PHARMACEUTICAL COMPOSITIONS HAVING SYNERGISTIC EFFECTS OF

NATURAL EXTRACTS FOR CANCER TREATMENT UNDERGOING CHEMOTHERAPY

Field of the invention

The invention relates to the fields of chemistry and pharmaceutical science with a special relation to pharmaceutical compositions having synergistic effects of natural extracts for cancer treatment undergoing chemotherapy.

Background of the invention

Sesamin is a major lignan as active ingredient of Sesamum indicum seeds. There have been numerous studied of anti-cancer properties of sesamin. The anti-cancer effects of sesamin have been mainly attributed to its anti-proliferation, proapoptotic, anti-inflammatory, anti-metastatic, anti- and pro-angiogenic, and pro-autophagocytic activities. Dietary supplementation of sesamin has been shown to inhibit chemically-induced mammary carcinogenesis in rats (Hirose et al, Anticancer Res. 12 (4)( 1992)). The previous studies show that sesamin inhibits proliferation in human MCF-7 breast cancer cells by inducing cell cycle arrest at G 1 phase (Yokota et al, Cancer Sci. 98(9) (2007)) and Sesamin has also been reported to inhibit matrix metalloproteinase-9 (MMP-9) activity (Lee et al, Inflammation. 34(3) (201 1)). Further, there have been several disclosed in patent prior art relating to sesamin as the component in the pharmaceutical compositions for cancer treatment. Refer to Taiwanese patent with publication number TWI423809B, it disclosed a pharmaceutical composition for treating or ameliorating a disease such as cancers or overactive bladder by inhibiting acetylcholine receptor activity. Said pharmaceutical compositions comprises an effective amount of a sesame extract and a pharmaceutical acceptable carrier. It might further comprise a chemotherapeutic agent. Moreover, there is also Taiwanese patent with publication number TW20061 8805A has been disclosed the use of sesamin as the hepatic cancer cell proliferation inhibitor by activating the cancer inhibition gene P53. These disclosed inventions show that the anti-cancer properties of sesame or sesamin extract has been well known knowledge and has been used in the actual industries.

Hesperidin is a natural bioflavonoid found abundantly in citrus fruits and have been widely used as a nutritional supplement. Furthermore, hesperidin has been also shown to be effective exhibits anti-inflammatory antibacterial and anti-oxidative properties. Jain and Parmar, Inflamm Res. 60(5) (201 1 ). There are further demonstrated to be an effective anti-cancer agent that has been shown to induce apoptosis in human hepatocellular carcinoma HepG2 cell. Banjerdpongchai et al, Tumor Biol. 637( 1 ) (2016) probably through caspase activation. From previous study dietary hesperidin inhibits carcinogenesis in the lung in rat model. Kamaraj et al, Fundam Clin Parmacol. 25( 1 ) (201 1).

In cancer or malignant patients, chemotherapeutic agent can prolong the patient’s life and potentially rid them of the cancer. For example, doxorubicin (DOX) is antibiotic of the anthracycline group are used in oncological diseases including lung cancer, breast cancer, and leucosis, are common and carry a high mortality rate. DOX non-covalently binds to DNA, blocking the synthesis of nucleic acid, but exert toxic effect in normal tissues and cells. Flowever, side effects are unpredictable and cause suffering physically to the patients. (Menna et al, Expert Opin Drug Saf. 1 1 (2012)). The most common side effects of chemotherapeutics agent are illness, easy bruising or breeding, weakened immune system and hair loss. Therefore, there are many studies in relation to using other substances that have been prove as medical safe to combine with chemotherapeutic agent to reduce its treatment dosage, but still efficiently treat the cancer patients. The present disclosed invention is combination of sesamin, hesperidin that bring several advantages for cancer therapy, such as synergistic therapeutic effect for improving therapeutic efficacy, minimized side effects and reduce overcoming drug resistance.

Summary of the invention

Pharmaceutical compositions having synergistic effects of natural extracts for cancer treatment undergoing chemotherapy is disclosed which synergistically to inhibit cancer cells growth. The pharmaceutical composition comprises at least sesamin, flavonoid compound that preferably hesperidin and chemotherapeutic agent. Development status the drug combination has been evaluated in human breast cancer model (in vitro cell culture assays) shown to be much more effective than individual treatments alone in terms of cytotoxicity.

The aim of the present disclosed invention is to increase cancer treatment efficacy, minimize the risk of resistance that finally lower drug concentrations are required to decrease the side effect in normal cells. Brief description of the drawings

Fig. l shown the structure of doxorubicin

Fig.2 shown the dose/response curves for doxorubicin alone (upper), hesperidin alone (middle) and sesamin alone (lower) on the breast cancer cells.

Fig.3 shown the dose/response curve for combination of hesperidin and sesamin on the breast cancer cells.

Fig.4 shown the dose/response curve indicating synergistic of hesperidin, sesamin and the chemotherapeutic agent (doxorubicin) on the breast cancer cells.

Detailed description of the invention

Advantageously, the present invention provides the composition for the synergistic treatment of cancer comprising administering a synergistically, the therapeutically effective amount of sesamin, flavonoid compound and chemotherapeuti c agents. The term“synergistic” means that the effect achieved with the method and composition of this invention is greater than the sum of the effects that result from the method and composition comprising sesamin or flavonoid compound or chemotherapeutic agent separately. Combination therapy using these agents should enhance the response rate of different cancers to these drugs. Further, this synergistic effect should significantly reduce side effects by permitting a lower therapeutic dose to be administered for oncological patients of these kind of cancer that is selected from leukemia, muscle tumor, bone cancer, lymphoma, melanoma, ovarian cancer, skin cancer, testicular cancer, stomach cancer, pancreatic cancer, kidney cancer, Breast cancer, prostate cancer, rectal cancer, cancer of the head and neck, brain cancer, esophageal cancer, bladder cancer, adrenocortical carcinoma, lung cancer, bronchial cancer, endometrial cancer, cervical cancer or liver cancer.

The sesamin can be selected from synthetic sesamin or extracted sesamin from sesame ( Sesamum indicum ) seeds wherein said sesamin is dissolved in organic solvent that can be selected from chloroform, methanol, ethanol, oil or DMSO or combination thereof, most preferably dissolved in DMSO.

The flavonoid compound can be selected from anthocyanins or chalcones or flavonones or flavones or flavonols or isoflavonoids or combination thereof wherein said flavonoid compound is preferably flavonones, and most preferably hesperidin. Said hesperidin can be selected from synthetic hesperidin or extracted hesperidin from citrus fruits. Moreover, said hesperidin is dissolved in the solvent that can be selected from chloroform, methanol, ethanol, oil, water or DMSO or combination thereof, most preferably dissolved in DMSO.

The chemotherapeutic agents is selected from the group consisting of paclitaxel, irinotecan HC1, docitaxel, doxorubicin, Danubicin (daunorubicin), epirubicin, 5-fluorouracil, melphalan, cis- platin, carboplatin, cyclophosphamide (cyclophosphamide), mitomycin, methotrexate, mitoxantrone, vinblastine, vincristine, ifosfamide, ghost Teniposide, etoposide, bleomycin, leucovorin, cytarabine, dactinomycin, a- Interferon alpha, streptozocin, prednisolone or procarbazine HC1. The most preferable of said chemotherapeutic agent according to this invention is doxorubicin that most preferably dissolved in DMSO.

Example

The human breast carcinoma cell line (MDA-MB231) were cultured in media containing 10% FBS. MDA-MB231 cells were plated in 96 well plates at a density of 5x10 ³ cells per well for overnight for testing with therapeutic composition designated herein as flavonoid compound (hesperidin), sesamin and known chemotherapeutic agent (doxorubicin) for 24 h. After 24 h, the incubated in 0.5 mg/ml MTT at 37°C for 4 h. A solubilization solution, 200 mΐ of DMSO (dimethyl sulfoxide), was added to each well and absorption values read at 540 on microplate reader. %Cell viability (ODtest/ODcontrol)xlOO

Doxorubicin is a member of anthracycline antibiotic family of chemotherapeutics. It acts by intercalating DNA so that DNA synthesis cannot occur. The tumor cells cannot reproduce and should become the dead cells. The chemical structure of said doxorubicin is provided in Fig. l .

The MDA-MB23 1 were tested against hesperidin or/and sesamin or/and doxorubicin individually at a various concentration and against the combination therapeutic containing all agents

The results

After 24-hour treatment with hesperidin alone or sesamin alone and doxorubicin alone are shown in Fig. 2. Doxorubicin demonstrated cytotoxicity on MDA-MB231 in a dose-dependent manner. In Fig 2 (Middle), a dose/response curve is shown for hesperidin alone on MDA-MB231 for 24 h using MTT assay. As shown in figure 2 Hesperidin treatment suppression cell growth in MDA- MB231 and the twenty percent inhibitory hesperidin dose was found to be 118.18 mM in 24 h. In Fig 2 (Lower), a standard dose/response curve is shown for sesamin alone on MDA-MB231 for 24 h using MTT assay. As shown in figure 2 sesamin treatment suppression cell growth in MDA-MB231 and the twenty percent inhibitory sesamin dose was found to be 154.12 mM in 24 h. The data of inhibiton concentration of doxorubicin, hesperidin and sesamin are given in tables 1.

Table 1 IC 10, IC20 and IC50 drug value in MDA-MB231 cells at 24 h

The same methods described above were used for this experiment this study was repeated four times once with exposing IC20 sesamin plus the increasing dose of hesperidin, once with IC2G of hesperidin plus the increasing dose of sesamin. The results show that exposing IC20 of hesperidin plus the increasing dose of sesamin a greater activity than exposing IC20 sesamin plus the increasing dose of hesperidin and compound alone in the same dose shown in figure 3. The data of inhibiton concentration of hesperidin combined with sesamin are given in tables 2

Table 2 IC10, IC20 and IC50 value of Hesperidin combined with sesamin in MDA-MB231 cells at 24 h

The same methods described above were used for this experiment this study was repeated four times once with exposing 50 mM hesperidin plus the increasing dose of doxorubicin, once with exposing 50 mM sesamin plus the increasing dose of doxorubicin and once exposing IC 10 of hesperidin and IC10 of sesamin plus the increasing dose of doxorubicin. The results show that exposing IC10 ofhesperidin I C 10 of sesamin plus the increasing dose of doxorubicin a greater activity than compound alone and combined with hesperidin and doxorubicin combined with sesamin and doxorubicin shown in figure 4. The experiment support that the synergy between the effect of hesperidin or sesamin and doxorubicin, inducing cell death in human breast cancer.The data of inhibiton concentration (IC) of Hesperidin or/and sesamin combined with drug (doxorubicin) in hesperidin and sesamin are given in tables 3.

Table 3 IC 10, IC20 and IC50 value of Hesperidin or/and sesamin combined with the drug (doxorubicin) in MDA-MB231 cells at 24 h

Although, this invention has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the present invention extends beyond the specifically disclosed embodiments to other alternative embodiments and/or uses of the invention and obvious modifications and equivalents thereof. In addition, while several variations of the invention have been shown and described in detail, other modifications, which are within the scope of this invention, will be readily apparent to those of skill in the art based upon this disclosure. It is also contemplated that various combinations or sub-combinations of the specific features and aspects of the embodiments may be made and still fall within the scope of the invention. It should be understood that various features and aspects of the disclosed embodiments can be combined with, or substituted for, one another in order to form varying modes of the disclosed invention. Therefore, it is intended that the scope of the present invention herein disclosed should not be limited by the particular disclosed embodiments described above, but should be determined only be a fair reading of the claims that follow.