredient having an immuno-enhancing action 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 yellow or tan pigment and 1.5 to 4.5% of several alkaloids. The main component of alkaloids is berberine. It also contains palmin, magnoflorine, guanidine, guatedine, jateorrizine, phellodendrine, candicine, menisperine, etc. High qualities include obakunone and obakulactone, -Sitosterol ( -Sitosterol) is known to contain. 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 soak (敗 醬), Matari (Patrinia scabiosaefolia FISCH.), Dukgal (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 by which the anti-tumor effect of these matari plants is caused. In addition, Matari strongly inhibits the development of Staphylococcus aureus, Streptococcus, etc. by in vitro tests, and has the ability to regenerate damaged liver cells to prevent their degeneration and to improve the circulation of the portal vein and promote the regeneration of liver cells. In addition, it acts on the central nervous system to make nerves safe, has a strong analgesic effect, and is known to have a blood pressure lowering effect and an antidiuretic effect.

Matari's root contains trace alkaloids such as essential oils, various saponins, carbohydrates and coumarin. 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, triterpene-based ursolic acid (ursolic acid) and oleanilic acid (oleanilic acid) is also contained. Ursolic acid is reported to inhibit carcinogenesis by TPA (12-O-teradecanoylphorball-13-acetate) and to inhibit metastasis by reducing the expression of matrix metalloproteinase-9 (MMP-9). Cha, HJ et al., Cancer Res. 56: 2281-2284, 1996 and Huang MT et al., Cancer Res. 54: 701-708, 1994). Dried seeds contain 19.4-19.9% 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 action including immune function revival and immunosupplement, which is not known to Hwangbaekpi and Matari so far, and the mixed water extract is also Compared to the case of using each extract of Hwangbaekpi and Matiri alone experimentally confirmed to exhibit a superior synergistic effect, 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 saturated vapor pressure (121) using a solvent such as water, such as constant or distilled water, by grinding the mixture of dried sulfur white skin and matari in the first step , Extract heat under 15 pounds / in a ² ).

The water used to extract the mixture of Hwangbaekpi and Matari 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, centrifugation of the extract in the second step to remove the precipitate and the extract is again saturated under high pressure, e.g. vapor pressure (121) , Coagulate the remaining protein by boiling under 15 pounds / in a ² ), and then remove by filtration by centrifugation or the like.

In the third step, the separated filtrate is extracted with an organic solvent such as chloroform, hexane, dichloromethane and 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 dissolved in water such as constant or distilled water again to about 1 to 5 The mixture is left at a low temperature of 18 to 24 hours and then the precipitate is filtered off and the filtrate is subjected to a physicochemical method, for example 1 to 5 Purification using talc at low temperature. Purified aqueous solution again 1 to 5 By lyophilization of the filtrate separated by filtration in a membrane filter apparatus at the water-soluble fraction (RI-W) of the mixed water extract according to the present invention is obtained.

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. If orally administered to adults, weight 1 5 to 50 per day , Preferably 10 to 30 It can be administered at the dose of. In addition, the extract according to the present invention may be administered or used in combination with a medicament such as an anticancer agent, an anti-inflammatory agent, an antiviral agent, or 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)

100 g of the powder ground by mixing the dried Phellodendron amurense RUPRCHT and Matari (Patrinia scabiosaefolia FISCH.) 1: 1 by weight, and distilled water 3000 Steam pressure by adding 121 After extraction for 40-60 minutes under 15 pounds / in a ² , the extract was aliquoted and the residue was removed. The extract is centrifuged to remove the precipitate, and the filtrate is 1500 in total. It was concentrated to heat and filtered. The filtrate was vapor pressure 121 , Precipitates containing coagulated protein produced by saturation under 15 pounds / in a ^{2 for} 15 minutes were removed by centrifugation and filtered. Put the filtrate into a separatory funnel and chloroform 400 Was added to elute the resin and fiber, and the chloroform layer was separated and removed. After the same operation was repeated twice, n-hexane 200 was again added to the aqueous layer. Was added to elute remaining resins, fibers and n-hexane soluble materials. 60 to 80 by separating the aqueous layer After heating, the solution was stirred with 500 g of talc, and then filtered under reduced pressure to remove talc. The filtrate was gradually filtered again, and the filtrate was lyophilized to be powdered. By this method, the mixed water extract (RI-P) is about 15000 from the mixture of Hwangbaekpi and Matari in a yield of about 15% (based on dry weight). Obtained.

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

1000 in mixed water extract RI-P obtained in Example 1 Fractions, distilled water 500 Dissolved in and the resulting solution about 1 After standing at low temperature for one day, 300 g of talc was added thereto, stirred, and filtered under reduced pressure to remove talc. About 5 purified filtrate The aqueous fraction of the filtrate passed through the membrane filter (Nucleopore) device slowly at a temperature of lyophilized and powdered. Water soluble fraction (RI-W) according to the invention in a yield (by dry weight) of about 80% by this method Obtained.

[Composition]

[Composition 1: Purification]

Lyophilized powdered mixed water extract 250 according to the present invention prepared in Example 1 Lactose 260 for Excipients And Avicel (microcrystalline cellulose) 35 Sodium Starch Glyconate 15 , L-HPC (Low-hydroxypropylcellulose) 80 The mixture was mixed into a U-type mixer and mixed for about 20 minutes. Magnesium stearate 10 as a lubricant after mixing is complete Was added and mixed for about 3 minutes, then tableted and film-coated in a conventional manner to give a single mixed water extract 250 A tablet containing was prepared.

[Composition 2: Syrup]

An appropriate amount of sucrose is dissolved in a certain amount of water, and paraoxy methylbenzoate 80 as a preservative therein. And paraoxypropylbenzoate 16 Was added and 4.5 g of the lyophilized powdered mixed water extract according to the present invention prepared in Example 1 was added to 60 To be completely dissolved, then cooled and distilled water is added to the total amount of 150 To be 1 Sugar Extract 30 A syrup containing was prepared.

[Composition 3: Capsule]

Lyophilized powdered mixed water extract 300 according to the present invention prepared in Example 1 Lactose 200 as a carrier Mixed with water and filled into hard gelatine capsules; mixed water extract 300 per capsule A capsule containing was prepared.

[Composition 4: Beverage Composition]

Lyophilized powdered mixed water extract 500 according to the present invention prepared in Example 1 Is dissolved in an appropriate amount of water, and vitamin C as an auxiliary ingredient, citric acid, sodium citrate, and high fructose as an auxiliary ingredient are added in an appropriate amount, and an appropriate amount of sodium benzoate is added as a preservative, and then water is added by adding water. To prepare a composition for a beverage.

Experimental Example 1

Effect of Mixed Water Extract on T Cell-Dependent Antibody Production Response 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 implanted with SRBC (Sheep Red Blood Cell) 2.4x10 ⁸ cells. Groups 3 to 6 were treated with SRBC as in group 2, and then the mixed water extract RI-P of yellowish skin and matari obtained in Example 1 according to the present invention for 3 days (day 0, day 1). , 2 days) weight 1 3, 10, 30, 100 per Each dose was administered orally. Four days after transplantation, the animal was sacrificed and the spleen was removed aseptically, then the splenocytes were separated and EBSS (Earle's Balanced Salt Solution) 3 Cells were freed using a plunger of a syringe in a Petri dish containing. Obtained cell suspension 15 Transferred to a cortical tube of 5 minutes, then the supernatant 2 Was taken and centrifuged at 1200 rpm for 10 minutes. Discard the supernatant and return the residue to EBSS 2 After suspension in, diluted by 30-fold with EBSS, this splenocyte dilution was used 100 μl each for one plaque forming cell assay. 100 μl of the splenocyte dilution thus obtained was washed with EBSS, SRBC (25 μl, target cells), guinea pig complement (25 μl) diluted twice with EBSS, and agarose (0.85% in EBSS, 350). [Mu] l) and pour into Petri dishes to solidify It was left in a CO ₂ incubator for about 1 hour. The modified Jerne Plaque Assay (Hwan M. Kim, et al., J. Toxicological Sciences 21: 41-45, 1996) was then used to count plaque and cell numbers, resulting in 10 ⁶ cells. It is represented by the number of sugar-producing cells. The average value of the measured results is shown in Table 1 and FIG.

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 in proportion to the concentration of the drug, resulting in 100 Of The administration group showed an increase of 307% in the number of antibody producing cells compared to the SRBC treatment group. 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

65 Balb / c mice weighing about 17 to 20 g were divided into 13 groups of 5 mice each to intraperitoneally transplanted 2.4 × ^{10 8} SRBC (Sheep Red Blood Cell). After SRBC transplantation, the first group was treated with physiological saline without further treatment as a control group, and the other groups were obtained with the same extracts of Hwangbaekpi skin extract obtained by the same method as Example 1 alone. Group 5) and Matari water extracts (Groups 6-9), and the mixed water extracts of the present invention (RI-P) obtained in Example 1 (Groups 10-13), respectively, for 3 days (0 days) from the first day of transplantation. Day, 1 day, 2 days) 3 per , 10 , 30 And 100 It was administered orally in the amount of. After 4 days, the animal was sacrificed and the spleen was removed aseptically, the tissue was chopped, and the spleen cells were isolated using a mesh to separate EBSS 3 Cells were liberated using a syringe plunger in a Petri dish containing. Cell suspension 15 Transferred to a cortical tube of 5 minutes, then the supernatant 2 Was taken and centrifuged at 1200 rpm for 10 minutes. Discard Supernatant and EBSS 2 After resuspended in, diluted 30-fold with EBSS was used for each plaque forming cell assay 100ul. 100 μl of the splenocyte dilution thus obtained was washed with EBSS, SRBC (25 μl, target cells), guinea pig complement diluted twice with EBSS (25 μl), and agarose (0.85% in EBSS, 350 μl) After mixing, pour into Petri dishes to solidify It was left in a CO ₂ incubator for about 1 hour. The number of antibody forming cells was then measured 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.

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-forming cells as compared to the case of administering each plant alone, that is, each extract of Hwangbaekpi and Matari. Therefore, it can be seen that the synergistic immune enhancing effect is excellent compared to the extract of each plant of the mixed water extract of the present invention.

Experimental Example 3

Effect of Mixed Water Extracts on T Cell Activation Responses (Mixed Immune Cell Responses)

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) antigens Aseptically remove spleen from two kinds of mice with different antigens, B6C3F1 (H-2k) and BDF1 (H-2d), and use fine mesh. To release the splenocytes. 2.5 x 10 free splenocytes, respectively cell/After adjusting so that B6C3F1 (H-2k) and BDF1 (H-2d) splenocytes were put into 100 µl of each well in a 96 well plate to finally 200 µl.

Herein, 0.01 to 100 µl / of the mixed water extract RI-P obtained in Example 1 according to the present invention. After treatment with a concentration of 37 And incubated for 3 days in a CO ₂ incubator. 1 μCi of [ ³ H] thymidine was added per well 18 hours before the end of the incubation. 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.

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 Extracts 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 splenocytes were released using a plunger of a 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 mixed with the mixed water extract (RI-P) obtained in Example 1 according to the present invention for 3 days from 0.01 to 100 µg /. After treatment at a concentration of 5 psi mixed gas (7% O ₂ , 10% CO ₂ , 83% N ₂ ) incubated using a rocking plate (rocking plate) in an incubator. After incubation, [ ³ H] thyminine was added to 5 μCi / Proliferation of immune cells was measured by measuring the degree of absorption by adding to the concentration of. The measured results are shown in Table 4 and FIG. 3.

As shown in Table 4 and the accompanying FIG. 3, it can be seen that the application of the mixed water extract of the present invention increased the proliferation of immune cells 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

100 and 300 of the mixed water extract RI-P of Hwangbaekpi and Matari prepared in Example 1 according to the present invention. Of After oral administration to mice at concentrations of 3 days, mice were sacrificed and spleen, thymus and bone marrow were isolated. Immune cells were separated from these organs by the same method as that of separating splenocytes in Experimental Example 2, followed by centrifugation. After discarding the supernatant, the cell precipitate was added to the erythrocyte hemolysis buffer (ACK buffer, 0.15M NH ₄ C1,0.01M KHCO ₃ , 0.1mM Na ₂ EDTA, pH 7.2) per organ. Treatment was carried out for 2 to 3 minutes each. After washing the immune cells with medium, the cell concentration was 10 ⁷ cells / After adjusting to 100 μl was dispensed per tube. 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 CD ₄ -FITC (Caltage Laboratories) and for CD8 T cells Ly ₂ -FITC (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 was increased in the spleen as shown in Table 4, and in the thymus, the composition ratio of T cells with Thy-1 antibody was increased. And CD4 T cells with CD4 antibody also increased. In the bone marrow, the proportion of B cells to which the B220 antibody was attached increased.

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 experiment, NIH-3T3 cell line distributed from National Cancer Institute, USA was used. All experiments were performed using cells passaged within 10 times after receiving the culture. The cells were cultured in a medium (R10) in which 10% FCS was added to RPMI1640 culture medium, and when performing SRB analysis, cells were cultured using RPMI1640 culture solution (R5) containing 5% FCS 5%, CO, 37 The culture was carried out under the conditions of. 4x104 cells / Cell culture solution containing the solution was dispensed into 96-well microplates and treated with 5% CO ₂ , 37 It was preincubated for about 24 hours under conditions of to stabilize before administration of the drug.

0.03 μg / ml of mixed water extract RI-P of yellowish white skin and matarium obtained in Example 1 according to the present invention From 3 µg / Cytotoxicity was measured by administration at various concentrations between (final concentration). After the addition of the mixed cell extract 5% CO _2, 37 Incubated for 48 hours under 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 FIG. 4, the mixed water extract according to the present invention had no effect on NIH-3T3 cells, which were normal 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 index indicating acute toxicity of drugs in order to confirm the safety of the mixed water extracts of P. alba and Matari according to the present invention. Obtained by the same method.

Thirty normal ICR mice (♀, 19 ± 1 g) were divided into five groups, A to E, 6 animals in one group, and weight 1 to group A. By increasing the drug homogeneously from administering the mixed water extract of the baekbaekpi and matari prepared in Example 1 according to the present invention 6g in group B, 9g in group C, 12g in group D and 15g in group E Oral administration and Behrens-Karber method [Reference: 高木 敬 次 陽 In addition, LD ₅₀ value by oral (po) administration of the mixed water extract of the present invention was measured in accordance with the present invention. The results are shown in Table 6 below.

As shown in the results described in Table 6 above, the weight of the mixed water extract of baekbaekpi and Matari according to the present invention 1 In the group administered orally at a high dose of 15 g per sugar, even one animal did not die, so the LD ₅₀ value obtained by oral administration of the mixed water extract was 15 g / weight. As mentioned above, it turned out that it is very safe for a living body. 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, all the 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.

All animals in each group were dissected and histopathologically examined under a microscope. That is, when administration of the mixed water extract of baekbaekpi and matari according to the present invention up to 15g per 1kg of the mouse body weight, the ideal findings by drug administration in the liver tissue was not observed (Fig. 5a). In addition, no abnormalities were observed in the kidney by drug administration (Fig. 5b), and no abnormal findings by drug administration were observed in cardiomyocytes of the heart (Fig. 5c). Abnormalities due to drug administration were not observed in other major organs such as 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, which is the maximum dose that can be administered to mice, and any organ damage, etc. It was determined to be a safe drug that does not cause toxicity.

Claims (15)

An immunoadjuvant composition containing a mixed water extract of baekbaekpi and matari plants. 2. The immunopotentiator composition according to claim 1, which is used to revive immune function inhibited by anticancer chemotherapeutic agents 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. The immunopotentiator composition according to claim 4, comprising a mixed water extract extracted by mixing Hwangbaekpi and Matari plants in a ratio of 1: 1 to 1: 2 by weight. The immunopotentiator composition according to claim 5, further comprising a mixed water extract obtained by mixing the baekbaekpi 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 according to 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 tablets, capsules, solutions, suspensions, syrups or food beverages. 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 after removing the water to separate the larvae lyophilized. 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 the baekpipi and matari plants. The method according to claim 10, wherein the extracting water in step 1) is used in a ratio of 7 parts by weight based on 1 part by weight of the mixture of the baekpipi 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.