WO2017086892A1

WO2017086892A1 - An extract for cancer treatment

Info

Publication number: WO2017086892A1
Application number: PCT/TR2016/050251
Authority: WO
Inventors: Halis SÜLEYMAN; Bahadir SÜLEYMAN; Durdu ALTUNER; Emine AKYÜZ TURUMTAY; Nihal CETIN; Saliha EKSI; Renad MAMMADOV
Original assignee: Süleyman Halis; Süleyman Bahadir; Altuner Durdu; Akyüz Turumtay Emine; Cetin Nihal; Eksi Saliha; Mammadov Renad
Priority date: 2015-11-16
Filing date: 2016-08-01
Publication date: 2017-05-26
Also published as: TR201514423A3

Abstract

The invention relates to medicaments used in the treatment of cancerous diseases in the health sector. The invention particularly relates to the use of the extract obtained from Usnea Longissima in the treatment of cancer diseases.

Description

DESCRIPTION

An Extract for Cancer Treatment

The Related Art

The invention relates to medicaments used in the treatment of cancer diseases in the health sector. The invention particularly relates to the use of the extract obtained from Usnea Longissima in the treatment of cancer diseases.

The Prior Art

Cancer is a disease developed by uncontrolled proliferation of normal cells and spread to the surrounding tissues via clonal metastasis and spread to the whole body via lymphs. Frequently seen cancer disease is one of the most common reasons for death. Cancer is still one of the most significant common health problems.

For reducing mortality and increasing survival, radioteraphy, chemotherapy, and surgical treatment are used in cancer treatment, mostly in combination with each other. Nowadays, medicaments used in cancer chemotherapy cannot treat cancer completely and the number of cancer-related deaths increase day by day. The medicaments used in cancer treatment have severe and lethal adverse effects. This restricts the success in cancer treatment. Although various studies are made about antineoplastic medicaments, desired level of success in cancer treatment has not been reached yet.

Antineoplastic medicaments can destroy pathologically proliferating cancer cells in the body as well as normal cells that proliferate rapidly. Therefore, most cancer medicaments have adverse effects on tissues such as bone marrow, blood cells, and other rapidly proliferating cells.

The improvements made about the subject in the related technical field are given below.

The patent No. EP1339420B1 relates to "A process of preparing aqueous plant extracts and the extracts obtained with this process." In the relevant invention, a method used for preparing aqueous extracts of vegetables, especially plants, and compositions obtained with this method and used in the field of medicine, especially in the treatment of immunosuppressant diseases such as cancer, tuberculosis, flu, common cold, and AIDS, or viral diseases such as hepatitis.

The patent No. CN101596227 (A) relates to "A herbal medicine for lung cancer treatment." Said herbal medicine consists of 19-21 % of elaeagnus glabra, 29-31 % of gnaphalium affine, 19-21 % usnea longissima, 19-21 % of cynoglossum, and 9-1 1 % of alangium root mixture.

As a result, the above said drawbacks and the inadequacy of the prior art solutions about the subject have necessitated an improvement in the related technical field.

Brief Description of the Invention

The invention relates to an extract for cancer treatment, which meets the above said requirements, eliminates all of the drawbacks, and brings some additional advantages.

The primary purpose of the invention is to provide a medicament with no lethal adverse effect for use in the treatment of cancer.

The purpose of the invention is to provide treatment of cancer diseases with the extract obtained from Usnea longissima.

A purpose of the invention is to develop a medicament for use in the treatment of cancer, which is easy to produce and administer, and which does not have any adverse effect.

Another purpose of the invention is to develop a cancer treatment medicament obtained from Usnea longissima by extraction method via a solvent substance (ethyl acetate, ethanol, and methanol etc. alcohols, water etc.).

Another purpose of the invention is to develop an extract for use in the treatment of lung cancer, obtained with extraction method from Usnea longissima via ethyl acetate solvent. Another purpose of the invention is to provide an ethyl acetate extract having anticancer activity, obtained with extraction method from Usnea longissima.

Another purpose of the invention is to obtain Usnea longissima ethyl acetate extract, which is not found in mixture form and can be used alone in cancer treatment.

In order to achieve the above said purposes, the invention consists of Usnea longissima extract enabling treatment of cancer diseases. In order to achieve the purposes of the invention, said extract is an extract obtained from Usnea longissima via extraction method in the presence of a solvent.

In order to achieve the purposes of the invention, said solvent includes alcohol, ethyl acetate, water, or other solvents.

In order to achieve the purposes of the invention, said extract, is an ethyl acetate extract obtained from Usnea longissima via liquid-liquid extraction method in the presence of ethyl acetate. In order to achieve the above said purposes, the invention is the use of Usnea longissima extract in cancer treatment.

The structural and characteristic features of the invention and all of its advantages shall be understood better with the figures and the detailed description given below in reference to the figures, and therefore, the assessment should be made by taking into account the said figures and detailed explanations.

Detailed Description of the Invention

In this detailed description, the preferred embodiments of an extract for cancer treatment according to the invention is only disclosed for better understanding of the subject without forming any limiting effect.

The invention relates to an extract prepared for use in the treatment of cancer diseases. Said extract is obtained via extraction from Usnea longissima. The ethyl acetate extract obtained from Usnea longissima via extraction (ULE) shows anticancer activity.

Usnea longissima showing anticancer activity is a lichen species. Lichens are not organisms on their own. They are symbiotic living beings formed of fungi (ascomycetes, bassidomycetes) and photosynthetic algae. Algae and fungi acting as a single individual within the structure of lichen can produce substances in this symbiotic life, which they cannot produce individually. These secondary metabolites produced by lichens are pulvic, prolichesterinic, physodic, lobaric, fumarprotosetrarik, and usnic acids; depsides; and depsidones etc. substances.

ULE has shown in vivo anticancer activity in the lung cancer tissue formed subdermally in Nude mice via A549 cell line (basal alveolar epithelial lung cancer cell). In the assay, the effect of the extract according to the invention on the cancer tissue formed by subcutaneous injection of lung cancer cell line (A549) in nude mice. In nude mice, after the tumour mass formed by subcutaneous injection of A549 cells reaches a certain size, 50 mg/kg per day dose of ULE is orally administered to a group for 30 days for treatment purpose. While the tumour mass increased from average of 69.3±14.3 mm³ to 5228±1320 mm³ in the non-treated group, on the other hand, in the treated group, the mass is reduced from 74.7±28.6 mm³ to 53.4±13.1 mm³.

The preparation method of the Usnea longissima extract according to the invention is given below. Usnea longissima extract is obtained by extraction method. In order to ensure extraction, 20 ml of methanol is added to 2 g of Usnea longissima sample and kept under reflux in a heater with magnetic stirrer at 50 for 2 hours. It is filtered through blue filter paper and the remaining part is washed twice with 5 ml of methanol. Afterwards methanol was evaporated and 20 ml boiling water (at 100 ^QC) is added. After cooling, 10 ml of n-hexane is added in order to prevent possible contamination in colon and the formed phase is taken. After this operation, liquid-liquid extraction is performed by adding 5 ml of each of diethyl ether, ethyl acetate, and n-butanole twice, respectively, on the aqueous phase. The solvents of the 10 ml samples taken into other balloons are evaporated and placed in dessicator. The resultant extracts are dissolved in 3 ml of methanol and diluted in appropriate rates with water. They are passed through 0.2 micron HPLC filters and their preparative HPLC analysis are made.

The extract obtained from Usnea longissima by ethyl acetate extraction method is in dry granulated powder form and it is used by reconstituting with distilled water before administration. The extract according to the invention show anticancer activity on lung cancer cells (A549) in cell culture assays.

Evaluation of the anticancer effect of Usnea longissima ethyl acetate extract via A549 cell implantation test in nude mice:

Preparation of the cells:

In this study, the lung cancer cell line A549 to be used for nude mice are started to be produced in vitro via passaging to T75 flasks in 1 x1 0³/ml concentration in an incubator at 37 ^<C supported by RPMI, 1 0% Fetal bovine serum (FBS), 1 00 μg/m\ streptomycin -1 00 U/ml penicilin, and 5% CO2. The cultures are aspirated moderately everyday and fed by new growth medium. When the cell density in the flasks reach 70-80%, the cells are trypsinized with 0.25% trypsin-EDTA and transferred into 50 ml falcon tubes containing cold medium. Afterwards, the cells precipitated at 600 rpm are washed 2 x times with colder RPMI, stained in 1 :1 ration with trypan blue, and counted on an inverted microscobe (Olympus inverted microscobe CK X 41 , 1 0 X).

Application of A549 lung cancer cell line to nude mice:

A549 cancer cells are placed in serum physiological suspension in the amount of 1 x1 0⁶ cells per 0.5 ml. Afterwards 6-7 weeks old 44 Nude mice are injected subcutaneously from the right flank with a suspension containing 0.5 ml A549. Tumour growth was assessed in mice by controlling three times a week. When the mean volume of the tumour became 72.03 mm³, among the tumour-growing Nude mice, the ones having similar tumour sizes are selected and separated into three assay groups as EG-50, CIG-2, and CG. The tumour volume is adjusted to be close to each other between the groups.

Administration of Usnea longissima ethyl acetate extract on Nude mice: 50 mg/kg of ULE is administered once a day orally via catheter to EG-50 (n-7) mouse group for 30 days. In the group CIG-2 (n-7), 2 mg/kg of cisplatin was intraperitoneally injected once in every 3 days (total of 10 doses). Distilled water was given as a solvent, orally via a catheter in the same volume (1 ml) to the CG (n-8) mouse group for 30 days. At the end of this period, the tumour volumes of all mouse groups were measured and the mice were sacrificed by decapitation after the operations are complete. Afterwards, the results from the EG-50 mouse group administered with ULE are compared with the results obtained from the control group (CG) having distilled water administered as solvent and the group CIG-2 having cisplatin administered . Tumour sizes were measured by means of a caliper and thus anticancer activity is evaluated.

ULE can be administered orally to rats and mice once a day in 50-100 mg/kg dose. It is reconstituted with distilled water prior to administration. It is recommended to be used for at least one month.

ULE according to the invention is obtained by preferably using ethyl acetate via extraction method. However, it is also possible to use Usnea longissima extracts obtained by dissolving ethanol, methanol etc. various alcohols or other solvents like water for the same purpose.

The extract of Usnea longissima obtained by dissolving with any solvent acts by reducing the number of A549 lung cancer cells in the cell culture and reducing the lung tumour mass formed in nude mice. It causes this effect by suppressing angiogenesis. The above-mentioned study shows the anticancer activity of Usnea longissima. Said anticancer activity can be obtained from the extracts of Usnea longissima formed using all solvents. The important point here is obtaining the extract from Usnea longissima. The extract is obtained by separating the substances found within Usnea longissima with the help of a solvent via extraction method.

Claims

1. The use of Usnea longissima extract in cancer treatment.

2. The use according to Claim 1 , characterized in that; said extract is an extract obtained from Usnea longissima via extraction method in the presence of a solvent.

3. The use according to Claim 1 , characterized in that; said extract, is an ethyl acetate extract obtained from Usnea longissima via liquid-liquid extraction method in the presence of ethyl acetate.