WO2004035073A1 - Composition for treating myeloid leukemia and increasing immunity comprising salicornia herbacea extracts - Google Patents

Composition for treating myeloid leukemia and increasing immunity comprising salicornia herbacea extracts Download PDF

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Publication number
WO2004035073A1
WO2004035073A1 PCT/KR2002/001933 KR0201933W WO2004035073A1 WO 2004035073 A1 WO2004035073 A1 WO 2004035073A1 KR 0201933 W KR0201933 W KR 0201933W WO 2004035073 A1 WO2004035073 A1 WO 2004035073A1
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Prior art keywords
composition
salicornia herbacea
juice
extracts
salicornia
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PCT/KR2002/001933
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French (fr)
Inventor
Chong-Kil Lee
Sun-A Im
Goo-Whan Kim
Kyungjae Kim
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Good Bio Ltd
Chong-Kil Lee
Sun-A Im
Goo-Whan Kim
Kim Jae-Heung
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Application filed by Good Bio Ltd, Chong-Kil Lee, Sun-A Im, Goo-Whan Kim, Kim Jae-Heung filed Critical Good Bio Ltd
Priority to PCT/KR2002/001933 priority Critical patent/WO2004035073A1/en
Priority to JP2004545003A priority patent/JP4327727B2/en
Priority to AU2002349558A priority patent/AU2002349558A1/en
Publication of WO2004035073A1 publication Critical patent/WO2004035073A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/21—Amaranthaceae (Amaranth family), e.g. pigweed, rockwort or globe amaranth
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants

Definitions

  • composition for treating myeloid leukemia and increasing immunity comprising Salicornia herbacea extracts
  • the present invention relates to a composition for treating myeloid leukemia or increasing immunity comprising Salicornia herbacea extracts.
  • Salicornia herbacea L. is a halophyte, which grows naturally in stock on foreshore of the west coast of Korea. Salicornia herbacea has an appearance of fossil plants and grows well in barren environment under which ordinary plants are unable to survive. As Salicornia herbacea grows in foreshore including large amounts of salt and various minerals, it is salty. Salt contained in Salicornia herbacea is necessary for metabolism to keep men's vitality. In addition, according to the research report by National Ocean and Fisheries Institute, Salicornia herbacea contains various kinds of useful organic substances, minerals and inorganic substance such as iron, calcium, magnesium and phosphorus.
  • Salicornia herbacea is very effective for intestinal cleaning via removal of fecal impaction and relieves edema due to inflammation and arthritis.
  • Salicornia herbacea exhibits superior effect against lumbago, diabetes mellitus, thyroiditis and bronchitis.
  • Salicornia herbacea contains various kinds of substances with noticeable efficacy. When exhausted with sweat, one can feel refreshment by chewing Salicornia herbacea, and Salicornia herbacea has been used as an edible plant for a long time.
  • Myeloid leukemia shows the highest incidence among leukemia and anticancer chemotherapy or radiotherapy have been used for the treatment since surgical operation could not be applied in leukemia.
  • a chemotherapeutic primarily has toxicity to bone marrow stem cells, and radiotherapy as well exhibits toxicity to bone marrow stem cells.
  • stem cell differentiates to macrophage in the order of monoblast, promonocyte, monocyte and macrophage.
  • stem cells transform into cancer cells in any one differentiation stage to macrophage.
  • Macrophage is a cell in which differentiation has been completed (end stage cell) and could not proliferate any more. Therefore, it can be expected that a drug differentiating a myeloid cancer cell to macrophage would be very useful as a novel drug for the treatment of leukemia, but such drug has not yet been developed for clinical use.
  • increasing immunity is no less important than any other remedy such as chemotherapy or radiotherapy.
  • increasing immunity is meant to improve our own defense capacity, it can be developed as remedy without intrinsic adverse effect such as toxicity to bone marrow stem cells, and further the facts that majority of cancer patients exhibit reduced immunity and the decline of immunity is inversely proportional to the cancer incidence reveal that immunopotentiation is essential for the prevention and treatment of cancer.
  • Macrophages are found in all living tissues and not only function as a scavenger of microorganisms and dead cells via strong phagocytosis but also are essentially involved in induction of specific immunity by acting as an antigen presenting cells.
  • the importance of macrophage in the induction of immune reaction is proved by the fact that majority of immunopotentiating agents developed so far exhibit immunity-increasing activity by primarily activating the function of macrophage.
  • a drug effective for activating the function of macrophage and for inducing differentiation of leukemia cell would be very useful as remedy without adverse effect such as toxicity to bone marrow stem cells, and for this reason, development of such drug has been urgent task in the art.
  • the inventors of the present invention have continued studies on the efficacy of Salicornia herbacea extracts and have found that
  • Salicornia herbacea extracts exhibit differentiation-inducing effect on a myeloid leukemia cell and macrophage -activating effect, and based on this, have completed the present invention.
  • the object of the present invention lies in providing a composition for treating myeloid leukemia, which exhibits superior therapeutic effect without adverse effect such as toxicity to bone marrow stem cells.
  • the present invention has another object providing an immunity-increasing composition with superior effect on activating the function of macrophage.
  • the present invention relates to a composition for treating myeloid leukemia or increasing immunity comprising Salicornia herbacea extracts.
  • the composition of the present invention as a leukemia-treating composition, can be administered alone or in combination with other therapeutics for leukemia.
  • composition of the present invention as an immunity-increasing composition, can be used for the treatment and prevention of various diseases by administering it alone or in combination with other drugs.
  • Salicornia herbacea extracts exhibit effects of inducing cell differentiation of myeloid leukemia, which has the highest incidence among leukemia, and of activating the function of macrophage. Macrophages are distributed in all living tissues, are important as scavengers of microorganisms and dead cells via strong phagocytosis, and further essentially involved in induction of specific immunity by acting as antigen presenting cells.
  • Salicornia herbacea extracts are extracted in a juice form and in a high molecular glycoprotein form.
  • the extracts in the juice form is obtained from the process comprising the following steps: i) Salicornia herbacea is washed with drinking water 2 to 3 times and passed through a crusher, ii) shattered Salicornia herbacea and its liquid from the crusher is wrapped with a hemp cloth and heated in an autoclave under no pressure for 1 to 2 hrs and boiled for 20 to 60 min at 80 to 110°C under 1 to 2 atmospheres, iii) boiled Salicornia herbacea and its juice are squeezed with a press and the juice only is packed using an automatic roll packaging machine, iv) the packed juice is subjected to sterilization with 100°C water for 5 to 30 min.
  • the roll packaging machine that can minimize the amount of foam occurring at the time of filling the juice into a pack. The less the foam, the lower amount of air to be included in the pack, and in the above case, prolonged storage without deterioration can be achieved.
  • the crusher used in extraction process has blades, which are made of titanium alloy and designed to cross-rotate, can shatter effectively tough and rough Salicornia herbacea harvested in a period of from the end of July to the beginning of September, and is made of material which is completely harmless to men even under long-lasting exposure to high concentration of salt.
  • the Salicornia herbacea extracts in the high molecular glycoprotein form are obtained from the process comprising the following steps: i) Salicornia herbacea is dried under hot air to obtain dried powder, ii) distilled water in an amount of 5 to 10 times to the weight of dried powder is added and heated at 90 to 100°C for 3 hrs, filtered to separate the water layer, and subjected to vacuum concentration at 35 to 45°C, iii) ethanol (1 to 5 folds) is added to the concentrate and left at a low temperature for 10 to 14 hrs to obtain a high molecular precipitate, and iv) the precipitate is recovered from centrifugation and subjected to lyophilization.
  • the immunity-increasing substance obtained as described above has a molecular weight of more than 30,000 dalton, and has galactose as a main monosaccharide and alanine as a main amino acid.
  • composition of the present invention as a leukemia-treating composition, can be administered alone or in combination with other leukemia therapeutics such as Imatinib or Tretinoin, and combination therapy can be more effective.
  • composition of the present invention activates the function of macrophage, and thus it can be used in treating and preventing various diseases by being administered alone or in combination with other drugs. For instance, when administered together with interferon- ⁇ , synergistic immunity- increasing effect is exhibited.
  • composition of the present invention for treating leukemia or increasing immunity can be administered 1 to 4 times a day depending on the age, sex and body weight of patients, and based on the forms of extracts, a unit dose can be preferably 10 to 200 ml for the juice form and 0.05 to 4.0 g for the high molecular glycoprotein form.
  • the composition of the present invention for treating leukemia or increasing immunity can comprise Salicornia herbacea extracts only, or can be formulated into varied preparations by using pharmaceutically acceptable carriers, excipients and/or additives. That is, the leukemia- treating and immunity-increasing composition of the present invention can comprise excipients such as lactose and starch, lubricants such as magnesium stearate, emulsifiers, suspending agents, tonicity-adjusting agents, stabilizers and buffers, and optionally can comprise sweeteners, intestinal absorption stimulating agents and/or flavors.
  • the composition according to the present invention can be formulated into oral or parenteral preparations.
  • the composition in the present invention can be formulated into oral preparations such as tablets, capsules, liquids and syrups, or parenteral preparation such as injections.
  • the composition of the present invention for treating leukemia or increasing immunity can be prepared in a drink form.
  • an amount equal to the unit dose of the above-mentioned juice or glycoprotein can be preferably diluted with distilled water to 10 to 80% by volume.
  • a mixture of liquid fructose and citric acid can be used.
  • sucrose or glucose instead of liquid fructose, and acetic acid or malic acid, instead of citric acid, can be used alone or in a mixture thereof.
  • the drink type composition of the present invention can be used for preventing and treating leukemia, or for increasing immunity.
  • composition in the present invention can be used for various kinds of foodstuffs in addition to drinks.
  • foodstuffs to which Salicornia herbacea extracts can be added tea and health food etc. can be enumerated.
  • Slicornia herbacea is safe to body since it has long been used as an edible plant, and thus there is no worry about negative effect such as cytotoxicity.
  • Fig. 1 represents morphological change after induction of differentiation of myeloid cancer cell by Salicornia herbacea juice (before (A) and after (B) treatment with Salicornia herbacea juice).
  • Fig. 2 shows morphological change after induction of differentiation of myeloid cancer cell by high molecular glycoprotein separated from Salicornia herbacea (before (A) and after (B) treatment with the high molecular glycoprotein).
  • Fig. 3 represents proliferation- inhibiting effect of Salicornia herbacea juice via induction of differentiation of myeloid cancer cell.
  • Fig. 4 illustrates proliferation-inhibition effect of high molecular glycoprotein separated from Salicornia herbacea via induction of differentiation of myeloid cancer cell.
  • Fig. 5 shows phagocytosis-increasing effect of Salicornia herbacea juice (before (A) and after (B) treatment with Salicornia herbacea juice).
  • Fig. 6 represents phagocytosis-increasing effect of high molecular glycoprotein separated from Salicornia herbacea (before (A) and after (B) treatment with the high molecular glycoprotein).
  • Fig. 7 shows stimulatory effect of Salicornia herbacea juice on the production of IL-l ⁇ .
  • Fig. 8 represents stimulatory effect on the production of IL-l ⁇ by high molecular glycoprotein separated from Salicornia herbacea.
  • Fig. 9 illustrates stimulatory effect of Salicornia herbacea juice on the production of TNF- ⁇ .
  • Fig. 10 represents stimulatory effect on the production of TNF- ⁇ by high molecular glycoprotein separated from Salicornia herbacea.
  • Fig. 11 shows stimulatory effect of Salicornia herbacea juice on the production of NO.
  • Fig. 12 represents stimulatory effect on the production of NO by high molecular glycoprotein separated from Salicornia herbacea.
  • Fig. 13 represents increase in NO production upon combined administration of high molecular glycoprotein separated from Salicornia herbacea and interferon- ⁇ .
  • Example 1 Preparation of Salicornia herbacea juice Salicornia herbacea was washed with drinking water 2 to 3 times, and shattered to pieces using a crusher. Salicornia herbacea liquid and pieces thus obtained was put into a hemp cloth bag and heated in autoclave for 1 hr under no pressure. The mixture was subjected to additional heating at 110°C for 25 min under 1,519.88 hPa (1.5 atm). The product thus obtained was pressed to obtain juice, and the juice was packed using an automatic packaging machine. The packed juice was sterilized with 100°C water for 10 min.
  • Salicornia herbacea was hot air-dried to obtain dried powder, and distilled water in an amount of 10 times to the weight of dried powder was added, heated at 100° C for 3 hrs and filtered to separate the water layer.
  • the water layer was subjected to vacuum concentration at 45°C, 1 to 2 folds ethanol was added and then left at low temperature for 12 hrs to obtain a precipitate.
  • the precipitate was recovered from centrifugation and subjected to lyophilization.
  • Salicornia herbacea extracts from Example 2 250.0 mg
  • Salicornia herbacea extracts from Example 2 were screened, mixed with lactose, starch and pregelatinated corn starch, and an adequate amount of purified water was added and granulated. The granules were dried, mixed with magnesium stearate and compressed to prepare tablets.
  • Salicornia herbacea extracts from Example 2 250.0 mg
  • Salicornia herbacea extracts from Example 2 were screened, mixed with starch and magnesium stearate, and filled in gelatin capsules to prepare capsules.
  • Salicornia herbacea extracts from Example 2 100.0 mg
  • Salicornia herbacea extracts from Example 2 was dissolved in an adequate amount of diluted sodium hydroxide to adjust pH value to 7.5 to 8.5, sodium chloride for injection BP was added to control the volume, and thoroughly mixed.
  • the solution was filled in a 2 ml-transparent- glass ample, and the ample was sealed by melting glass and sterilized in an autoclave to prepare an injection.
  • Salicornia herbacea juice 10 ml was diluted with distilled water to 80 ml, and liquid fructose (7 to 10% by weight) and citric acid (0.1 to 0.2% by weight) were added to prepare 100 ml of mixed drinks.
  • High molecular glycoprotein 0.1 g was diluted with distilled water to 80 ml, and liquid fructose (7 to 10% by weight) and citric acid (0.1 to 0.2% by weight) were added to prepare 100 ml of mixed drinks.
  • Test sample was dissolved in water, filtered with a micropartition system (MPS-1) to estimate the molecular weight of active substance. As a result, it could be confirmed that the active substance has the molecular weight larger than 30,000 daltons.
  • MPS-1 micropartition system
  • test sample was treated with 6N HC1 at 100°C for 4 hrs to hydrolyze amino sugars, and further subjected to treatment with 2M trifluoroacetic acid at 100°C for 4 hrs to hydrolyze neutral sugars.
  • Analysis condition Bio-LC DX-300 (Dionex, USA), Carbopac PAl column (4.5 x 50 mm) and PED2 detector with integrated amperometry. Results are summarized in Table 1.
  • composition of amino acids included in the test sample was analyzed according to PICO-tag method via hydrolysis and PITC labeling.
  • HPLC analysis condition Waters 996 photodiode array detector (PDA, 254 nm), Waters Pico-tag column (3.9 x 300 mm, 4 ⁇ m). Results are summarized in Table 2.
  • Myeloid cancer cells once differentiated to macrophage do not further proliferation.
  • the cells were added to a 96-well microtiter plate at a concentration of 2 x 10 4 cells/well concentration, and the extracts from Example 1 was diluted to 1, 1/3, 1/9, 1/27, 1/81, 1/243 and 1/729 folds and added in an amount of 0.05% by volume based on culture solution, and the extracts from Example 2 was added at concentrations of 200, 70, 20, 7, 2.5, 0.8 and 0.3 ⁇ g/ml and incubated for 2 days.
  • [3H]-thymidine was added to cell culture solution, further incubated for 6 hrs, and the cells were harvested, and the amount of [3H] -thymidine incorporated into the cells was measured with a microbetacounter.
  • juice or high molecular glycoprotein of Salicornia herbacea showed concentration- dependent proliferation-inhibiting effect against cancer cells.
  • juice or high molecular glycoprotein of Salicornia herbacea inhibits proliferation of myeloid cancer cells, Raw 264.7 by inducing their differentiation.
  • Myeloid cancer cells Raw 264.7, was added to 2-well chamber slide at a concentration of 1 x 10 5 cells/ml, the extracts from Examples 1 and 2 were added to culture solution in an amount of 0.05% by volume based on culture solution and 10 ⁇ g/ml, respectively, and the cells was allowed to differentiate into macrophages by incubating for 48 hrs.
  • Fig. 5 before (A) and after (B) treatment with Salicornia herbacea juice
  • Fig. 6 before (A) and after (B) treatment with high molecular glycoprotein
  • juice or high molecular glycoprotein (10 ⁇ g/ml) of Salicornia herbacea remarkably enhanced phagocytosis to sheep erythrocytes. Therefore, it is expected that juice or high molecular glycoprotein of Salicornia herbacea of the present invention will exhibit anti-cancer effect by stimulating induction of immune response via significantly increasing phagocytosis.
  • a test sample was added to myeloid cancer cells, Raw 264.7, and incubated for 48 hrs to induce macrophage differentiation. That is, cells were added to a 24-well plate at a concentration of 5 x 10 5 cells/well, and the extract from Example 1 was diluted to 1, 1/3, 1/9, 1/27 and 1/81 folds, added in an amount of 0.05% by volume based on culture solution, and the extract from Example 2 was added at concentrations of 100, 33, 11, 3.7 and 1.2 ⁇ g/ml, respectively. After 48 hrs of incubation, culture supernatant was harvested. The content of IL-l ⁇ contained in the culture supernatant was determined with ELISA kit (R&D company). As a result, as can be confirmed from Figs.
  • juice or high molecular glycoprotein of Salicornia herbacea exhibited concentration-dependent stimulation effect on the production of IL-l ⁇ . Accordingly, it is expected that juice or the high molecular glycoprotein of Salicornia herbacea will exhibit anti-cancer effect by allowing macrophage to induce the production of IL-l ⁇ .
  • mice were added to myeloid cancer cells, Raw 264.7, and subjected to 48 hrs of incubation, leading to differentiation to macrophage. That is, cells were added to a 24-well plate at a concentration of 5 x 10 5 cells/well, the extract from Example 1 was diluted to 1, 1/3, 1/9, 1/27, 1/81 and 1/243 folds, respectively, added to the wells in an amount of 0.05% by volume based on culture solution, and the extract from Extract 2 was added at concentrations of 100, 33, 11, 3.7, 1.2 and 0.4 ⁇ g/ml, respectively. After 48 hrs of incubation, culture supernatant was harvested. The content of TNF- ⁇ contained in the culture supernatant was determined with ELISA kit from Pharmingen.
  • a test sample was added to myeloid cancer cells, Raw 264.7, and incubated for 48 hrs to induce macrophages differentiation. That is, cells were added to a 24-well plate at a concentration of 5 x 105 cells/well, the extract from Example 1 was diluted to 1, 1/3, 1/9, 1/27, 1/81, 1/243 and 1/729 folds, respectively, added to the wells in an amount of 0.05% by volume based on culture solution, and the extract from Example 2 was added at concentrations of 100, 33, 11, 3.7, 1.2, 0.4 and 0.14 ⁇ g/ml, respectively. After 48 hrs of incubation, culture supernatant was harvested.
  • juice or high molecular glycoprotein of Salicornia herbacea exhibited concentration-dependent stimulation of the production of NO. Therefore, it is expected that juice or high molecular glycoprotein of Salicornia herbacea of the present invention will exhibit anti-cancer effect by allowing macrophage to induce the production of NO.
  • Myeloid cancer cells Raw 264.7, were placed on a 24-well culture plate at a concentration of 5 x 10 5 cells/well, and the extract from Example 2 was added to the wells at concentrations of 100, 33, 11, 3.7, 1.2 and 0.4 ⁇ g/ml, respectively, and as another set, after the addition of the extract from Example 2 at the same concentrations described above, 10 units/ml of interferon- ⁇ was added, respectively. After 48 hrs of incubation, culture supernatant was harvested.
  • Salicormia herbacea extracts exhibit differentiation-inducing effect for myeloid cancer cells and macrophage-activating effect. Therefore, the present invention can provide a composition for treating myeloid leukemia, which has superior therapeutic effect without adverse effects such as toxicity to bone marrow stem cells and a composition for increasing immunity that can be used for prevention and treatment of various diseases.

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Abstract

The present invention relates to a composition for treating myeloid leukemia or increasing immunity comprising Salicornia herbacea extracts. The present invention can provide a composition for treating myeloid leukemia that exhibits superior therapeutic effect without adverse effects such as toxicity to bone marrow stem cells and a composition for increasing immunity that can be used for prevention and treatment of various diseases.

Description

Composition for treating myeloid leukemia and increasing immunity comprising Salicornia herbacea extracts
Technical Field The present invention relates to a composition for treating myeloid leukemia or increasing immunity comprising Salicornia herbacea extracts.
Background Art Salicornia herbacea L. is a halophyte, which grows naturally in stock on foreshore of the west coast of Korea. Salicornia herbacea has an appearance of fossil plants and grows well in barren environment under which ordinary plants are unable to survive. As Salicornia herbacea grows in foreshore including large amounts of salt and various minerals, it is salty. Salt contained in Salicornia herbacea is necessary for metabolism to keep men's vitality. In addition, according to the research report by National Ocean and Fisheries Institute, Salicornia herbacea contains various kinds of useful organic substances, minerals and inorganic substance such as iron, calcium, magnesium and phosphorus. Salicornia herbacea is very effective for intestinal cleaning via removal of fecal impaction and relieves edema due to inflammation and arthritis. Salicornia herbacea exhibits superior effect against lumbago, diabetes mellitus, thyroiditis and bronchitis. Salicornia herbacea contains various kinds of substances with noticeable efficacy. When exhausted with sweat, one can feel refreshment by chewing Salicornia herbacea, and Salicornia herbacea has been used as an edible plant for a long time.
Myeloid leukemia shows the highest incidence among leukemia and anticancer chemotherapy or radiotherapy have been used for the treatment since surgical operation could not be applied in leukemia. However, a chemotherapeutic primarily has toxicity to bone marrow stem cells, and radiotherapy as well exhibits toxicity to bone marrow stem cells. It is known that stem cell differentiates to macrophage in the order of monoblast, promonocyte, monocyte and macrophage. In case of myeloid leukemia, stem cells transform into cancer cells in any one differentiation stage to macrophage. Macrophage is a cell in which differentiation has been completed (end stage cell) and could not proliferate any more. Therefore, it can be expected that a drug differentiating a myeloid cancer cell to macrophage would be very useful as a novel drug for the treatment of leukemia, but such drug has not yet been developed for clinical use.
In cancer treatment, increasing immunity is no less important than any other remedy such as chemotherapy or radiotherapy. As increasing immunity is meant to improve our own defense capacity, it can be developed as remedy without intrinsic adverse effect such as toxicity to bone marrow stem cells, and further the facts that majority of cancer patients exhibit reduced immunity and the decline of immunity is inversely proportional to the cancer incidence reveal that immunopotentiation is essential for the prevention and treatment of cancer.
Macrophages are found in all living tissues and not only function as a scavenger of microorganisms and dead cells via strong phagocytosis but also are essentially involved in induction of specific immunity by acting as an antigen presenting cells. The importance of macrophage in the induction of immune reaction is proved by the fact that majority of immunopotentiating agents developed so far exhibit immunity-increasing activity by primarily activating the function of macrophage.
Therefore, in treating leukemia, a drug effective for activating the function of macrophage and for inducing differentiation of leukemia cell would be very useful as remedy without adverse effect such as toxicity to bone marrow stem cells, and for this reason, development of such drug has been urgent task in the art.
The inventors of the present invention have continued studies on the efficacy of Salicornia herbacea extracts and have found that
Salicornia herbacea extracts exhibit differentiation-inducing effect on a myeloid leukemia cell and macrophage -activating effect, and based on this, have completed the present invention.
Therefore, the object of the present invention lies in providing a composition for treating myeloid leukemia, which exhibits superior therapeutic effect without adverse effect such as toxicity to bone marrow stem cells. In addition, the present invention has another object providing an immunity-increasing composition with superior effect on activating the function of macrophage.
Disclosure of the Invention
The present invention relates to a composition for treating myeloid leukemia or increasing immunity comprising Salicornia herbacea extracts. The composition of the present invention, as a leukemia-treating composition, can be administered alone or in combination with other therapeutics for leukemia.
Additionally, the composition of the present invention, as an immunity-increasing composition, can be used for the treatment and prevention of various diseases by administering it alone or in combination with other drugs.
Salicornia herbacea extracts exhibit effects of inducing cell differentiation of myeloid leukemia, which has the highest incidence among leukemia, and of activating the function of macrophage. Macrophages are distributed in all living tissues, are important as scavengers of microorganisms and dead cells via strong phagocytosis, and further essentially involved in induction of specific immunity by acting as antigen presenting cells.
In the present invention, Salicornia herbacea extracts are extracted in a juice form and in a high molecular glycoprotein form.
First, the extracts in the juice form is obtained from the process comprising the following steps: i) Salicornia herbacea is washed with drinking water 2 to 3 times and passed through a crusher, ii) shattered Salicornia herbacea and its liquid from the crusher is wrapped with a hemp cloth and heated in an autoclave under no pressure for 1 to 2 hrs and boiled for 20 to 60 min at 80 to 110°C under 1 to 2 atmospheres, iii) boiled Salicornia herbacea and its juice are squeezed with a press and the juice only is packed using an automatic roll packaging machine, iv) the packed juice is subjected to sterilization with 100°C water for 5 to 30 min.
In case of packaging the juice, as substantial amount of foam occurs during packaging, it is preferable to use the roll packaging machine that can minimize the amount of foam occurring at the time of filling the juice into a pack. The less the foam, the lower amount of air to be included in the pack, and in the above case, prolonged storage without deterioration can be achieved.
The crusher used in extraction process has blades, which are made of titanium alloy and designed to cross-rotate, can shatter effectively tough and rough Salicornia herbacea harvested in a period of from the end of July to the beginning of September, and is made of material which is completely harmless to men even under long-lasting exposure to high concentration of salt.
The Salicornia herbacea extracts in the high molecular glycoprotein form are obtained from the process comprising the following steps: i) Salicornia herbacea is dried under hot air to obtain dried powder, ii) distilled water in an amount of 5 to 10 times to the weight of dried powder is added and heated at 90 to 100°C for 3 hrs, filtered to separate the water layer, and subjected to vacuum concentration at 35 to 45°C, iii) ethanol (1 to 5 folds) is added to the concentrate and left at a low temperature for 10 to 14 hrs to obtain a high molecular precipitate, and iv) the precipitate is recovered from centrifugation and subjected to lyophilization. The immunity-increasing substance obtained as described above has a molecular weight of more than 30,000 dalton, and has galactose as a main monosaccharide and alanine as a main amino acid.
The composition of the present invention, as a leukemia-treating composition, can be administered alone or in combination with other leukemia therapeutics such as Imatinib or Tretinoin, and combination therapy can be more effective.
The composition of the present invention, as an immunity- increasing composition, activates the function of macrophage, and thus it can be used in treating and preventing various diseases by being administered alone or in combination with other drugs. For instance, when administered together with interferon-γ, synergistic immunity- increasing effect is exhibited.
The composition of the present invention for treating leukemia or increasing immunity can be administered 1 to 4 times a day depending on the age, sex and body weight of patients, and based on the forms of extracts, a unit dose can be preferably 10 to 200 ml for the juice form and 0.05 to 4.0 g for the high molecular glycoprotein form.
The composition of the present invention for treating leukemia or increasing immunity can comprise Salicornia herbacea extracts only, or can be formulated into varied preparations by using pharmaceutically acceptable carriers, excipients and/or additives. That is, the leukemia- treating and immunity-increasing composition of the present invention can comprise excipients such as lactose and starch, lubricants such as magnesium stearate, emulsifiers, suspending agents, tonicity-adjusting agents, stabilizers and buffers, and optionally can comprise sweeteners, intestinal absorption stimulating agents and/or flavors. In addition, the composition according to the present invention can be formulated into oral or parenteral preparations. For example, the composition in the present invention can be formulated into oral preparations such as tablets, capsules, liquids and syrups, or parenteral preparation such as injections.
The composition of the present invention for treating leukemia or increasing immunity can be prepared in a drink form. For the drink type, an amount equal to the unit dose of the above-mentioned juice or glycoprotein can be preferably diluted with distilled water to 10 to 80% by volume. As additives, a mixture of liquid fructose and citric acid can be used. Further, sucrose or glucose, instead of liquid fructose, and acetic acid or malic acid, instead of citric acid, can be used alone or in a mixture thereof. The drink type composition of the present invention can be used for preventing and treating leukemia, or for increasing immunity.
The composition in the present invention can be used for various kinds of foodstuffs in addition to drinks. As foodstuffs to which Salicornia herbacea extracts can be added, tea and health food etc. can be enumerated.
Slicornia herbacea is safe to body since it has long been used as an edible plant, and thus there is no worry about negative effect such as cytotoxicity.
Brief Explanation of Drawings
Fig. 1 represents morphological change after induction of differentiation of myeloid cancer cell by Salicornia herbacea juice (before (A) and after (B) treatment with Salicornia herbacea juice).
Fig. 2 shows morphological change after induction of differentiation of myeloid cancer cell by high molecular glycoprotein separated from Salicornia herbacea (before (A) and after (B) treatment with the high molecular glycoprotein). Fig. 3 represents proliferation- inhibiting effect of Salicornia herbacea juice via induction of differentiation of myeloid cancer cell.
Fig. 4 illustrates proliferation-inhibition effect of high molecular glycoprotein separated from Salicornia herbacea via induction of differentiation of myeloid cancer cell. Fig. 5 shows phagocytosis-increasing effect of Salicornia herbacea juice (before (A) and after (B) treatment with Salicornia herbacea juice).
Fig. 6 represents phagocytosis-increasing effect of high molecular glycoprotein separated from Salicornia herbacea (before (A) and after (B) treatment with the high molecular glycoprotein).
Fig. 7 shows stimulatory effect of Salicornia herbacea juice on the production of IL-lβ.
Fig. 8 represents stimulatory effect on the production of IL-lβ by high molecular glycoprotein separated from Salicornia herbacea.
Fig. 9 illustrates stimulatory effect of Salicornia herbacea juice on the production of TNF-α.
Fig. 10 represents stimulatory effect on the production of TNF-α by high molecular glycoprotein separated from Salicornia herbacea. Fig. 11 shows stimulatory effect of Salicornia herbacea juice on the production of NO.
Fig. 12 represents stimulatory effect on the production of NO by high molecular glycoprotein separated from Salicornia herbacea.
Fig. 13 represents increase in NO production upon combined administration of high molecular glycoprotein separated from Salicornia herbacea and interferon- γ.
Best mode for carrying out the invention
The present invention will be described in more detail by the following Examples. The following Examples illustrate the present invention, yet do not intend to limit the scope of the present invention.
Example 1: Preparation of Salicornia herbacea juice Salicornia herbacea was washed with drinking water 2 to 3 times, and shattered to pieces using a crusher. Salicornia herbacea liquid and pieces thus obtained was put into a hemp cloth bag and heated in autoclave for 1 hr under no pressure. The mixture was subjected to additional heating at 110°C for 25 min under 1,519.88 hPa (1.5 atm). The product thus obtained was pressed to obtain juice, and the juice was packed using an automatic packaging machine. The packed juice was sterilized with 100°C water for 10 min.
Example 2'- Separation of high molecular glycoprotein from Salicornia herbacea
Salicornia herbacea was hot air-dried to obtain dried powder, and distilled water in an amount of 10 times to the weight of dried powder was added, heated at 100° C for 3 hrs and filtered to separate the water layer. The water layer was subjected to vacuum concentration at 45°C, 1 to 2 folds ethanol was added and then left at low temperature for 12 hrs to obtain a precipitate. The precipitate was recovered from centrifugation and subjected to lyophilization. Preparation Example 1 : Tablets
Salicornia herbacea extracts from Example 2 250.0 mg
Lactose BP 250.0 mg Starch BP 30.0 mg
Pregelatinated corn starch BP 15.0 mg
Magnesium stearate 1.0 mg
Salicornia herbacea extracts from Example 2 were screened, mixed with lactose, starch and pregelatinated corn starch, and an adequate amount of purified water was added and granulated. The granules were dried, mixed with magnesium stearate and compressed to prepare tablets.
Preparation Example 2-' Capsules
Salicornia herbacea extracts from Example 2 250.0 mg
Starch 1500 100.0 mg
Magnesium stearate BP 1.0 mg
Salicornia herbacea extracts from Example 2 were screened, mixed with starch and magnesium stearate, and filled in gelatin capsules to prepare capsules. Preparation Example 3: Syrup
Salicornia herbacea extracts from Example 2 100.0 g
Sucrose 637.5 g Sodium carboxymethylcellulose 2.0 g
Methylparaben 0.28 g
Propylparaben 0.12 g
Ethanol 20 ml
Sodium carboxymethylcellulose solution (sodium carboxymethylcellulose was dissolved in 400 ml of purified water) was added to sucrose solution (sucrose was dissolved in 500 ml of purified water) and mixed. Methylparaben and propylparaben were added and dissolved to the mixture, ethanol was added, and purified water was added to 1000 ml. The screened Salicornia herbacea extracts from Example 2 were suspended with the solution to prepare syrup.
Preparation Example 4'- Injections
Salicornia herbacea extracts from Example 2 100.0 mg
Diluted sodium hydroxide pH 7.5 to 8.5
Sodium chloride for injection BP max. 2 ml
Salicornia herbacea extracts from Example 2 was dissolved in an adequate amount of diluted sodium hydroxide to adjust pH value to 7.5 to 8.5, sodium chloride for injection BP was added to control the volume, and thoroughly mixed. The solution was filled in a 2 ml-transparent- glass ample, and the ample was sealed by melting glass and sterilized in an autoclave to prepare an injection.
Preparation Example 5: Drinks
Salicornia herbacea juice 10 ml was diluted with distilled water to 80 ml, and liquid fructose (7 to 10% by weight) and citric acid (0.1 to 0.2% by weight) were added to prepare 100 ml of mixed drinks.
Preparation Example 6- Drinks
High molecular glycoprotein 0.1 g was diluted with distilled water to 80 ml, and liquid fructose (7 to 10% by weight) and citric acid (0.1 to 0.2% by weight) were added to prepare 100 ml of mixed drinks.
Experimental Example 1 : Physico-chemical characteristics of high molecular glycoprotein extracted from Salicornia herbacea Physico-chemical characteristics of high molecular glycoprotein extracted in Example 2 were determined.
(1) Estimation of the molecular weight
Test sample was dissolved in water, filtered with a micropartition system (MPS-1) to estimate the molecular weight of active substance. As a result, it could be confirmed that the active substance has the molecular weight larger than 30,000 daltons.
(2) Composition of sugar
As pretreatment for determining composition of monosaccharides contained in a test sample, the test sample was treated with 6N HC1 at 100°C for 4 hrs to hydrolyze amino sugars, and further subjected to treatment with 2M trifluoroacetic acid at 100°C for 4 hrs to hydrolyze neutral sugars. Analysis condition: Bio-LC DX-300 (Dionex, USA), Carbopac PAl column (4.5 x 50 mm) and PED2 detector with integrated amperometry. Results are summarized in Table 1.
Table 1. Composition of monosaccharides contained in the test sample
Figure imgf000016_0001
(3) Composition of amino acids
Composition of amino acids included in the test sample was analyzed according to PICO-tag method via hydrolysis and PITC labeling. HPLC analysis condition: Waters 996 photodiode array detector (PDA, 254 nm), Waters Pico-tag column (3.9 x 300 mm, 4 μm). Results are summarized in Table 2.
Table 2. Composition of free amino acids contained in the test sample
Figure imgf000017_0001
Figure imgf000018_0001
Experimental Example 2: Differentiation-inducing effect on myeloid cancer cells
Differentiation-inducing activity of the extracts was tested on myeloid cancer cells, Raw 264.7. The cells were put into 24-well culture dish to 5 x 105 cells/well concentration, and the extracts from Examples 1 and 2 were added to 0.05% by volume based on culture solution, 10 μg/ml, respectively. After 48 hrs of incubation, microscopic observation was carried out. As a result, as can be seen from Fig. 1 (before (A) and after (B) treatment with Salicornia herbacea juice) and Fig. 2 (before (A) and after (B) treatment with high molecular glycoprotein), it was confirmed that juice or high molecular glycoprotein of Salicornia herbacea (10 μg/ml) induces differentiation of myeloid cancer cells, resulting in the change of their shape to be similar to fibroblast, the enhancement of adhesiveness to plastic surface, and the formation of many vacuoles.
Myeloid cancer cells once differentiated to macrophage do not further proliferation.
Experimental Example 3: Proliferation-inhibiting effect on myeloid cancer cells
Differentiation-inducing activity on myeloid cancer cells, Raw 264.7, of the extracts according to the present invention was observed.
The cells were added to a 96-well microtiter plate at a concentration of 2 x 104 cells/well concentration, and the extracts from Example 1 was diluted to 1, 1/3, 1/9, 1/27, 1/81, 1/243 and 1/729 folds and added in an amount of 0.05% by volume based on culture solution, and the extracts from Example 2 was added at concentrations of 200, 70, 20, 7, 2.5, 0.8 and 0.3 μg/ml and incubated for 2 days. To determine proliferation degree of the cells, [3H]-thymidine was added to cell culture solution, further incubated for 6 hrs, and the cells were harvested, and the amount of [3H] -thymidine incorporated into the cells was measured with a microbetacounter. As a result, as can be seen from Figs. 3 and 4, juice or high molecular glycoprotein of Salicornia herbacea showed concentration- dependent proliferation-inhibiting effect against cancer cells.
Therefore, it was confirmed that juice or high molecular glycoprotein of Salicornia herbacea inhibits proliferation of myeloid cancer cells, Raw 264.7 by inducing their differentiation.
Experimental Example 4: Phagocytosis-stimulating effect
Myeloid cancer cells, Raw 264.7, was added to 2-well chamber slide at a concentration of 1 x 105 cells/ml, the extracts from Examples 1 and 2 were added to culture solution in an amount of 0.05% by volume based on culture solution and 10 μg/ml, respectively, and the cells was allowed to differentiate into macrophages by incubating for 48 hrs. 50 μl of sensitized sheep erythrocytes [antigen against sheep erythrocytes was diluted (a ratio of 1:256), added, reacted at 37°C for 30 min, washed twice with phosphate buffered saline (PBS), and suspended in phosphate buffered saline (PBS) to 1%] were added and incubated at 37°C for 1 hr. Uningested sheep erythrocytes were removed with ACK lysis buffer and phagocytosed sheep erythrocytes were stained with Wright and Giemsa solution and subjected to microscopic observation.
As a result, Fig. 5 (before (A) and after (B) treatment with Salicornia herbacea juice) and Fig. 6 (before (A) and after (B) treatment with high molecular glycoprotein) demonstrated that juice or high molecular glycoprotein (10 μg/ml) of Salicornia herbacea remarkably enhanced phagocytosis to sheep erythrocytes. Therefore, it is expected that juice or high molecular glycoprotein of Salicornia herbacea of the present invention will exhibit anti-cancer effect by stimulating induction of immune response via significantly increasing phagocytosis.
Experimental Example 5: Stimulation effect on the production of IL-lβ
A test sample was added to myeloid cancer cells, Raw 264.7, and incubated for 48 hrs to induce macrophage differentiation. That is, cells were added to a 24-well plate at a concentration of 5 x 105 cells/well, and the extract from Example 1 was diluted to 1, 1/3, 1/9, 1/27 and 1/81 folds, added in an amount of 0.05% by volume based on culture solution, and the extract from Example 2 was added at concentrations of 100, 33, 11, 3.7 and 1.2 μg/ml, respectively. After 48 hrs of incubation, culture supernatant was harvested. The content of IL-lβ contained in the culture supernatant was determined with ELISA kit (R&D company). As a result, as can be confirmed from Figs. 7 and 8, juice or high molecular glycoprotein of Salicornia herbacea exhibited concentration- dependent stimulation effect on the production of IL-lβ. Accordingly, it is expected that juice or the high molecular glycoprotein of Salicornia herbacea will exhibit anti-cancer effect by allowing macrophage to induce the production of IL-lβ.
Experimental Example 6: Stimulation effect on the production of TNF-α A test sample was added to myeloid cancer cells, Raw 264.7, and subjected to 48 hrs of incubation, leading to differentiation to macrophage. That is, cells were added to a 24-well plate at a concentration of 5 x 105 cells/well, the extract from Example 1 was diluted to 1, 1/3, 1/9, 1/27, 1/81 and 1/243 folds, respectively, added to the wells in an amount of 0.05% by volume based on culture solution, and the extract from Extract 2 was added at concentrations of 100, 33, 11, 3.7, 1.2 and 0.4 μg/ml, respectively. After 48 hrs of incubation, culture supernatant was harvested. The content of TNF-α contained in the culture supernatant was determined with ELISA kit from Pharmingen.
As a result, as can be seen from Figs. 9 and 10, juice or high molecular glycoprotein of Salicornia herbacea exhibited concentration- dependent stimulation effect on the production of TNF-α. Accordingly, it is expected that juice or high molecular glycoprotein of Salicornia herbacea will exhibit anti-cancer effect by allowing macrophage to induce the production of TNF-α. Experimental Example !'■ Stimulation effect on the production of NO
A test sample was added to myeloid cancer cells, Raw 264.7, and incubated for 48 hrs to induce macrophages differentiation. That is, cells were added to a 24-well plate at a concentration of 5 x 105 cells/well, the extract from Example 1 was diluted to 1, 1/3, 1/9, 1/27, 1/81, 1/243 and 1/729 folds, respectively, added to the wells in an amount of 0.05% by volume based on culture solution, and the extract from Example 2 was added at concentrations of 100, 33, 11, 3.7, 1.2, 0.4 and 0.14 μg/ml, respectively. After 48 hrs of incubation, culture supernatant was harvested. To determine the content of NO contained in the culture supernatant, 50 μl of cell culture supernatant and an equal amount of Griess reagent (0.5% sulfonylamide, 0.05% A -(l-naphthyl)- ethylenediamine dihydrochloride in 2.5% H3P04) were mixed on a 96- well microtiter plate, reacted at room temperature for 5 min, and subjected to detection at 550 nm by ELISA reader.
As a result, as can be seen from Figs. 11 and 12, juice or high molecular glycoprotein of Salicornia herbacea exhibited concentration- dependent stimulation of the production of NO. Therefore, it is expected that juice or high molecular glycoprotein of Salicornia herbacea of the present invention will exhibit anti-cancer effect by allowing macrophage to induce the production of NO.
Experimental Example 8: Effect of the extracts of the present invention upon combined administration with interferon-γ
Myeloid cancer cells, Raw 264.7, were placed on a 24-well culture plate at a concentration of 5 x 105 cells/well, and the extract from Example 2 was added to the wells at concentrations of 100, 33, 11, 3.7, 1.2 and 0.4 μg/ml, respectively, and as another set, after the addition of the extract from Example 2 at the same concentrations described above, 10 units/ml of interferon-γ was added, respectively. After 48 hrs of incubation, culture supernatant was harvested. To determine the content of NO contained in the culture supernatant, 50 μl of cell culture supernatant and an equal amount of Griess reagent (0.5% sulfonylamide, 0.05% ΛMl-naphthyl)-ethylenediamine dihydrochloride in 2.5% H3P04) were mixed on a 96-well microtiter plate, reacted at room temperature for 5 min, and subjected to detection at 550 nm by ELISA reader.
As a result, as can be seen in Fig. 13, high molecular glycoprotein separated from Salicornia herbacea stimulated the production of NO, when administered in combination with interferon-γ. The NO production-enhancing effect of the extract of the present invention upon combined administration with interferon-γ was more obvious when the concentration of high molecular glycoprotein separated from Salicornia herbacea was very low.
Industrial applicability
As can be confirmed from the above-mentioned Experimental Examples, Salicormia herbacea extracts exhibit differentiation-inducing effect for myeloid cancer cells and macrophage-activating effect. Therefore, the present invention can provide a composition for treating myeloid leukemia, which has superior therapeutic effect without adverse effects such as toxicity to bone marrow stem cells and a composition for increasing immunity that can be used for prevention and treatment of various diseases.

Claims

1. A composition for treating myeloid leukemia or increasing immunity comprising Salicornia herbacea extracts.
2. The composition in Claim 1, characterized in that said Salicornia herbacea extracts is obtained from the process comprising the following steps: i) Salicornia herbacea is washed with drinking water 2 to 3 times and passed through a crusher, ii) shattered Salicornia herbacea and its liquid from the crusher is wrapped with a hemp cloth and heated in an autoclave under no pressure for 1 to 2 hrs and boiled for 20 to 60 min at 80 to 110°C under 1 to 2 atmospheres, iii) boiled Salicornia herbacea and its juice are squeezed with a press and the juice only is packed using an automatic roll packaging machine, and iv) the packed juice is subjected to sterilization with 100°C water for 5 to 30 min.
3. The composition in Claim 1, characterized in that said Salicornia herbacea extracts has high molecular glycoprotein separated from the process comprising the following steps: i) Salicornia herbacea is hot air-dried to obtain dried powder, ii) distilled water in an amount of 5 to 10 times to the weight of dried powder is added and heated at 90 to 100°C for 3 hrs, filtered to separate the water layer, and subjected to vacuum concentration at 35 to 45°C, iii) 1 to 5 folds ethanol is added and left at low temperatures for 10 to 14 hrs to obtain a high molecular precipitate, and iv) the precipitate is recovered from centrifugation and subjected to lyophilization.
4. The composition in any one of Claims 1 to 3, characterized in that the composition is formulated in a drink form.
5. A method for treating myeloid leukemia comprising administering a therapeutically effective amount of the composition in Claim 1.
6. A method for increasing immunity comprising administering a therapeutically effective amount of the composition in Claim 1.
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