GB2544360A - Use of mogrosides or the pharmaceutically acceptable salts thereof in the manufacture of a tumor radiation sensitizing agent - Google Patents

Abstract

Use of mogrosides or pharmaceutically acceptable salts thereof in the manufacture of a tumor radiation sensitizing agent. Preferably the mogrosides have the following general structure: wherein, R and R1 are glucose residues. More preferably, the mogroside is mogroside II, mogroside III, mogroside IV, mogroside V or mogroside VI. The radiation sensitizing agent may comprise the mogroside and an acceptable carrier, wherein the acceptable carrier is a compatible solid, liquid filler or gel substance. The tumor sensitizing agent may up-regulate the expression of anti-cancer P53 gene and/or down-regulate the expression of Bcl-2 protein. Preferably the tumor is selected from liver, lung or cervical cancer. The tumour radiation sensitizing agent may increase the sensitivity of tumors to radiotherapy while the side effects of radiation therapy can be reduced.

Description

USE OF MOGROSIDES OR THE PHARMACEUTICALLY ACCEPTABLE SALTS THEREOF IN THE MANUFACTURE OF A TUMOR RADIATION SENSITIZING AGENT

TECHNICAL FIELD

The present invention relates to the use of mogrosides, and more specifically, to the use of mogrosides or the pharmaceutically acceptable salts thereof in the manufacture of a tumor radiation sensitizing agent.

BACKGROUND

Tumors include benign and malignant tumors. Benign tumors which can generally be healed by removing through operation, do relatively less harm to human body. In contrast, malignant tumors greatly threaten the health of human body. Currently, malignant tumor has become a disease of high incidence and high mortality in China. Presently, the major methods for treating malignant tumors include radiation therapy, chemotherapy, and resection. Radiation therapy, after its development for about a hunched of years, has become a mature and effective means for treating tumors. Radiation therapy is required for the treatment of more than halt' of patients with malignant tumors at various stages.

Although the treatment of malignant tumors by radiation therapy has a reliable effect and well-defined adverse response and thus enables the control of the most of tumors to an extent, it has following disadvantages: (1) While damaging or killing malignant tumor cells, radiation therapy may also more or less cause damage to a number of normal cells, and lead to a certain local or systemic damage and response. That is, the treatment of malignant tumors by radiation therapy comes with side effects, such as, swelling local tissues, dermahemia, pigmentation, keratosis fcllicularis, dry skin peeling, loss of hair or the like; malaise, nausea, anorexia, insomnia, reduction of leukocyte, and the like. (2) The treatment of radiation therapy alone only has effect on the malignant tumors sensitive to radiation. However, there are many malignant tumors not sensitive to radiation. In this case, the increase of the radiation dose may help to increase the sensitiveness to radiation, but may also damage or kill a large number of normal cells, resulting in irreversible damage to patients. Thus, when malignant tumors are not sensitive to radiation, radiation therapy may have to be abandoned.

Mogrosides refer to a variety of triterpene compounds extracted from the fruit of fructus momordicae. It not only has the characteristic flavor of fractus momordicae, but also as a triterpene glucoside, has a very high sweetness which is about 300 times of that of sucrose, and results in no calory. Therefore, mogrosides are generally used as food additives, such as sweetener, flavor, for use in the industries of beverage, candy, food, and the like, for improving the taste and flavor of a product. Up to now, it has not been reported that mogrosides can be used as an active ingredient for the manufacture of a tumor radiation sensitizing agent.

In vie w of above, the present invention is made.

SUMMARY OF THE INVENTION

An object of the present invention is to provide the use of mogrosides or the pharmaceutically acceptable salts thereof in the manufacture of a sensitizing agent for the chemotherapy of tumors.

In order to achieve the above-mentioned object, following technical solutions are utilized:

Use of mogrosides or the pharmaceutically acceptable salts thereof in the manufacture of a tumor radiation sensitizing agent.

Radiation therapy can not be used for the treatment of malignant tumors when it often comes with side effects, or when some of malignant tumors are not sensitive to radiation. The present inventors found by investigation that mogrosides have a sensitizing effect on the radiation therapy of tumors, and thus can be used for the manufacture of a radiation sensitizing agent. Up to now, such use of mogrosides has not been reported.

Preferably, the mogrosides have a structure of following formula:

wherein, R and R( are glucose residues including any of

Preferably, the mogroside is mogroside V, which has a CAS number of 88901-36-4, a molecular formula of C60H102O29, and a chemical structure of:

Preferably, the mogroside is mogroside VI, which has a CAS number of 89590-98-7, a molecular formula of 066¾ 12()34, and a chemical structure of:

Preferably, the mogroside is mogroside III, which has a CAS number of 130567-83-8, a molecular formula of C48H820i9, and a chemical structure of:

Preferably, the mogroside is mogroside II. which has a CAS number of 88901-38-6, a molecular formula of C42.H72O14, and a chemical structure of:

Preferably, the mogroside is mogroside IV, which has a CAS number of 89590-95-4, a molecular formula of C54H92O2.1, and a chemical structure of:

Preferably, the radiation sensitizing agent comprises mogrosides or the pharmaceutically acceptable salts thereof and an acceptable carrier(s) which is(are) one or more compatible solid or liquid filler or gel substances. The acceptable carriers are suitable for human use, and have a purity high enough and a toxicity low enough. The term “compatible” as used herein means that the components are combinable with each other and with the mogrosides of the present invention, without an significantly reducing efficacy. The acceptable carriers include one or more of cellulose and the derivatives thereof, gelatin, talc, solid lubricants, calcium sulphate, vegetable oils, polyols, emulsifying agents, wetting agents, colorants, flavors, stabilizing agents. antioxidants, preservatives, and pyrogen-free water.

Preferably, the radiation sensitizing agent is in the form of any of oral liquid formulation, granule, tablet, powder, pill, capsule, delayed-release agents, dropping pill, or oral disintegration agent.

The administration manner of the tumor radiation sensitizing agent produced from mogrosides is not particularly limited. The representative administration forms include: oral, injection, intratumoral and topical administration.

The solid dosage forms for oral administration include any of capsules, tablets, pills, powders, or granules. In these solid dosage forms, active compounds are mixed with at least one conventional inert excipient or carrier, such as dicalcium phosphate or sodium citrate, or with following ingredients: (a) fillers or solubilizing agents, e.g., lactose, starch, sucrose, mannitol, glucose or silicic acid; (b) binders, e.g., hydroxymethyl cellulose, alginates, gelatin, polyethylene pyrrolidone, sucrose, and acacia gum; (c) humectants, e.g., glycerol; (d) disintegration agents, e.g., agar, calcium carbonate, potato starch, or cassava starch, alginates, some complexed silicates, and sodium carbonate; (e) retarding solvents, e.g. paraffin; (f) absorption accelerating agents, e.g., quartemary ammonium compounds; (g) wetting agents, e.g. cetanol and glycerol monostearate; (h) sorbents, e.g., kaolin; and (i) lubricants, e.g., talc, calcium stearate, magnesium stearate, solid PEG, sodium lauryl sulphate, or the combinations thereof.

Solid dosage forms, such as tablets, dragees, capsules, pills and granules, can be made from coating and shell materials, such as casing and other materials well known in the art which may comprise opacifying agents. The release of the active compounds or compounds from the composition may be performed in a certain section of alimentary tract in a delayed-release manner. Examples of the embedding components which may be used are polymers and wax-like substances. If necessary; active compounds may be formed into microcapsules together with one or more of the excipients as described above.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. The liquid dosage forms can further comprise, in addition to the active compound, commonly used inert diluents, such as water or other solvents, solubilizing agents and emulsifying agents, e g., ethanol, ethyl carbonate, isopropanol, 1,3-butanediol ethyl acetate, propanediol, dimethyl carboxamide and oils, especially cotton seed oil, peanut oil, com germ oil, olive oil, castor oil and sesame oil or the mixtures thereof.

The t umor radiation sensitizing agent may further comprise, in addition to these inert diluents, adjuvants, such as wetting agents, emulsifying agents and suspending agents, sweetener, flavor and perfume.

The suspension may further comprise, in addition to the active compound, suspending agents, e.g., ethoxylated isooctadecanol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, methanol aluminum and agar or the mixtures thereof.

The tumor radiation sensitizing agent for parenteral injection may comprise physiologically acceptable sterile aqueous or water-free solutions, dispersions, suspensions or emulsions, and sterile powders that can be redissolved into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

The dosage forms of the tumor radiation sensitizing agent for topical administration include ointments, powders, patches, sprays and inhalants. The active ingredients are mixed with physiologically acceptable carriers and any preservatives, buffers, or if necessary, optional propellant under sterile conditions.

The present tumor radiation sensitizing agent can be administrated either alone or in combination with other pharmaceutically acceptable compounds.

When using the present tumor radiation sensitizing agent, a safe and effective amount of the drug is administrated to mammals, such as human, in need thereof, with the dose being a dose considered to be pharmaceutically effective.

For the range of every dose, the daily dose for e.g., a person of 65 kg body weight, is generally 100 ‘" 400 mg. Specific doses also depend on various factors such as the route of administration, health conditions of patients.

Preferably, the present invent ion provides the use of the tumor radiation sensitizing agent for the manufacture of a medicament capable of up-regulating the expression of anti-cancer P53 gene and/or down-regulating the express of Bel-2 protein. p53, as an important anti-cancer gene, may cause the apoptosis of tumor cells so as to prevent tissue cells from turning to cancer cells. P53 is also capable of facilitating the repair of the defects of cell genes. Bel-2, an anti-apoptosis protein, is capable of inhibiting the programmed apoptosis of cells. It has been shown that, p53 and Bcl~2 also play a key role in the sensitization during radiation therapy. A sensitizing effect on radiation therapy may be achieved by increasing the expression of p53 or decreasing the expression of Bcl-2. In the present invention, the present inventors found that, mogrosides may up-regulate the expression of anti-cancer P53 gene or down-regulate the expression of Bcl-2 protein in tumor cells. This finding reveals the action mechanism of mogrosides as a radiation sensitizing agent: mogrosides may facilitate the apoptosis of tumor cells by increasing the expression of anti-cancer gene p53 or decreasing the expression of Bcl-2, so as to improve the sensitivity of radiation.

The present invention has following advantages as compared to the prior art: (1) providing the use of mogrosides for the manufacture of a tumor radiation sensitizing agent, which has not been reported yet; and (2) providing the action mechanism of mogrosides as radiation sensitizing agent: mogrosides may improve the sensitivity of radiation by increasing the expression of anti-cancer gene p53 or decreasing the expression of Rcl-2 in tumor cells.

DESCRIPTION OF FIGURES

The figures required for the description of Examples or the prior art are briefly described below to illustrate the technical solutions of the Examples of the present invention or those in the prior art: in more detail.

Fig. 1 shows the results of the test on the regulation of the expression of P53 and Bcl-2 proteins in Hep-G2 hepatocyte cancer by mogroside II;

Fig. 2 shows the effect of various treatments (control, drug only, radiation only, and the combination of drug and radiation) on the apoptosis of Hep~G2 liver cancer cells;

Fig. 3 shows the effect of various treatments (control, drug only; radiation only, and the combination of drug and radiation) on the morphology of Hep-G2 liver cancer cells;

Fig. 4 shows the results of the test on the regulation of the expression of P53 and Bcl-2 proteins in A549 lung cancer cells by mogroside V;

Fig. 5 shows the effect of various treatments (control, drug only, radiation only, and the combination of drug and radiation) on the apoptosis of A549 lung cancer cells;

Fig. 6 shows the effect of various treatments (control, drug only, radiation only, and the combination of drug and radiation) on the morphology of A549 lung cancer cells.

DETAILED DESCRIPTION

The embodiments of the present invention will be described in detail by reference to following Examples, which, as will be appreciated by those skilled in the art, are only used to illustrate the present invention, and should not be considered in any way as limiting the scope of the present invention. The Examples for which no specific conditions were indicated below were performed under conventional conditions or in accordance with the manufacturer’s instructions. The agents or instruments used for which the manufacturers were not indicated below are commercial available products.

Mogrosides used in the present invention may be produced as follows: 1) The fruit of Grosvenor momordica is smashed and weighed. To the smashed fruit, water is added at a mass ratio of the smashed fruit to water of 1:6-1:8 and stirred at 80-9533 for 1-3 hours for extraction. The suspension is then centrifuged to collect supernatant with precipitate being subjected to stirring and extraction as above repeatedly tor 1-4 times. The supernatants from each centrifugation are combined to give an extract solution, 2) To the extract solution, a flocculant is added to remove tannins and soluble proteins from the extract solution, giving a clear aqueous solution. 3) The aqueous solution is absorbed on XAD-16 resin, which is then eluted with 30-50% ethanol, to give a water-ethanol mixture solution rich in mogrosides. 4) The water-ethanol mixture solution is concentrated under reduced pressure, during which ethanol is recovered, to give a paste-like extract which is then weighed. To the extract, deionized water was added at an amount which is 3-6 times by mass of that of the extract to dilute the extract, yielding an aqueous solution of crude mogrosides. 5) The aqueous solution of crude mogrosides is decolorized by using Diaion PA resin. Flow through is collected to give an enriched solution. Separation by purification using semi-preparative liquid chromatography is then performed to yield mogrosides having a purity of greater than 98%,

For step 1), the extraction is performed by stirring at 80-95 °C for 1-3 hours, preferably 2 hours. The supernatant is obtained by centrifugation, and the precipitate is subjected to extraction by stirring as above repeatedly for 1-4 times, preferably 1 time. The supernatant for each time is collected. The more times repeated, the more the extract solution is obtained.

For step 2), the flocculant used is chitosan, which, as an organic polymeric flocculant, is commonly used in the art.

For step 3), the specific XAD-16 resin is Amber! he XAD-16 nonionic macroporous resin, which is generally used for absorption of small molecules such as antibiotics and terpenes.

For step 5), the specific Diaion PA resin is anion exchange resin porous-Diaion PA series from Mitsubishi Chemical Corporation, which are mainly used for the deeolorization of mogrosides. The semi-preparative liquid chromatography is performed under following conditions: reverse phase Cl8 column (which is a nonpolar column) as chromatography column, UV detection wavelength: 214+2.0 nm; acetonitrile-water as mobile phase, elution with the gradient of 40% acetonitrile at 0-20 min, 40-60% acetonitrile at 20-40 min, and 60% acetonitrile at 40-60 min; flow rate: 1.0 mL/min; column temperature: 25°C. Samples are collected in accordance with different retention times. The more the number of glycosyl residues contained in a mogroside, the shorter the retention time is (and thus the faster the peak appears). Mogroside VI (22min), mogroside V (27min), mogroside IV (35min), mogroside III (41 min), and mogroside II (46min), which will be described in detail hereinafter, are sequentially collected.

Example 1

This Example demonstrated that the sensitizing effect of mogroside II, which has a C AS number of 88901-38-6 and a molecular formula of C47H72O14, on the radiation of liver cancer cells. 1. The effect of mogroside II on P53 and Bcl-2 in Hep-G2 liver cancer cells was detected by using Western Blotting.

Hep-G2 cells were cultured in the present of various concentrations of the drug (OpmolTC, lOpmol L"1, SOpmol L'1, and ΟΟμηιοΗ./1) for 24 h. Then, the cell culture was stopped, removed by suction, and the cells were washed with PBS (0.01 mol L"1, pH 7.4). A lysis buffer containing PMSF was added at 50 pL/well, and the cells were lysed in ice-bath for 30 min. Subsequently, the lysate was centrifuged at 14000 r-min'1 for 10 min to give total proteins. The concentration of proteins was measured by using BC A colorimetry. 50 pg of the total proteins were separated by electrophoresis on 12% SDS-PAGE, and then electronically transferred to PVDF film, which was then blocked with 5% skim milk (containing 0.1% Tween 20) for 1 h. Antibodies against to p53, Bcl-2, and β-actin respectively were added as primary antibodies and incubated at 4°C overnight (β-actin as a loading control). The film was then washed with TBS-T tor 3 times, 5 min for each time. Subsequently, secondary' antibodies labeled with IIRP were added and incubated at room temperature for 1 h. The film was then washed with rinsing solution (TBS-T) for 3 times, 10 min for each time. Then, ECL was added and incubated in dark for 5 min. The expression levels of P53, Bcl-2, and β-actin were determined by development by using fluorescence image analyzer, scanning, and analyzing. The results were shown in Fig. 1.

As can be seen from Fig. 1 (concentrations of the drug (from left to right, Qpmol-L" , lOpmol-L' , SOpmolL', and 60pmol-L~)), the expression of Bcl-2 protein in Hep-G2 liver cancer cells treated with mogroside II w'as significantly decreased and was negatively correlated with, the concentration of mogroside II, while the expression level of P53 was significantly upregulated and was positively correlated with the concentration of mogroside II.

As can be seen, mogrosides have an effect of upregulating p53 and downregulating Bcl-2 in tumor cells. Therefore, mogroside II has a potential for increasing the sensitivity of tumor cells to radiation therapy, and thus can be prepared into tumor radiation sensitizing agents. 2. The effect of the control group, drug-only group, radiation-only group, and combination group (radiation + drug) on the apoptosis of liver cancer cells.

The effect of mogroside II on the apoptosis of liver cancer cells was determined by flow cytometry. The cells cultured in the respective groups were collected, washed twice with 200 pL of cold PBS, and collected again. 50 pL of Binding Buffer was added to resuspend the cells. 2 pL of Annexin V-F1TC was added and mixed and 5 pL of PI was added and mixed. The mixture was allowed to stand in dark at room temperature for 10 min. Then, flow cytometry was carried out. The results were shown in Fig. 2.

As can be seen from Fig. 2, both the drug-only group and the radiation-only group showed a significant inhibitory effect on the growth of the tumor cells. The combination group showed the best inhibitory effect. In th is group, the apoptosis of the liver cancer cells was sign ificantly increased, showing that mogroside II had a sensitizing effect. 3. The effect of the control group, drug-only group, radiat ion-only group, and combination group (radiation t drug) on the morphology of liver cancer cells.

The effect of the radiation-only group, drug-only group, and combination group (radiation + drug) on the morphology of liver cancer cells was determined by tunel staining. The cells was washed once with PBS, immobilized with 4% paraform for 30 minutes, and then washed once with PBS. PBS (containing 0.1% Triton X-100) was added and incubated in ice-bath for 2 minutes to break membrane. Washing with PBS was then performed once. 0.3% H2O2 in methanol was added and incubated at room temperature for 20 minutes to inactivate endogenous peroxidases. Washing with PBS was then performed three times. To the samples, 50pl of a solution containing biotin label was added, and incubated at 37°C for 60 minutes. Washing with PBS was then performed once. 0.2ml of labeling reaction quenching solution was added and incubated at room temperature for 10 minutes. Washing with PBS was then performed three times. To the samples, 50pl of Streptavidin-FIRP working solution were added and incubated at room temperature for 30 minutes. Washing with PBS was then performed three times. 0,4 ml of DAB developing solution was added and incubated at room temperature for 15 minutes. Washing with PBS was then performed three times. The nucleoli of the cells were stained with hematoxylin. Washing with PBS was then performed three times. The cells were observed directly under microscope. The results were shown in Fig. 3.

After tunel staining, normal cancer cells were not stained, the apoptotic cancer cells turned into brown and darker in color, and the apoptotic liver cancer cells became smaller with their nucleoli shrinking. As can be seen from Fig, 3, the drug-only group, the radiation-only group, and the combinat ion group all showed a facilitating effect on the apoptosis of liver cancer cells. The apoptosis of liver cancer cells in the combination group was most significant,. In this group, the nucleoli of the liver cancer cells were stained into dark brown. These results also demonstrated that mogroside II had a sensitizing effect for radiation. 4. Colony formation assay

The sensitivity of the cells for radiation was determined by using colony formation assay. Hep-G2 cells were diluted to lxl04/mL and added to a 96-well plate at lOOpL/well. The cells were divided into radiation-only group and combination group (drug + radiation group). Before radiation, the cells in the drug + radiation group were treated with mogroside II at 10 pmol/L for 24 h. Afterward, at room temperature, single radiation was performed by using 6 MV-X ray at a dose of OGy, 2Gy, 4Gy, 6Gy, and 8 Gy, respectively. The radiation was carried out under following conditions: 6 MV-X ray, room temperature, radiation area 15 cm* 15 cm, with 1.5 cm equivalent tissue filler. After the radiation, the cell culturing medium was changed, and the culture was continued for 2 weeks after which supernatant in each well was removed. Then, the cells were immobilized with formaldehyde and stained by using Giemsa staining. The number of the colonies containing 50 cells or more was counted under inverted microscope. The results were recorded to calculate cell’s survival rate. Survival fraction SF2 = (average OD value of experiment group / average OD value of blank control group) χ 100%, and sensitivity enhancing rate SER SF of radiation control group / (SF of drug-only group + radiation-only group). The parameters associated with radiation sensitivity (D0, Dq, N, and K) were calculated by using multitarget-single hitting model SF = l-(l-e'D/D0)N. The assay was repeated for 3 times to obtain an average value. The related radiobiological parameters were calculated by curve fitting by using prism 5 software.

Table 1. The survival fraction of liver cancer cells after radiation at different doses.

Table 2. The effect of mogroside 0 on the radiation sensitivity of Hep-G2 cells.

The results showed that D0, ST2, Dq, and N for the combination group are significantly smaller than those for the radiation-only group. Do, as an important parameter in multitarget theory, indicated the single radiation dose required for killing 63% of cells. A smaller D0 means that the drug increases the sensitivity of cells to radiation. After the administration of the drag, D0 was reduced from 2.32 to 1.68, showing that mogroside II increased the sensitivity of liver cancer cells to radiation. SI 2 can directly reflect the effect of mogroside II on cell’s sensitivity. After the administration of the drug, SF2 was reduced to 39.23%, showing the sensitizing effect of mogroside II on liver cancer cells. Dq (quasi-threshold dose) reflected the ability of cells to repair subiethal damage. A smaller value of Dq indicates a reduced ability of liver cancer cells to repair subiethal damage. The results showed that mogroside II had a sensitizing effect on Hep-G2 liver cancer cells in the radiation sensitizing assay.

Example 2

This Example demonstrates the sensitizing effect of mogroside V, which has a CAS number of 88901-36-4 and a molecular formula of C60H102O29, on the radiation of lung cancer cells. 1. The effect of mogroside V on P53 and Bcl-2 in lung cancer cells.

The effect of mogroside V on P53 and Bcl-2 was detected by using Western Blotting. A549 cells were cultured in the presence of various concentrations of the drug (ΟμπιοΙΤ/1, ΙΟμηιοΗ/1, SOgmolT/1, and όΟμηιοΗ/1) for 24 h. Then, the cell culture was stopped, removed by suction, and the cells were washed with PBS (0.01 mol l/1, pH 7.4). A lysis buffer containing PMSF was added at 50 pL/well, and the cells were lysed in ice-bath for 30 min. Subsequently, the lysate was centrifuged at 14000 rmirw for 10 min to give total proteins. The concentration of proteins was measured by using BCA colorimetry. 50 pg of the total proteins were separated by electrophoresis on 12% SDS-PAGE, and then electronically transferred to PVDF film, which was then blocked with 5% skim milk (containing 0.1% Tween 20) for 1 h. Antibodies against to p53, Bcl-2, and β-actin respectively' were added as primary antibodies and incubated at 4°C overnight (β-actin as a loading control). The film was then washed with TBS-T for 3 times, 5 min for each time. Subsequently, secondary antibodies labeled with HRP were added and incubated at room temperature for 1 h. The film was then washed with rinsing solution (TBS-T) for 3 times, 10 min for each time. Then, ECL was added and incubated in dark for 5 min. The expression levels of P53, Bcl-2, and β-actin were determined by development by using fluorescence image analyzer, scanning, and analyzing. The results were shown in Fig. 4.

As can be seen from Fig. 4 (concentrations of'the drag (from left to right, OpmolTT1, lOpmolTT1, SOpmolL·3, and bOnmol IT1)), the expression of Bcl-2 protein in A549 cells treated with mogroside V was significantly decreased and was negatively correlated with the concentration of mogroside V, while the expression level of P53 was significantly upregulated and was positively correlated with the concentration of mogroside V.

In the Example, mogroside V had an ability of upregulating p53 and downregulating Bcl~2 in tumor cells. Therefore, mogrosides have a potential for increasing the sensitivity of tumor cells to radiation therapy, and thus can be prepared into tumor radiation sensitizing agents. 2. The effect of the control group, drug-only group, radiation-only group, and combination group (radiation + drag) on the apoptosis of lung cancer cells.

The effect of mogroside V on the apoptosis of lung cancer cells was determined by flow cytometry. The cells cultured in the respective groups were collected, washed twice with 200 pL of cold PBS, and collected again. 50 pL of Binding Buffer was added to resuspend the cells. 2 pL of Annexin V-FITC was added and mixed and 5 pL of PI was added and mixed. The mixture was allowed to stand in dark at room temperature for 10 min . Then, flow cytometry was carried out. The results were shown in Fig. 5.

As can be seen from Fig. 5, both the drug-only group and the radiation-only group showed a significant inhibitory effect on the growth of the tumor cells. The combination group showed the best inhibitory effect. In this group, the apoptosis of the liver cancer cells was significantly increased, showing that mogroside V has a sensitizing effect. 3. The effect of the control group, drag-only group, radiation-only group. and combination group (radiation + drug) on the morphology of lung cells.

The A549 cells were divided into the groups and cultured. After that, the waste fluid was removed by suction. To each well, 0.5 niL of immobilizing solution was added. The cells were immobilized for 25 min and then washed twice with PBS (each for 3 min). Hoechst 33258 staining solution was then added and the cells were stained at room temperature in dark for 20 min. The change in the morphology of the cells was observed by using fluorescence microscope. The results were shown in Fig. 6.

As can be seen from Fig. 6, the nucleoli of the cells in the control group were intact, uniformly stained, had dispersed fluorescence, and showed no sign of cell apoptosis, while in both of the drug-only group and the radiation-only group, there appeared apoptotic cells which showed partially particulate fluorescence. The effect of the radiation-only group is better than that of the drug-only group. In the combination group, there appeared a large number of crimpy apoptotic cells, demonstrating that the lung cancer cells in the combination group were strongly suppressed. 4. Colony formation assay

The sensitivity of the cells for radiation was determined by using colony formation assay. A549 cells were diluted to 1 fO'VmL and added to a 96-well plate at 100pL/well. The cells were divided into radiation-only group and combination group (drug + radiation group). Before radiation, the cells in the drug + radiation group were treated with mogroside V at 10 μιηοΙ/L for 24 h. Afterward, at room temperature, single radiation was performed by using 6 MV-X ray at a dose of OGy, 2Gy, 4Gy, 6Gy, and 8 Gy, respectively. The radiation was carried out under following conditions: 6 MV-X ray, room temperature, radiation area 15 cmx'15 cm, with 1.5 cm equivalent tissue filler. After the radiation, the cell culturing medium was changed and the culture was cont inued for 2 weeks after which the supernatant in each well was removed. Then, the cells were immobilized with formaldehyde and stained by using Giemsa staining. The number of the colonies containing 50 cells or more was counted under inverted microscope. The results were recorded to calculate cell’s survival rate. Survival fraction SF2 = (average 01) value of experiment group / average OD value of blank control group) x 100%, and sensitivity enhancing rate SER = SF of radiation control group / (SF of drug-only group + radiation-only group). The parameters associated with radiation sensitivity (Do, Dq, N, and K) were calculated by using multitarget-single hitting model SF = i~(i-e‘])/1)0)N. The assay was repeated for 3 times to obtain an average value. The related radiobiological parameters were calculated by curve fitting by using prism 5 software.

Table 3. The survival fraction of lung cancer cells after radiation at different doses.

Table 4. The effect of mogroside V on the radiation sensitivity of A549 cells.

The results show that D0, SF2, Dq, and N for the combination group are significantly smaller than those for the radiation-only group. D0, as an important parameter in multitarget theory, indicates the single radiation dose required for killing 63% of cells. A smaller Do means that the drug increases the sensitivity of cells to radiation. After the administration of the drug, D0 was reduced from 2.33 to 1.91, showing that mogroside V increased the sensitivity of lung cancer cells to radiation. SF2 can directly reflect the effect of mogroside V on cell’s sensitivity. After the administration of the drug, SF2 was reduced to 47.23%, showing the sensitizing effect of mogroside V on lung cancer cells. Dq (quasi-threshold dose) reflects the ability of cells to repair sublethal damage. A smaller value of Dq indicates a reduced ability of lung cancer cells to repair sublethal damage. The results showed that mogroside V had a sensitizing effect on A549 lung cancer cells in the radiation sensitizing assay.

Example 3

This Example demonstrated that the sensitizing effect of mogroside VI, which has a CAS number of 89590-98-7 and a molecular formula of C66H132O34, on the radiation of cervical cancer Hela cells.

Colony formation assay

The sensitivity of the cells for radiation was determined by using colony formation assay. Hela cells were diluted to 1 χ 104/mL. and added to a 96-well plate at 100pL/well. The cells were divided into radiation-only group and combination group (drug + radiation group). Before radiation, the cells in the drug + radiation group were treated with mogroside VI at 10 μ rnol/L for 24 h. Afterward, at room temperature, single radiation was performed by using 6 MV-X ray at a dose of OGy, 2Gy, 4Gy, 6Gy, and 8 Gy, respectively The radiation was carried out under following conditions: 6 MV-X ray, room temperature, radiation area 15 cm* 15 cm, with 1.5 cm equivalent tissue filler. After the radiation, the cell culturing medium was changed and the culture was continued for 2 weeks after which the supernatant in each well was removed.

Then, the cells were immobilized with formaldehyde and stained by using Giemsa staining. The number of the colonies containing 50 cells or more was counted under inverted microscope. The results were recorded to calculate cell’s survival rate. Survival fraction SF?. = (average OD value of experiment group / average OD value of blank control group) χ 100%, and sensitivity enhancing rate SER = SF of radiation control group / (SF of drug-only group + radiation-only group). The parameters associated with radiation sensitivity (D0, Dq, N, and K) were calculated by using multitarget-single hitting model SF = 1-(1 -e‘D/D0)N. The assay was repeated for 3 times to obtain an average value. The related radiobiological parameters were calculated by curve fitting by using prism 5 software.

Table 5. The survival traction of Flela cells after radiation at different doses.

Table 6. The effect of mogroside VI on the radiation sensitivity of Hela cells.

The results showed that D0, SF2, Dq, and N for the combination group were significantly smaller than those for the radiation-only group. D0, as an important parameter in multitarget theory, indicates the single radiation dose required for killing 63% of cells. A smaller D0 means that the drug increases the sensitivity of cells to radiation. After the administration of the drag, Do was reduced from 4.0! to 3.57, showing that mogroside VI increased the sensitivity of Hela cells to radiation. SF2 can directly reflect the effect of mogroside VI on cell’s sensitivity. After the administration of the drug, SF2 was reduced, showing the sensitizing effect of mogroside VI on the cervical Hela cancer cells. Dq (quasi-threshold dose) reflects the ability of cells to repair sublethal damage. A smaller value of Dq indicates a reduced ability of cervical cancer cells to repair sublethal damage. The results showed that mogroside VI has a sensitizing effect on cervical cancer Hela cells in the radiation sensitizing assay.

Application Example 1

Mogroside IV was used for the preparation of an oral tablet having a tumor radiation sensitizing effect. 500g of mogroside I V was mixed with an appropriate amount of dextrin and then distilled water to give a damp mass, which was then prepared into dry granules by using a granulator, sterilized, and compressed into semi-product tablets have a diameter of' 1 cm by using a compressor. Subsequently, the semi-product tablets were sterilized by radiation under UV light for 15 minutes to provide oral tablets, each containing 60 mg of mogroside IV.

Inclusion criteria: Kamofsky Performance Score >70; pathologically diagnosed as non-small cell lung cancer; clinical phase: phases ΤΙ'ΉΙΙ; no obvious malfunction of heart, lung, liver, and kidney. 80 non-small cell lung cancer patients were divided randomly into two groups: 39 patients for a sensitizing group, to whom, the oral tablets were administrated from Day 1 of the radiation therapy, 3 times a day and one tablet (containing 60 mg of mogroside IV) for each time, until the end of the radiation therapy; and 41 patients for a control (radiation-only) group. For both of the sensitizing group and the control group, conventional fractional radiation therapy was used for the radiation process, using 15MVX ray, at a total dose of 66"-70Gy/33'"- 35f, for 6~7 weeks.

Table 7. The recent comparison of the effect of the radiation therapy in the sensitizing group and the control group.

1) P<0,05 as compared to the control group.

Note: The parentheses contain percentages.

As can be seen from Table 5, the criteria for evaluating therapeutic effect, including completely relieved (CR), partially relieved (PR), and objectively effective (CR+PR), showed that the effect in the sensitizing group was significantly better than that in the control group, demonstrating that mogroside IV had a sensitizing effect.

Although the present invention has been illustrated and described with reference to specific Examples, it would be appreciated that many other modifications and changes can be made without deviating from the spirit and scope of the present invention. Thus, this means that the claims encompass all of those modifications and changes falling within the scope of the present invention.

Claims (10)

1. Use of mogrosides or the pharmaceutically acceptable salts thereof in the manufacture of a tumor radiation sensitizing agent.

2. The use of mogrosides or the pharmaceutically acceptable salts thereof in the manufacture of a tumor radiation sensitizing agent according to claim 1, characterized in that, the mogrosides have a structure of following general formula:

wherein, R and Ri are glucose residues including any of

or

3. The use of mogrosides or the pharmaceutically acceptable salts thereof in the manufacture of a tumor radiation sensitizing agent according to claim 2, characterized in that, the mogroside is mogroside V, which has a CAS number of 88901-36-4 and a molecular formula of C60H102O29.

4. The use of mogrosides or the pharmaceutically acceptable salts thereof in the manufacture of a tumor radiation sensitizing agent: according to claim 2, characterized in that, the mogroside is mogroside VI, which has a CAS number of 89590-98-7 and a molecular formula of C66H112O34.

5. The use of mogrosides or the pharmaceutically acceptable salts thereof in the manufacture of a tumor radiation sensitizing agent according to claim 2, characterized in that the mogroside is mogroside III, which has a CAS number of 130567-83-8 and a molecular formula of C48H82Oi9.

6. The use of mogrosides or the pharmaceutically acceptable salts thereof in the manufacture of a tumor radiation sensitizing agent according to claim 2, characterized in that, the mogroside is mogroside II, which has a CAS number of 88901-38-6 and a molecular formula of C42H720]4,

7. The use of mogrosides or the pharmaceutically acceptable salts thereof in the manufacture of a tumor radiation sensitizing agent according to claim 1, characterized in that, the mogroside is mogroside IV, which has a CAS number of 89590-95-4 and a molecular formula of C54H92O24.

8. The use of mogrosides or the pharmaceutically acceptable salts thereof in the manufacture of a tumor radiation sensitizing agent according to any one of claims 1-7, characterized in that, the radiation sensitizing agent comprises the mogroside and an acceptable carrier; and the acceptable carrier is one or more compatible solid or liquid filler or gel substances, including one or more of cellulose and the derivatives thereof, gelatin, talc, solid lubricant, calcium sulphate, vegetable oils, polyols, emulsifying agents, wetting agents, colorants, flavors, stabilizing agents, antioxidants, preservatives, and pyrogen-free water.

9. The use of mogrosides or the pharmaceutically acceptable salts thereof in the manufacture of a tumor radiation sensitizing agent according to claim 8, characterized in that, the radiation sensitizing agent is in the form of any' of oral liquid formulation, granule, tablet, powder, pill, capsule, delayed-release agents, dropping pill, or oral disintegration agent.

10. The use of mogrosides or the pharmaceutically acceptable salts thereof in the manufacture of a tumor radiation sensitizing agent according to claim 1, characterized in that, the tumor radiation sensitizing agent is capable of up-regulating the expression of anti-cancer P53 gene and/or down-regulating the express of Bcl-2 protein, preferably the tumor is selected from the group consisting of liver cancer, lung cancer and cervical cancer.