WO2007091744A1

WO2007091744A1 - Pharmaceutical composition containing an extract of ulmus davidiana planch for the prevention and treatment of glioma

Info

Publication number: WO2007091744A1
Application number: PCT/KR2006/000465
Authority: WO
Inventors: Cheorl-Ho Kim; In Seon Lee; Dong-Soo Kim
Original assignee: Industry-Academic Cooperation Foundation, Keimyung University
Priority date: 2006-02-09
Filing date: 2006-02-09
Publication date: 2007-08-16

Abstract

The present invention relates to an extract of Ulmus davidiana Planch, which shows a therapeutic activity for glioma and a pharmaceutical composition for the treatment of glioma comprising the extract.

Description

PHARMACEUTICAL COMPOSITION CONTAINING AN

EXTRACT OF ULMUS DAVIDIANA PLANCH FOR THE

PREVENTION AND TREATMENT OF GLIOMA

Technical Field

[1] The present invention relates to a pharmaceutical composition containing an extract of Ulmus davidiana Planch for the prevention and treatment of glioma.

[2]

Background Art

[3] Gliomas are a class of tumor that develops from glial (neuroepithelial or support) cells. Astrocytes, ependymal, and oligodendroglial cells are all examples of glial cells that compose the supportive tissue of the brain. Gliomas comprise nearly one-half of primary brain tumors and one-fifth of all primary spinal cord tumors.

[4] Malignant gliomas are one of the most devastating tumors that can affect any given individual. Nevertheless, this past decade, major advances in the fields of molecular biology and cellular biology, as well as genomics, have begun to improve our current understanding of malignant gliomas. Grade IV gliomas, often referred to as Glioblastoma Multiforme or "GBM", possess multiple genetic and chromosomal abnormalities which cause these tumors to grow rapidly.

[5] The presenting symptoms of a glioma depend on the location of the tumor within the brain and its rate of growth. Common symptoms include: headaches, seizures, difficulty speaking, weakness/paralysis in one part of the body or face, difficulty with vision, impairment of sensation, impairment of balance, nausea/vomiting, behavioral changes, and impairment of memory or thinking. The clinical course of an untreated malignant glioma is characterized by relentless invasive growth and, even with treatment, near universal recurrence.

[6] Traditional treatment options for malignant gliomas include: surgery, radiation therapy, and chemotherapy. Open surgery, through a window cut into the skull(craniotomy), is the primary form of treatment for malignant gliomas. The goal of surgery is to remove as much of the visible tumor as possible without damaging normal neurological functions. Radiation therapy and chemotherapy are widely used as adjuvant treatments following surgery. Both therapies have a growth-suppressant effect on the tumor and represent side effects.

[7] Apoptosis characterized morphologically by chromatin condensation and cell shrinkage, can be induced by the alternation of various signaling pathways and is also an essential and complex regulated process for balancing cell number in animal de- velopment and adult homeostasis. However, in most cell lines, an amplification of the death signal via the mitochondrial pathway is required for apoptosis. Mitochondria play a pivotal role in apoptosis of mammalian cells by releasing apoptogenic proteins into the cytoplasm. One apoptogenic molecule, cytochrome c, binds to and induces oligomerization of Apaf-1, a cytoplasmic protein in the presence of dATP. When Apaf-1 oligomerizes, pro-caspase-9, one of death-driving proteolytic enzymes known as caspases, is recruited and autoactivated. Activated caspase-9 cleaves and activates effector caspases such as caspase-3, which then cleave a variety of cellular proteins and cause cell death. Mitochondrial membrane permeability during apoptosis is regulated directly by the Bcl-2 family of proteins. Members of the Bcl-2 family constitute a group of proteins that play important roles in apoptosis regulation. So far, over two dozen homologues of Bcl-2 have been identified. These proteins are classified into two subfamilies having opposing roles in apoptosis regulation. Members from one subfamily such as Bcl-2, BcI-XL, and Bcl-w promote cell survival while members from the other subfamily such as B ax, Bid, and Bak promote cell death. Among the various Bcl-2 homologues identified to date, Bcl-2, BcI-XL and Bax represent the most well characterized members. The major function of Bcl-2 family members is to control directly mitochondrial membrane permeability and thereby regulate release of apoptogenic factors from the intermembrane space into the cytoplasm. The major molecular players of the apoptotic process are largely the same in glioma cells, non-transformed glial and non-glial cells.

[8] Traditional oriental medicinal herbs are widely known to be effective in the treatment of many diseases without making serious side effects. However, The cytotoxic activity of the crude extract or the isolated compounds of traditional oriental medicinal herbs effective for glioma have not yet been demonstrated.

[9]

Disclosure of Invention Technical Problem

[10] Accordingly, it is an object of the present invention to provide an extract of Ulmus davidiana Planch, which has an activity of anti-glioma.

[11] It is another object of the present invention to provide a process for preparing above extract to treat glioma.

[12] It is another object of the present invention to provide a pharmaceutical composition comprising the above extract and a pharmaceutically acceptable carrier for the prevention and treatment of glioma without making side effects.

[13] It is another object of the present invention to provide a use of above extract for the preparation of pharmaceutical composition to treat glioma. [14] It is another object of the present invention to provide a health care food comprising above extract and sitologically acceptably additive to prevent and treat glioma.

[15]

Technical Solution

[16] In accordance with on aspect of the present invention, there are provided an extract of Ulmus davidiana Planch, which shows an effective therapeutic activity against glioma, and a pharmaceutical composition for the treatment of glioma which comprises the extract and a pharmaceutically acceptable carrier.

[17] In accordance with the present invention, it has been found that an extract of Ulmus davidiana Planch induce mitochondria-mediated apoptosis in U87 glioblastoma cells and the ability to activate DNA fragmentation and cytochrome c release.

Advantageous Effects

[18] Safty and efficacy of the extract of Ulmus davidiana Planch are well established through a long history of human use.

[19] Accordingly, inventive extract of Ulmus davidiana Planch and inventive pharmaceutical composition containing the extract represent high level of cytotoxic effect and have no side effect like radiotheraphy and usual chemotherapy.

[20]

Brief Description of the Drawings

[21] The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the accompanying drawings.

[22] Fig. 1 depicts a flow chart which shows fractionation of Ulmus. davidiana Planch.

[23] Fig. 2 shows the effects of three fractions of Ulmus davidiana Planch on cell proliferation of U87 glioblastoma. A; The cytotoxic effects of the methanol fraction(obtained from last step) at various concentrations were determined using XTT assay kit. B; Cell viability in the control(l) and presence of the methanol fraction(2) or chloroform fraction(3), and hexane fraction(4) are measured as described in the Material and Methods section. Data levels are mean values and SEM obtained from three replicates.

[24] Fig. 3 shows the effect of methanol fraction on cultured U87 glioblastoma cells.

Cells were treated methanol fraction of Ulmus davidiana Planch at 0(A), l(B), 5(C), and 20mg/ml(D) for 24h.

[25] Fig. 4 shows the effects of methanol fraction of Ulmus davidiana Planch on the

DNA fragmentation and cytochrome c release from mitochondria of U87 glioblastoma cells. To evaluate effects of methanol fraction of Ulmus davidiana Planch on the DNA fragmentation(A) and cytochrome c release(B), cells were treated without(l) or with(2) lOmg/ml methanol fraction of Ulmus davidiana Planch for 24h. Its effects on the release of cytochrome c were analyzed by immunoblotting. As a control for protein amount, GAPDH was measured.

[26] Fig. 5 shows effects of Bcl-2 family proteins expression in mitochondrial fraction of

U87 glioblastoma cells by methanol fraction of Ulmus davidiana Planch. Bcl-2 family protein, Bcl-2 and Bax was examined by detection of protein levels in mitochondria fractions of U87 glioblastoma cells treated with 10mg/ml methanol fraction of Ulmus davidiana Planch for 0-60min and detected by western blot.

[27]

Best Mode for Carrying Out the Invention

[28] Preparation of the methanol extract of Ulmus davidiana Planch

[29] The dried stem barks of UD were collected in Kyungju city, the Republic of Korea, and the sample are kept in the herbarium of the College of Oriental Medicine, Dongguk University. The plant samples were pulverized with a grinder and extracted 3 times with Il of methanol, respectively, at 70⁰C for 5h. The extracts were filtered through a 10mm cartridge paper and lyophilized. The methanol extract(lθg) was suspended in methanol(500ml) and extracted by 500ml of chloroform. The chloroform fraction was removed and the remained methanol fraction(upper phase) was re- extracted by 500ml of hexane. The final remaining methanol fraction was concentrated under reduced pressure using a rotary evaporator and then freeze-dried. For the bioassay test, samples were dissolved in methanol in lmg/ml and further diluted in DMSO, medium or buffer solution.

[30] The yield of methanol extract at first step was 3.05%(10g; liophilized weight).

Then, the methanol extract was re-extracted using Chloroform(CHCl ) to yield 2.98g(liophilized weight) of extract. At last, it was extracted using hexane to yield 1.67g(liophilized weight).

[31]

Mode for the Invention

[32] Hereinafter, the present invention is further described in detail with referencing the examples provided below. However, those examples are only provided for illustrative purposes, by no means limiting the scope of the present invention.

[33]

[34] Example 1: Preparation of the water extract of Ulmus davidiana Planch

[35] The dried stem barks of UD were collected in Kyungju city, the Republic of Korea, and the sample are kept in the herbarium of the College of Oriental Medicine, Dongguk University. The plant samples were pulverized with a grinder and extracted 3 times with 1.5/ of distilled water, respectively, for 5h under reflux. The resulting extract was filtered, concentrated under reduced pressure and lyophilized to yield 11.2g(3.4%).

[36]

[37] Experimental Example 1: Cell culture, treatment, and morphological assessment

[38] U87 glioblastoma cell line was obtained from the American Type Culture

Collection( ATCC). These cell lines were grown in Dulbecco modified Eagle medium(DMEM) containing IOOU of penicillin per ml and 10% fetal bovine serum(FBS) at 37°C in 5% CO -air. To treat with methanol fraction of UD extract, cells were seeded at 2X10 /100-mm culture dish in DMEM containing FBS and grown to sub-confluence. Cells were rinsed with phosphate -buffered saline (PBS) and then incubated in serum-free medium for Ih. The methanol fraction of extract was added in various doses(0, 1, 5, 20mg/ml) to triplicates and cells were then incubated. After 24h, cells were harvested and analyze. To determined morphological changes, cells were photographed at x 100 using bright-field optics after 24h of incubation with methanol fraction. Many morphological changes were induced in methanol fractions of 5mg/ml and 20mg/ml(Fig. 3).

[39]

[40] Experimental Example 2: XTT proliferation assay

[41] The effect of the methanol extracts from the stem bark of UD on proliferation of

U87 glioblastoma cell was investigated using a commercially available proliferation kit(XTT II, Boehringer Mannheim, Mannheim, Germany). Briefly, the cells were plated in 96-well culture plates at a density of 10,000 cells per well in DMEM culture medium and allowed to attach for 2h. The methanol fraction of extract was added to various final concentrations(and control: 0mg/ml) in triplicates. And, each 10mg/ml of the methanol fraction, chloroform fraction and hexane fraction of extract was added in triplicates. After 24h of culture, 50ml of XTT reaction solution (sodium 3 - [l-(phenyl-aminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzenesulfonic acid hydrate and N-methyl dibenzopyrazine methyl sulfate; mixed in proportion 50:1) was added to the wells. The optical density was read at 490 nm wavelength in an ELISA plate reader after 4h incubation of the plates with XTT in an incubator (37°C and 5% CO -air). All determinations were confirmed using replication in at least three identical experiments.

[42] The results were represented in Fig. 2. Methanol fraction has high level of cytotoxicity as compared with Chloroform fraction and hexane fraction(Fig. 2B). And, methanol fraction was represented cytotoxic effect in concentration-dependent manner(Fig. 2A).

[43] [44] Experimental Example 3: DNA fragmentation

[45] After treated with methanol fraction of UD extract for 24h, U87 glioblastoma cells were harvested by centrifugation at 2000 g for 5min and washed one time with ice cold PBS. After this step, all operations were carried out on ice. Cell pellets were gently re- suspended in 500ml lysis buffer[50mM Tris-HCl(pH 8.0), 2OmM EDTA (pH 8.0), 1% NP-40, and 20ml of 10% SDS] and incubated at 65°C for 30min. After that, 100ml of 8M potassium acetate was added to the suspended mixtures and incubated on ice bath for Ih. The lysates were centrifuged at 10,000 g for lOmin and the supernatants were transfered to a new eppendorf tube. Finally, DNA was extracted with phenol/ chloroform/isoamyl alcohol(25:24:l (v/v/v)) and precipitated using ethanol. To perform the DNA fragmentation assay, sample was dissolved in 20ml TE buffer(10mM Tris-HCl and ImM EDTA, pH 8.0) and incubated at 37°C for 30min with ImI of RNase A(10mg/ml) and ImI of proteinase K(100mg/ml). Electrophoresis was carried out on 1.5% agarose gel. The gel was stained with 5mg/ml ethidium bromide and photographed under UV conditions.

[46] DNA fragments of 200-1400bp were detected in MeOH fraction treatment group(Fig. 4A).

[47]

[48] Experimental Example 4: Antibodies and immunoblot analysis

[49] The anti-cytochrome c(Pharmingen, San Diego, CA), anti-Bcl-2 (Pharmingen, San

Diego, CA) and anti-GAPDH(Santa Cruz, CA) antibodies are mouse monoclonal antisera. The anti-Bax(Δ21) (Santa Cruz, CA) antibody is rabbit polyclonal antisera.

[50] To evaluate effects of methanol fraction of Ulmus davidiana Planch on the cytochrome c release, cells were treated without or with 10mg/ml methanol fraction of Ulmus davidiana Planch for 24h. Its effects on the release of cytochrome c were analyzed by immunoblotting. As a control for protein amount, GAPDH was measured.

[51] Level of cytochrome c secreted from mitochondria was increased by treating with methanol fraction of of Ulmus davidiana Planch(Fig. 4B).

[52] U87 glioblastoma cells by methanol fraction of Ulmus davidiana Planch. Bcl-2 family protein, Bcl-2 and Bax was examined by detection of protein levels in mitochondria fractions of U87 glioblastoma cells treated with 10mg/ml methanol fraction of Ulmus davidiana Planch for 0-60min and detected by western blot.

[53] The level of Bcl-2 protein was decreased in 60min after treatment of methanol fraction of Ulmus davidiana Planch. And, the level of Bax protein was increased in 30min after treatment of methanol fraction of Ulmus davidiana Planch(Fig. 5).

[54] Western blotting analysis was performed by the method described previously [Kim

KS, Lee SD, Kim KH, KiI SY, Chung KH, Kim CH 2005. Suppressive effects of a water extract of Ulmus davidiana Planch (Ulmaceae) on collagen-induced arthritis in mice. Journal of Ethnopharmacology. 97, 65-71]. Briefly, cell lysates were prepared, electrotransferred, and immunoblotted with antibodies. Bands were visualized by ECL and exposure to X-ray film.

[55]

[56] Densitometric and statistical analysis

[57] The intensity of the bands obtained from western blotting studies was estimated with Gel-Print System (Core Bio Corp., Seoul, KOREA). The values are expressed as means+SE.

[58]

Industrial Applicability

[59] As mentioned above, since inventive extract of Ulmus davidiana Planch showed cytotoxic activity to a glioblastoma cell, and various activity to induce morphological changes in a glioblastoma cell, to activate DNA fragmentation and cytochrome c release, to decrease Bcl-2, an anti-apoptotic protein, and to increase Bax, pro-apoptotic protein, it can be used as pharmaceutical composition to prevent or treat glioma.

[60] While the invention has been described with respect to the above specific embodiments, it should be recognized that various modifications and changes may be made to the invention by those skilled in the art which also fall within the scope of the invention as defined by the appended claims.

[61]

Claims

[1] a pharmaceutical composition containing an extract of Ulmus davidiana Planch for the prevention and the treatment of glioma. [2] The composition of claim 1, wherein said extract is prepared by methanol extraction. [3] The composition of claim 1, wherein said extract is prepared by methanol extraction in first step, and MeOH: Chloroform extraction in second step, and

MeOH:hexane extraction in third step. [4] Use of the extract of Ulmus davidiana Planch for preparation of pharmaceutical composition to treat glioma. [5] A healthcare food comprising extract of Ulmus davidiana Planch, and a sito- logically acceptable additive, for the prevention and the treatment of glioma.