Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
2000-09-08
2002-07-16
Peselev, Elli (Department: 1623)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C536S123100, C536S127000
Reexamination Certificate
active
06420348
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a novel pectic substance and a purification method thereof. More particularly, the present invention relates to pectic polysaccharides isolated from
Angelica gigas
Nakai, which are immuno-stimulative, and a purification method thereof. Also, the present invention is concerned with use of the pectic polysaccharides in the treatment or prophylaxis of immune-related disease and in basic immunology research.
BACKGROUND ART
The immuno-stimulating agents derived from natural substances are used for the treatment of cancers, AIDS and chronically infected states by enhancing immunoresponses or restoring lowered immune functions. Extensive research on fungi, bacidiomycota and medicinal herbs has been made to obtain immuno-stimulating agent. Particularly, the polymer fractions of the natural substances showed immuno-stimulation, such as anticancer activity, anticomplementary activity, and induction of lymphocyte proliferation. Glucan polysaccharides, such as lentinan, PSK, and schzophyllan, derived mainly from mushrooms, are useful to be clinical usefulness in therapies of cancer.
It is reported in oriental medical documents that Angelica, belonging to family Umbelliferae, can be used for the treatment of gynecological diseases (anaemia, interruption in blood circulation, etc) by virtue of anodynic effect. From
Angelica gigas
Nakai, found only in Korea, coumarin substances, such as decursin, decursinol, nodakenetin, umbelliferon, nodakenin, and &bgr;-sitosterol, have been extracted, thus far.
Pharmaceutical research on the herb showed that the ether extract of
Angelica gigas
Nakai made the enucleated intestinal canals and uterus of rabbit excited. Free decursin and decursinol were reported to have a paralysis action on the enucleated intestinal canals of rabbit, a suppressive action on the enucleated heart of frog, and a breath-suppression and blood-depression action on rabbit. Recently, decursin have been found to have in vitro therapeutic activity against cancers and to enhance the activity of protein kinase C.
DISCLOSURE OF THE INVENTION
Bearing the above-mentioned situations in mind, the present inventors repeated thorough and intensive research with the aiming of finding a novel immuno-stimulating entity in
Angelica gigas
Nakai and finally, isolated a polysaccharide component which is immuno-stimulative and active against cancer.
Therefore, it is an object of the present invention to provide pectic polysaccharides which are immuno-stimulative and anticancer-effective.
It is another object of the present invention to provide a method for purifying the polysaccharides from
Angelica gigas
Nakai.
It is a further object of the present invention to provide information for use of the polysaccharides in the treatment or prophylaxis of cancers and immune-related diseases and in the recovery of immune functions.
It is still a further object of the present invention to provide information for use of angelan as an immuno-stimulating agent useful for basic immunology research.
In accordance with the present invention, the pectic polysaccharides which are purified by refluxing
Angelica gigas
Nakai in hot water and subjecting the hot water extract to ion exchange chromatography, are immuno-stimulative and shows anticancer activity.
BEST MODES FOR CARRYING OUT THE INVENTION
A detailed description will be given of a purification method of angelan, a pectic polysaccharide, from
Angelica gigas
Nakai and the physicochemical characteristics thereof, below.
Sliced roots of
Angelica gigas
Nakai were refluxed with hot water for 1 hr, followed by filtering the water through a four-folded gauze and Whatman filter paper. The filtrate was mixed with three volumes of ethanol and allowed to stand at 4° C. for 3 hours to give precipitates. They were obtained as brown polymers by centrifugation. From the fact that the polymers can easily obtained by ethanol precipitation and there are no denaturated protein precipitates even if solubilized precipitates in water are boiled for 20 min, the polymer fractions are assumed to contain a large amount of non-proteinous polymeric materials. Most of the colored material is absorbed in DEAE-cellulose, an anion exchange resin. Using the DEAE-cellulose absorption method, acidic and neutral fractions can be obtained. The acidic fraction was named angelan. They both are polysaccharides with a small amount of proteins. Angelan 10 KD in molecular weight was chemically characterized as a single substance as certified by the analytical high performance liquid chromatography method (HPLC).
Pectic polysaccharides compose most of the immuno-stimulating fraction of the hot water extract, amounting 85-90% of the total weight. The immuno-stimulating fraction also contains 7-8% of proteins and 15.5-68% of uronic acid. The fraction is also rich in calcium ion and magnesium ion in combination with other inorganic compounds including iron, aluminum, manganese, zinc, potassium, phosphorus, sulfur, etc.
Even after treatment with proteinase K, angelan has almost constant immune activity, showing that the immuno-stimulation principle of angelan resides in the polysaccharides alone or in combination with the inorganic compounds.
Quantitative and qualitative analyses for the sugars of angelan can be performed using thin layer chromatography (TLC) and ion exchange HPLC, respectively. The data demonstrate that the polysaccharides of angelan comprises galacturonic acid, galactose and arabinose as major components and mannose, rhamnose and xylose as minor components. That is, the composition of angelan, which consists mainly of galacturonic acid, arabinose and galactose, is typical of pectic polysaccharides which exist in a wide range of plants. Therefore, the major component of the immuno-stimulating principle of angelan is pectic polysaccharides and the immuno-stimulation of angelan is attributed to the pectic polysaccharides combined with the inorganic compounds. The immuno-stimulative polysaccharides isolated from
Angelica gigas
Nakai is 10 kDa or less in molecular weight.
Specific pathogen free mice for immune test were obtained from Korea Research Institute of Bioscience and Biotechnology, Korea. Investigation was done on the influence of angelan on the immune functions of the splenocytes including B cells, T cells and macrophages. Based on the data, the following conclusions were made.
The cellular targets in the angelan-induced immunopotentiation are divided into two types, i.e., direct and indirect targets. The direct exposure of angelan to splenocytes increases the expression of cytokines. The expression of IL-6, which is produced by activated macrophages, is enhanced by angelan. For IFN-&ggr;, which is related to natural killer cells, a significant production increase can be induced by angelan. Also, IL-2 and IL-3, which are secreted from helper T cells, are expressed at an elevated level by angelan. However, the time-dependent analysis showed some differences in the onset time of angelan's action among the four cytokines. IL-6 and IFN-&ggr; respond rapidly to angelan and the increased expression levels are maintained thereafter. In contrast, the production of IL-2 and IL-4 is weakly enhanced at delayed times. IL-2 production gradually increases and IL-4 production is affected for only a few hours after treatment with angelan. Accordingly, this suggests that macrophages and natural killer cells, which produce IL-6 and IFN-&ggr; respectively, are primary cellular targets directly affected by angelan whereas helper T cells are indirectly affected by angelan.
Angelan has an direct influence on B cells. Similar to LPS, which is a B cell mitogen, angelan causes a high level of mitogenic proliferation of B cells. When total splenocyte populations including B cells, T cell and macrophages are treated with angelan, they are proliferated at a significant increased level. In the absence of T cells and macrophages, angelan also augments the proliferation of B cells whereas the B cell-depleted populations are not affected, which su
Ahn Kyungseop
Han Sangbae
Hong Namdoo
Kim Hwanmook
Kim Ikhwan
Buckwalter Brian L.
Korea Institute of Science and Technology
Mathews, Collins Shepherd & McKay, P.A.
Peselev Elli
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