AU2016204180B2 - Mogrosides and the salts thereof, the preparing method and the use of the same and pharmaceutical compositions comprising the mogrosides and the salts thereof - Google Patents
Mogrosides and the salts thereof, the preparing method and the use of the same and pharmaceutical compositions comprising the mogrosides and the salts thereof Download PDFInfo
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Abstract
FIl50363AU The present invention provides mogrosides and the salts thereof, the preparing method and the use of the same, and pharmaceutical compositions comprising the mogrosides and the salts thereof In one aspect, the present invention provides a process for producing mogrosides and the salts thereof, comprising the steps of: obtaining an extract solution; obtaining a clear aqueous solution; obtaining a water-ethanol mixed solution rich in mogrosides; obtaining an aqueous solution of crude mogrosides; and obtaining the mogrosides and the salts thereof. In another aspect, the present invention provides mogrosides and the salts thereof produced by the above-mentioned process. In another aspect, the present invention provides the use of the mogrosides and the salts thereof in the manufacture of a chemotherapy synergist for the treatment of tumors. In a further aspect, the present invention provides a pharmaceutical composition comprising the mogrosides and the salts thereof and a pharmaceutically acceptable carrier. The mogrosides and the salts thereof of the present invention may increase the sensitivity of tumor cells to chemotherapy drugs and reduce the dose of the chemotherapy drugs.
Description
The present invention provides mogrosides and the salts thereof, the preparing method and the use of the same, and pharmaceutical compositions comprising the mogrosides and the salts thereof. In one aspect, the present invention provides a process for producing mogrosides and the salts thereof, comprising the steps of: obtaining an extract solution; obtaining a clear aqueous solution; obtaining a water-ethanol mixed solution rich in mogrosides; obtaining an aqueous solution of crude mogrosides; and obtaining the mogrosides and the salts thereof. In another aspect, the present invention provides mogrosides and the salts thereof produced by the above-mentioned process. In another aspect, the present invention provides the use of the mogrosides and the salts thereof in the manufacture of a chemotherapy synergist for the treatment of tumors. In a further aspect, the present invention provides a pharmaceutical composition comprising the mogrosides and the salts thereof and a pharmaceutically acceptable carrier. The mogrosides and the salts thereof of the present invention may increase the sensitivity of tumor cells to chemotherapy drugs and reduce the dose of the chemotherapy drugs.
FI150363AU
2016204180 21 Jun 2016
MOGROSIDES AND THE SALTS THEREOF, THE PREPARING METHOD AND THE USE OF THE SAME AND PHARMACEUTICAL COMPOSITIONS COMPRISING THE MOGROSIDES AND THE SALTS THEREOF
TECHNICAL FIELD
The present invention relates to use of compounds extracted from a plant in the manufacture of a chemotherapy synergist for the treatment of tumors. More specifically, the present invention relates to mogrosides and the salts thereof, the preparing method and the use of the same and pharmaceutical compositions comprising the mogrosides and the salts thereof.
BACKGROUND
Chemotherapy is important means for treating tumor diseases. However, some of the drugs for chemotherapy have great side effects, which bring pains to patients. For example, vincristine, methotrexate, cisplatin, cyclophosphamide and the like may lead to toxic side effects such as pain at injection sites, vein occlusion, gastrointestinal response, marrow suppression, peripheral neuropathy. Further, the cytotoxicity of chemotherapy drugs may even lead to complications such as infection and hemorrhage in patients. For example, cisplatin, which is a cell cycle non-specific agent, has cytotoxicity and may inhibit the complicated process of DNA of tumor cells and damage the structures on the membranes of the cells. After the administration of cisplatin to patients, the patients may have following adverse effects: marrow suppression, gastrointestinal response, nephrotoxicity, neurotoxicity, allergic response, electrolyte disorder, and the like.
Tumor chemotherapy sensitizers are agents which may increase the sensitivity of tumor cells to chemotherapy drugs and reduce the dosage of the chemotherapy drugs. In order to reduce the toxic side effects of chemotherapy drugs, to improve the therapeutic effect of the drugs, and to
FI150363AU
2016204180 21 Jun 2016 reduce the occurrence of drug-resistance, the combined use with suitable chemotherapy sensitizers has become an important method for the treatment of tumors. For example, in clinical, cisplatin is used in combination with 5-Fu and zeocin for the treatment of esophageal cancer to improve the therapeutic effect of the drugs.
Therefore, there has always been a need for a tumor chemotherapy synergist which has less or even no adverse effect. The sensitivity of a tumor to a drug is closely related to the expression of its related genes. It has been reported that, the expression of Bax may increase the sensitivity of pancreatic cancer to chemotherapy drugs such as gemcitabine and 5-FU and radiotherapy. Further, it has been found that, the expression of Bax gene in tumor tissues such as gastric cancer and liver cancer is significantly down-regulated, suggesting that the expression of Bax may increase the sensitivity of tumors to radiotherapy and chemotherapy (Wei HE, Yuxia LI, Wei LI. Influence of Bax gene transfection on apoptosis and chemotherapy sensitivity of A549 cell line. Cancer Research on Prevention and Treatment, 2014, 06: 515-518). Bcl-2 protein, the product of proto-oncogene Bcl-2, is a cell survival promoting factor. Bcl-2 of the Bcl-2 gene family is the most important apoptosis inhibiting gene. It has been reported that, a high expression level of Bcl-2 may significantly inhibit the apoptosis of tumor cells induced by chemotherapy drugs, resulting in resistance of tumor cells to a variety of chemotherapy drugs (Tingfang ZHOU, Yingzhi ZHUANG, Jianguo CAO. Rosiglitazone enhances the inhibition effect of cisplatin on proliferation of human lung adenocarcinoma cells. Chinese Pharmacological Bulletin, 2005, 01: 88-91).
Multidrug resistance (MDR) denotes a phenomenon in which while tumor cells have resistance to one anti-tumor drug, they also show resistance to other anti-tumor drugs having different structures and target sites. It has been demonstrated by extensive studies that, the over expression of ERK pathway is positively correlated with the resistance of tumors to chemotherapy
2016204180 11 Dec 2017 drugs. The study on ERK signaling pathway provided medieine support for handling the resistance of tumors to drugs in clinical (Wei LIANG, Zhi-xia CHEN, Hui-jun WANG, Song-po WANG. Research on the role of ERK signaling pathway in the reversing effect of Zhizhen Recipe on multidrug resistance in human colorectal cancer cell line HCT-8/VCR. Chin JIntegr Trad West Med Dig, 2015, 05: 315-320). ERK is highly expressed in most of acute leukemia cells. For example, the anti-apoptosis property of K562 cell line is related to the high basal activity of ERK. The inhibition of the activity of ERK leads to the apoptosis of K562. Suppression of ERK pathway reduces the expression level of phosphorylated ERK 1/2 proteins and thus down-regulates the activity of telomerase, which can improve the sensitivity of drug-resistant cells such as leukemia cells and ovarian cancer cells to chemotherapy drugs (Dengju LI, Yaozhen ZHANG, Wei HUANG, Fankai MENG. To inhibit ERK for enhancing chemotherapy sensitivity of drug-resistant cell lines of leukemia and ovarian carcinoma. Journal of Experimental Hemotology, 2003, 11(6): 595-599).
To solve above-mentioned problems in the prior art, the present invention provides a new solution.
Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.
Throughout this specification the word comprise”, or variations such as comprises or comprising, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
J
2016204180 11 Dec 2017
SUMMARY OF THE INVENTION
To solve the above-mentioned problems in the prior art, the present invention provides mogrosides and the salts thereof, the preparing method and the use of the same, and pharmaceutical compositions comprising the mogrosides and the salts thereof According to the present invention, the mogrosides and the salts thereof may up-rcgulalc the expression of Bax and down-regulate the expression of Be I-2, or may down-regulale the expression of p-ERK, and thus increase the sensitivity of tumor cells to chemotherapy drugs and reduce the dosage of the chemotherapy drugs without other toxic side effects or adverse response.
One aspect relates to the use of the mogrosides and the salts thereof in the manufacture of a chemotherapy synergist for the treatment of tumors, wherein the mogrosides and the salts thereof are represented by the following general formula:
wherein, R is a H atom, meial ion, or n molecules of glucose, and RT is a metal ion or n molecules of glucose, each n > 1, and wherein the mogrosides and the salts thereof are able to up-regu!ate the expression of Bax and down-regulate the expression of Be 1-2, or down-reguiate the expression of p-ERk.
In another aspect, the present invention provides a process for producing
FI150363AU
2016204180 21 Jun 2016 mogrosides and the salts thereof, comprising the steps of:
1) smashing the fruit of Grosvenor momordica and weighing, adding water to the smashed fruit at a mass ratio of the smashed fruit to water of 1:6-1:8 and stirring at 80-95°C for 1-3 hours for extraction, then centrifuging to collect supernatant, subjecting the precipitate to the stirring for extraction and the centrifuging, repeatedly for 1-4 times, and combining supernatants from each centrifugation to give an extract solution;
2) adding a flocculant to the extract solution to remove tannins and soluble proteins from the extract solution to give a clear aqueous solution;
3) absorbing the aqueous solution on XAD-16 resin, which is then eluted with 30-50% ethanol, to give a water-ethanol mixed solution rich in mogrosides;
4) concentrating the water-ethanol mixed solution under reduced pressure and recovering ethanol to give a paste-like extract, weighing the paste-like extract, adding deionized water to the extract at an amount which is 3-6 folds by mass of that of the extract to dilute the extract, yielding an aqueous solution of crude mogrosides; and
5) decolorizing the aqueous solution of crude mogrosides by using Diaion PA resin, collecting flow through to give an enriched solution, then performing separation by using semi-preparative liquid chromatography to yield mogrosides and the salts thereof having a purity of greater than 98%.
In another aspect, the present invention provides mogrosides and the salts thereof produced by the above-mentioned process, wherein, the mogrosides and the salts thereof are represented by the following general formula:
FI150363AU
2016204180 21 Jun 2016 \-νΟΗ •Ζ**
RO
wherein, R is a Η atom, metal ion, or n molecules of glucose, and R1 is a metal ion or n molecules of glucose, each n > 1.
Alternatively, the mogrosides are represented by the following formulae:
QH OH
Of-: 05· hc\.
HO' OH
I *
0.. A .,OH
..0
HO. 1
HO/,
Λ ..
ΟΗ
Η § Η : · Ύΐΐ
HCA 'Ύ· 'Ό'^'-- ΟΗ
ΗΟ i
ΟΗ
ΌΗ mogroside VI ΟΗ ΟΗ
ΟΗ ΟΗ ’<\ ····...α . ,Ο
I
ΗΟ' ,Α„ .0 ΗΟ V ΟΗ * I
Ο. ‘., ΟΗ
S- . Ο.
ο ο —( ..---. χ.
HCT V ΟΗ
ΌΗ
Η©>·. A
...... Ά J
ΟΗ ηο*/'Ά\γ '
ΟΗ mogroside V
FI150363AU
2016204180 21 Jun 2016
MO.
OH OH
V-,0
OH OH ,-Y .0 HO- γ
Z'X x
, 1
HOHO' y OH
0,. ..4-. ,,OH r i .OH
HO
HO'
XJ
H: i H
.. Π ,Y i Ϊ
-I-/
OH mogroside IV
OH
HO,. ,Λ .OH
ΗΌ,.
HO,, .1, y'’
OH
HO. ,.-. -H U H <Ύ'Χχ1 i/'
o.
'ΌΗ
HO*'
OH mogroside III.
In another aspect, the present invention provides the use of the mogrosides and the salts thereof in the manufacture of a chemotherapy synergist for the treatment of tumors.
Alternatively, the chemotherapy synergist includes chemotherapy sensitizers and resistance reversing agents.
Further, the mogrosides and the salts thereof may regulate the expression of genes related to sensitivity of drugs and drug-resistance in tumor cells.
Further, the mogrosides and the salts thereof may up-regulate the expression of Bax and down-regulate the expression of Bcl-2, or may down-regulate the expression of p-ERK.
Alternatively, the tumor is selected from leukemia, liver cancer, lung cancer, blood cancer, colon adenocarcinoma, lymphoma, breast cancer, pancreatic cancer, laryngeal cancer, melanoma or cervical cancer.
In a further aspect, the present invention provides a pharmaceutical
FI150363AU
2016204180 21 Jun 2016 composition comprising the mogrosides and the salts thereof and a pharmaceutically acceptable carrier.
Alternatively, the pharmaceutically acceptable carrier is selected from fillers, binders, disintegrants, lubricants, absorption promoters, absorbing carriers, flavors, sweeteners, diluents, excipients or wetting agents.
The mogrosides and the salts thereof, the preparing method and the use of the same and pharmaceutical compositions comprising the mogrosides and the salts thereof according to the present invention have at least one of the following benefits:
(1) Effect of chemotherapy synergist. The mogrosides and the salts thereof of the present invention show significant sensitizing and resistance reversing effect in tumor chemotherapy. As used herein, sensitizing effect includes the effect of increasing the sensitivity of non-drug-resistant tumor cells to chemotherapy drugs, and the effect of reversing the drug-resistance of drug-resistant tumor cells. The mogrosides and the salts thereof of the present invention have chemotherapy sensitizing effect and drug-resistance reversing effect by regulating Bax and Bcl-2 proteins and inhibiting ERK signaling pathway in tumor cells, and thus may significantly suppress the proliferation of tumor cells and effectively reduce the dosage of chemotherapy drugs so as to improve therapeutic effect. They would thus be useful in practical applications.
(2) Convenient and wide use. The mogrosides and the salts thereof of the present invention may be used as it is or as a pharmaceutical composition, may be administrated orally or by injection, may be used in combination with chemotherapy drugs such as anti-metabolic agents, transcription inhibiting agents, mitosis inhibiting agents and DNA replication interfering agents, and can be used in various tumors such as leukemia, oral cavity cancer, breast cancer, liver cancer, lung cancer, cervical cancer or pancreatic cancer.
THE DESCRIPTION OF FIGURES
FI150363AU
2016204180 21 Jun 2016
Fig. 1 illustrates the results of the western blotting of Example 1 of the present invention, showing the regulation of Bax and Bcl-2 proteins in PANC-1 pancreatic cancer cells by mogroside VI.
Fig. 2 illustrates the results of Example 1 of the present invention, showing the suppression of pancreatic cancer by the combination of mogroside VI and gemcitabine.
Fig. 3 illustrates the results of the western blotting of Example 2 of the present invention, showing the regulation of Bax and Bcl-2 proteins in liver cancer cells by mogroside V.
Fig. 4 illustrates the results of Example 2 of the present invention, showing the suppression of liver cancer by the combination of mogroside V and cisplatin.
Fig. 5 illustrates the results of the western blotting of Example 3 of the present invention, showing the regulation of p-ERKl/2 proteins in leukemia drug-resistant strain HL-60/ADR cells by mogroside IV.
Fig. 6 illustrates the results of the western blotting of Example 4 of the present invention, showing the regulation of p-ERK proteins in breast cancer MCF-7ADR cells by mogroside III.
DETAILED DESCRIPTION
The exemplary Examples of the present invention will be described below in detail with reference to accompanying figures. These exemplary Examples are provided such that those skilled in the art may clearly understand the present invention and carry out the present invention according to the description provided herein. The figures and specific examples are not intended to limit the present invention, and the scope of the present invention is defined by the claims.
In one aspect, the present invention provides a process for producing mogrosides and the salts thereof, comprising the steps of:
1) smashing the fruit of Grosvenor momordica and weighing, adding
FI150363AU
2016204180 21 Jun 2016 water to the smashed fruit at a mass ratio of the smashed fruit to water of 1:6-1:8 and stirring at 80-95°C for 1-3 hours for extraction, then centrifuging to collect supernatant, subjecting the precipitate to the stirring for extraction and the centrifuging, repeatedly for 1-4 times, and combining supernatants from each centrifugation to give an extract solution;
2) adding a flocculant to the extract solution to remove tannins and soluble proteins from the extract solution to give a clear aqueous solution;
3) absorbing the aqueous solution on XAD-16 resin, which is then eluted with 30-50% ethanol, to give a water-ethanol mixed solution rich in mogrosides;
4) concentrating the water-ethanol mixed solution under reduced pressure and recovering ethanol to give a paste-like extract, weighing the paste-like extract, adding deionized water to the extract at an amount which is 3-6 folds by mass of that of the extract to dilute the extract, yielding an aqueous solution of crude mogrosides; and
5) decolorizing the aqueous solution of crude mogrosides by using Diaion PA resin, collecting flow through to give an enriched solution, then performing separation by using semi-preparative liquid chromatography to yield mogrosides and the salts thereof 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 the stirring for extraction and the centrifuging, repeatedly for 1-4 times, preferably for 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 Amberlite XAD-16 nonionic macroporous resin, which is generally used for absorption of small molecules such as antibiotics and terpenes.
FI150363AU
2016204180 21 Jun 2016
For step 5), the specific Diaion PA resin is an anion exchange resin porous-Diaion PA series from Mitsubishi Chemical Corporation, which are used for the decolorization 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±2.0nm; 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, mogroside V, mogroside IV, and mogroside III, which will be described in detail hereinafter, are sequentially collected.
In another aspect, the present invention provides mogrosides and the salts thereof produced by the above-mentioned process, wherein, the mogrosides and the salts thereof are represented by the following general formula:
wherein, R is a H atom, metal ion, or n molecules of glucose, and R1 is a metal ion or n molecules of glucose, each n > 1.
When R is 3 molecules of glucose and R1 is 3 molecules of glucose, the mogroside is mogroside VI, which has a CAS number of 89590-98-7, a molecular formula of C66H112O34, and a chemical structure of:
FI150363AU
2016204180 21 Jun 2016
OH OH Λ ·ά ho· γ
Οχ ΗΟ, 1 : -'Κ Ό
I
ΟΗ<
ΟΗ
HQ
QH OH
WO' 'γ· ΟΙ
Q Λ. .C
ΟΗ ο^Ό · OH
ΗΟ*^ ' - '''OH OH mogroside VI
When R is 2 molecules of glucose and R1 is 3 molecules of glucose, the mogroside is mogroside V, which has a CAS number of 88901-36-4, a molecular formula of C60H102O29, and a chemical structure of:
OH OH HOV... -χχ>
OH OH
HO oh
A »
| - | |||
| . ' ’X' .· < | .O | ... | |
| ho γ CA | HO,. „ 'x. | Γ-.....9...... HO | |
| 1 | . A J R | ) ' OH | |
| HO. | A. '9 |
,ο„
OH
..... ,sS' - , ..- St N X xX,
HO γ' tri γ 'OH '
ΌΗ oh mogroside V
When R is 2 molecules of glucose and R1 is 2 molecules of glucose, the mogroside is mogroside IV, which has a CAS number of 89590-95-4, a molecular formula of C54H92O24, and a chemical structure of:
FI150363AU
2016204180 21 Jun 2016
HO
OH OH
A.. O
Ϊ
Z'X x
, 1
HOHO
HO'
OH
A J.
Λ
OH OH
HO ''γ° OH
0,. ..-1 X ,,OH
T .OH
1./ mogroside IV
When R is 1 molecule of glucose and R1 is 2 molecules of glucose, the mogroside is mogroside III, which has a CAS number of 130567-83-8, a molecular formula of C48H820i9, and a chemical structure of:
OH
HO.
HO. | /
HO,. A, OH OHC,.-A.o o.
’ J. 'x. '
ΌΗ
HO,.
H
ΎΪΎ /Ah ho OH
OH
OH mogroside III
When R is 1 molecule of glucose and R1 is 1 molecule of glucose, the mogroside is mogroside salt A, which has a chemical structure of:
HQx
HO.. 1 ϊ Y J
HO^V O'/
-Au <
h°.·. .. -/A
3H .i:
- .--X-.. V ONa
,......
/ OH mogroside salt A
In another aspect, the present invention provides the use of the
FI150363AU
2016204180 21 Jun 2016 mogrosides and the salts thereof in the manufacture of a chemotherapy synergist for the treatment of tumors. The chemotherapy synergist includes chemotherapy sensitizers and resistance reversing agents. They may be used in various tumors cells such as leukemia, liver cancer, lung cancer, blood cancer, breast cancer, skin cancer, pancreatic cancer, laryngeal cancer, melanoma, cervical cancer, oral cavity cancer, lymphoma, and colon cancer. As used herein, sensitizing effect includes the effect of increasing the sensitivity of non-drug-resistant tumor cells to chemotherapy drugs, and the effect of reversing the drug-resistance of drug-resistant tumor cells.
Current studies are being focused on increasing the sensitivity of tumor cells to chemotherapy drugs to induce apoptosis of the tumor cells. The mogrosides and the salts thereof of the present invention are shown by the results of experiments to be able to regulate the expression of the genes related to sensitivity and resistance to drugs in tumor cells. Specifically, the mogrosides and the salts thereof can be used to regulate Bax and Bcl-2 proteins and to inhibit ERK signaling pathway in tumor cells. That is, the mogrosides and the salts thereof can up-regulate the expression of Bax and down-regulate the expression of Bcl-2 or can down-regulate the expression of p-ERK. According to the present invention, the mogrosides have a killing effect on in vitro cultured tumor cells such as pancreatic cancer, lung cancer, blood cancer, liver cancer, gastric adenocarcinoma, lymphoma, and melanoma. Up to now, it has not been reported that mogrosides can be used for the manufacture of tumor chemotherapy drug sensitizer and multidrug-resistance reversing agents.
The tumor is selected from leukemia, liver cancer, lung cancer, blood cancer, colon adenocarcinoma, lymphoma, breast cancer, pancreatic cancer, laryngeal cancer, melanoma or cervical cancer.
The mogrosides and the salts thereof of the present invention can be used as it is, or can be used in a form of pharmaceutical composition. Thus, in another aspect, the present invention provides a pharmaceutical composition
FI150363AU
2016204180 21 Jun 2016 comprising the mogrosides and the salts thereof as active ingredients and a pharmaceutically acceptable carrier.
The pharmaceutically acceptable carrier may be various commonly used pharmaceutical excipients, such as fillers (anhydrous lactose, starch, lactose beads, glucose), binders (microcrystalline cellulose), disintegrants (crosslinked carboxymethyl starch sodium, croscarmellose sodium, low-substituted hydroxypropyl cellulose, crosslinked PVP), lubricants (magnesium stearate), absorption promoters, absorbing carriers, flavors, sweeteners, diluents, excipients, wetting agents, or the like.
The pharmaceutical composition can be produced by standard methods in the art and can be administrated intestinally, non-intestinally, or topically. Oral formulations include tablets, granules, capsules, suspensions, solutions, and the like. The formulations for non-intestinal administration include injections. The formulations for local administration include creams, ointments, patches, sprays, and the like.
The above-mentioned pharmaceutical formulations may be administrated orally, sublingually, transdermally, intramuscularly, subcutaneously, mucocutaneously, transurethrally, intravaginally, or intravenously.
The mogrosides and the salts thereof of the present invention and/or the pharmaceutical compositions comprising the same can be used for increasing the sensitivity of the above-mentioned various tumor cells to chemotherapy. The mogrosides and the salts thereof and/or the pharmaceutical composition comprising the same may be used in combination with chemotherapy drugs, resulting in synergistic effect even at a lower dose, significant inhibition of the proliferation of pancreatic cancer cells, and the reduction of the dosage of chemotherapy drugs, and thus improved therapeutic effect on patients and practical value. The chemotherapy drugs include, but not limited to, anti-metabolic agents, transcription inhibiting agents, mitosis inhibiting agents and DNA replication interfering agents.
The anti-metabolic agents are cell cycle specific agents and include, but
FI150363AU
2016204180 21 Jun 2016 not limited to, 5-FU, methotrexate, and the like. The transcription inhibiting agents include, but not limited to, doxorubicin, daunorubicin, actinomycin, plicamycin, and the like. The mitosis inhibiting agents include, but not limited to, paclitaxel, vincristine, docetaxel, and the like. The DNA replication interfering agents include, but not limited to, antibiotics, zeocin, topotecan, irinotecan, cisplatin, and the like.
The method for chemotherapy sensitization by using the mogrosides and the salts thereof of the present invention and/or the pharmaceutical compositions comprising the same is to administrate chemotherapy drugs and an effective (in increasing sensitivity and reducing toxicity) amount of the mogrosides and the salts thereof and/or the pharmaceutical compositions comprising the same to patients in need thereof.
The dosage of the mogrosides and the salts thereof of the present invention and/or the pharmaceutical compositions comprising the same may vary depending on administration routes, the age and weight of the patient, and the type and severity of the tumor to be treated. In other words, the dosage may be adjusted in accordance with administration routes, the age and weight of the patient, and the type and severity of the tumor to be treated. A daily dose may be 0.005-200 mg/kg, via single administration or separated administrations.
The present invention will be further described below in detail through specific Examples.
Example 1. The sensitizing effect of mogroside VI on pancreatic cancer.
1. The preparation of mogroside VI, comprising following steps:
1) The fruit of Grosvenor momordica was smashed and weighed. To the smashed fruit, water was added at a mass ratio of the smashed fruit to water of 1:6 and stirred at 95°C for 2 hours for extraction. The suspension was then centrifuged to collect supernatant, and the precipitate was subjected to the stirring for extraction and the centrifuging repeatedly for 2 times. The
FI150363AU
2016204180 21 Jun 2016 supernatants from each centrifugation were combined to give an extract solution.
2) To the extract solution, chitosan as a flocculant was added to remove tannins and soluble proteins from the extract solution, giving a clear aqueous solution.
3) The aqueous solution was absorbed on XAD-16 resin, which was then eluted with 50% ethanol, to give a water-ethanol mixed solution rich in mogrosides.
4) The water-ethanol mixed solution was concentrated under reduced pressure, during which ethanol was recovered, to give a paste-like extract which was then weighed. To the extract, deionized water was added at an amount which was 4-folds 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 was decolorized by using Diaion PA resin. Flow through was collected to give an enriched solution. Separation by using semi-preparative liquid chromatography was then performed to yield mogroside VI having a purity of greater than 98%. The semi-preparative liquid chromatography was performed under following conditions: reverse phase Cl8 column (a nonpolar column) as chromatography column, UV detection wavelength: 214nm; acetonitrile-water as mobile phase, elution with the gradient of 40% acetonitrile at 0-20 min, 50% acetonitrile at 20-40 min, and 60% acetonitrile at 40-60 min; flow rate: 1.0 mL/min; column temperature: 25°C. The eluent at a retention time of 22min was collected to provide mogroside VI.
2. The effect of mogroside VI on Bax and Bcl-2 in pancreatic cancer PANC-1 cells.
The effect of mogroside VI on the expression of Bax and Bcl-2 proteins in PANC-1 cells was determined by using western blotting. PANC-1 tumor cells were seeded into a 6-well plate and treated with the agent at different concentrations for 24h. Then, the tumor cells were collected and lysed and
FI150363AU
2016204180 21 Jun 2016 the total proteins of the cells were extracted. The concentration of the proteins was measured by BCA method, using BSA as a standard. 50gg of each sample was loaded for electrophoresis separation. After the electrophoresis separation, the proteins were transfer onto PVDF membrane, which was then blocked with 5% skim milk at room temperature for lh, incubated with primary antibody at 4°C overnight, washed with TBST (3xl5min). Then, a corresponding secondary antibody was added and incubated at room temperature for 45min. The excessive secondary antibodies were washed with TBST (3xl5min). Subsequently, the PVDF membrane was placed on a preservative film, and covered with ECL working solution on its surface and incubated at room temperature for 5min. The expression levels of Bax, Bcl-2, and β-actin proteins were detected by sensitization and scanning.
It has been shown that the expression of Bax in tumors may increase the sensitivity of tumor cells to chemotherapy drugs, and that the expression of Bax may increase the sensitivity of pancreatic cancer to chemotherapy drugs such as gemcitabine and 5-FU. Thus, increasing the expression of Bax may effectively improve the effect of chemotherapy. Bcl-2 family is a family important for the apoptosis of cells. The down-regulation of Bcl-2 gene may facilitate the apoptosis of tumor cells. The expression levels of Bcl-2 and Bax proteins in various tumor cells were determined by using western blotting. The results showed that increasing the concentration of mogroside VI may up-regulate the expression of Bax protein and down-regulate the expression of Bcl-2 protein in tumor cells. Fig. 1 illustrates the results of the western blotting of Example 1 of the present invention, showing the regulation of Bax and Bcl-2 proteins in PANC-1 pancreatic cancer cells by mogroside VI. Referring to Fig. 1 (concentrations of the drug (from left to right, Ogmol-L1, lOgmol-L1, 30gmoTL'1, and όΟμπιοΤΕ'1)), the western blotting results showed that, mogroside VI may up-regulate Bax protein and down-regulate Bcl-2 protein in PANC-1 pancreatic cancer cells, suggesting that mogroside VI may
FI150363AU
2016204180 21 Jun 2016 increase the sensitivity of PANC-1 pancreatic cancer cells to chemotherapy drugs.
3. The inhibition of tumor cells by drugs.
PANC-1 pancreatic cancer cells in log phase were adjusted to have a concentration of 1χ 106 cells/L, and seeded into a 96-well cell culture plate (100 pL/well) and pre-cultured for 24h. According to the treatment received, the cells were divided into following groups: blank control group, mogroside only group, drug only group, and combination group. The wells were added with lOOpL of cell culture containing the agents at different concentrations. Tumor cells cultured with DMSO were used as control group. After culturing the cells for 24h, 15pL of 5mg/mL MTT solution was added to each well. After continuing to culture for 4h, the culture was stopped. The culture medium was removed. 150 pL of DMSO was added to each well and shaken on a shaking table for lOmin to dissolve crystals. The absorbance value of each well was detected at 490nm by using an ELISA reader. OD values were used to calculate inhibition rate.
The cells were divided into following groups: (1) blank control group, no agent added; (2) mogroside only group, cells treated with mogroside VI alone for 24h; (3) drug only group: cells treated with gemcitabine alone for 24h; (4) combination group, cells treated with mogroside VI (lOpmol/L) in combination with gemcitabine for 24h. Tig. 2 illustrates the results of Example 1 of the present invention, showing the inhibition of pancreatic cancer by the combination of mogroside VI and gemcitabine. Referring to Fig. 2, the results of the inhibition test on tumor cells PANC-1 showed that, the inhibitory effect of the combination group (mogroside VI + gemcitabine) is significant better than that of the drug only group (gemcitabine) (P<0.05).
4. Determination of sensitivity to drugs.
The inhibition rate of pancreatic cancer cells by the different groups was determined by using MTT method as described above. The test was repeated for 3 times. Then, the effect of the combined use of the agents was analyzed
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2016204180 21 Jun 2016 according to Kim’s formula:
Kim’s formula: Q=E(a+B)/(Ea+Eb —Εα·Εβ), in which E(A+B) represents the inhibition rate after the administration of the combination of Agent A and Agent B, EA represents the inhibition rate after the administration of Agent A alone, and EB represents the inhibition rate after the administration of Agent B alone. Q value is used to evaluate the effect of the combination of the two agents. The results were shown in Table 1.
Table E The inhibitory effect of mogroside VI in combination with gemcitabine on the proliferation of PANC-1 pancreatic cancer cells.
| Concentration of Mogroside VI (pmol/L) | Concentration of Gemcitabine (pmol/L) | Inhibition rate (%) | Q value | Synergistic effect |
| 10 | 0 | 14.21 | ||
| 0 | 0.01 | 4.33 | ||
| 0 | 0.1 | 13.12 | ||
| 0 | 1 | 29.12 | ||
| 0 | 10 | 39.66 | ||
| 0 | 50 | 57.32 | ||
| 0 | 100 | 68.22 | ||
| 10 | 0.01 | 21.31 | 1.19 | ++ |
| 10 | 0.1 | 33.25 | 1.31 | ++ |
| 10 | 1 | 53.52 | 1.36 | ++ |
| 10 | 10 | 81.67 | 1.69 | ++ |
| 10 | 50 | 88.76 | 1.4 | ++ |
| 10 | 100 | 95.61 | 1.26 | ++ |
Q = 0.85-1.15, indicates that the combined use of the two agents results in an additive effect (+);
Q = 1.15-2.0, indicates that the combined use of the two agents results in a synergistic effect with an increased effect of the drug (++);
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Q > 2, indicates that the combined use of the two agents results in a synergistic effect with a significantly increased effect of the drug (+++);
Q < 0.85, indicates an antagonistic effect (-).
The combined administration of a lower dose of mogroside VI and gemcitabine may increase the sensitivity of pancreatic cancer cells to the chemotherapy drug, and thus increase the inhibitory effect of the drug on pancreatic cancer cells. The combination group showed a more significant inhibitory effect on the proliferation of the tumor cells as compared to the drug only group. To estimate the effect of the combined use of the two agents, we tested the effect of the combination of 10 pmol/L of mogroside VI and gemcitabine at various concentrations. The results showed that, the Q value for each combination of mogroside VI and gemcitabine was greater than 1.15, indicating that the combined use of the two agents resulted in a synergistic effect and an increased effect of the drug. The combined administration of 10 pmol/L of mogroside VI and 10 pmol/L of gemcitabine resulted in an inhibition rate of PANC-1 of 81.67%, as compared to an inhibition rate of 39.66% when gemcitabine was administrated alone, and a Q value of 1.69 which indicated a significant synergistic effect. In view of above, the combined use of mogroside VI (even at a lower dose) and the chemotherapy drug may result in a synergistic effect, not only significantly inhibiting the proliferation of pancreatic cancer cells, but also effectively decreasing the dose of the chemotherapy drug, and thus resulting in an increased therapeutic effect on patients and having a practical application value.
Example 2. The sensitizing effect of mogroside V on liver cancer cell SMMC-7721.
1. The preparation of mogroside V, comprising following steps:
1) The fruit of Grosvenor momordica was smashed and weighed. To the smashed fruit, water was added at a mass ratio of the smashed fruit to water of
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1:8 and stirred at 88°C for 3 hours for extraction. The suspension was then centrifuged to collect supernatant, and the precipitate was subjected to the stirring for extraction and the centrifuging one more time. The supernatants from each centrifugation were combined to give an extract solution.
2) To the extract solution, chitosan as a flocculant was added to remove tannins and soluble proteins from the extract solution, giving a clear aqueous solution.
3) The aqueous solution was absorbed on XAD-16 resin, which was then eluted with 40% ethanol, to give a water-ethanol mixed solution rich in mogrosides.
4) The water-ethanol mixed solution was concentrated under reduced pressure, during which ethanol was recovered, to give a paste-like extract which was then weighed. To the extract, deionized water was added at an amount which was 3-folds 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 was decolorized by using Diaion PA resin. Flow through was collected to give an enriched solution. Separation by using semi-preparative liquid chromatography was then performed to yield mogroside V having a purity of greater than 98%. The semi-preparative liquid chromatography was performed under following conditions: reverse phase Cl8 column (a nonpolar chromatography column) as chromatography column, UV detection wavelength: 214nm; acetonitrile-water as mobile phase, elution with the gradient of 40% acetonitrile at 0-20 min, 50% acetonitrile at 20-40 min, and 60% acetonitrile at 40-60 min; flow rate: 1.0 mL/min; column temperature: 25°C. The eluent at a retention time of 27min was collected to provide mogroside V.
2. The effect of mogroside V on Bax and Bcl-2 in liver cancer cells.
The effect of mogroside V on the expression of Bax and Bcl-2 proteins was determined by using western blotting. SMMC-7721 cells were seeded into a 6-well plate and treated with mogroside V at different concentrations for
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24h. Then, the tumor cells were collected and lysed and the total proteins of the cells were extracted. The concentration of the proteins was measured by BCA method, using BSA as a standard. 50gg of each sample was loaded for electrophoresis separation. After the electrophoresis separation, the proteins were transfer onto PVDT membrane, which was then blocked with 5% skim milk at room temperature for lh, incubated with primary antibody at 4°C overnight, washed with TBST (3><15min). Then, a corresponding secondary antibody was added and incubated at room temperature for 45min. The excessive secondary antibodies were washed with TBST (3><15min). Subsequently, the PVDT membrane was placed on a preservative film, and covered with ECL working solution on its surface and incubated at room temperature for 5min. The expression levels of Bax, Bcl-2, and β-actin proteins were detected by sensitization and scanning.
Up-regulating the expression of Bax may increase the sensitivity of tumor cells to chemotherapy drugs. Thus, increasing the expression of Bax may effectively improve the effect of chemotherapy. Bcl-2 family is a family important for the apoptosis of cells. The down-regulation of Bcl-2 gene may facilitate the apoptosis of tumor cells.
Fig. 3 illustrates the results of the western blotting of Example 2 of the present invention, showing the regulation of Bax and Bcl-2 proteins in liver cancer cells by mogroside V. Referring to Fig. 3 (concentrations of the drug (from left to right, Ogmol-L1, lOgmol-L1, 30gmoTL'1, and όΟμπιοΤΕ'1)), the western blotting results showed that, mogroside V may up-regulate Bax protein and down-regulate Bcl-2 protein in SMMC-7721 liver cancer cells, and that increasing the concentration of mogroside V may up-regulate Bax protein and down-re gulate Bcl-2 protein in SMMC-7721 liver cancer cells, suggesting that mogroside V may increase the sensitivity of SMMC-7721 liver cancer cells to chemotherapy drugs.
3. The inhibition of SMMC-7721 tumor cells by drugs.
SMMC-7721 liver cancer cells in log phase were adjusted to have a
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2016204180 21 Jun 2016 concentration of 1χ 106 cells/L, and seeded into a 96-well cell culture plate (100 pL/well) and pre-cultured for 24h. According to the treatment received, the cells were divided into following groups: blank control group, mogroside only group, drug only group, and combination group. The wells were added with lOOpL of cell culture containing the agents at different concentrations. Tumor cells cultured with DMSO were used as control group. After culturing the cells for 24h, 15pL of 5mg/mL MTT solution was added to each well. After continuing to culture for 4h, the culture was stopped. The culture medium was removed. 150 pL of DMSO was added to each well and shaken on a shaking table for lOrnin to dissolve crystals. The absorbance value of each well was detected at 490nm by using an ELISA reader. OD values were used to calculate inhibition rate.
The cells were divided into following groups: (1) blank control group, no agent added; (2) mogroside only group, cells treated with mogroside V alone for 24h; (3) drug only group: cells treated with cisplatin alone for 24h; (4) combination group, cells treated with mogroside V (lOpmol/L) in combination with cisplatin for 24h. Tig. 4 illustrates the results of Example 2 of the present invention, showing the inhibition of liver cancer by the combination of mogroside V and cisplatin. Referring to Fig. 4, the results of the inhibition test on liver cancer cells showed that, the inhibitory effect of the combination group (mogroside V + cisplatin) is significant better than that of the drug only group (cisplatin) (P<0.05).
4. Determination of sensitivity to drugs.
The inhibition rate of SMMC-7721 cells by the different groups was determined by using MTT method as described above. The test was repeated for 3 times. Then, the effect of the combined use of the agents was analyzed according to Kim’s formula:
Kim’s formula: Q=E(a+B)/(Ea+Eb —Εα·Εβ), in which E(A+B) represents the inhibition rate after the administration of the combination of Agent A and Agent B, EA represents the inhibition rate after the administration of Agent A
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2016204180 21 Jun 2016 alone, and EB represents the inhibition rate after the administration of Agent B alone. Q value is used to evaluate the effect of the combination of the two agents. The results were shown in Table 2.
Table 2. The inhibitory effect of mogroside V in combination with cisplatin on the proliferation of SMMC-7721 liver cancer cells.
| Concentration of Mogroside V (gmol/L) | Concentration of Cisplatin (gmol/L) | Inhibition rate (%) | Q value | Synergistic effect |
| 5 | 0 | 12.33 | ||
| 0 | 1 | 1.21 | ||
| 0 | 2 | 5.4 | ||
| 0 | 4 | 13.19 | ||
| 0 | 8 | 44.28 | ||
| 0 | 16 | 86.24 | ||
| 5 | 1 | 19.57 | 1.46 | ++ |
| 5 | 2 | 25.36 | 1.97 | ++ |
| 5 | 4 | 51.79 | 2.17 | +++ |
| 5 | 8 | 78.93 | 1.54 | ++ |
| 5 | 16 | 98.26 | 1.01 | + |
Q = 0.85-1.15, indicates that the combined use of the two agents results in an additive effect (+);
Q = 1.15-2.0, indicates that the combined use of the two agents results in a synergistic effect with an increased effect of the drug (++);
Q > 2, indicates that the combined use of the two agents results in a synergistic effect with a significantly increased effect of the drug (+++);
Q < 0.85, indicates an antagonistic effect (-).
The combined administration of a low dose of mogroside V and cisplatin may increase the sensitivity of liver cancer cells to the chemotherapy drug, and
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2016204180 21 Jun 2016 thus increase the inhibitory effect of the drug on liver cancer cells. The combination group showed more a significant inhibitory effect on the proliferation of the tumor cells as compared to the drug only group. To estimate the effect of the combined use of the two agents, we tested the effect of the combination of 5 gmol/L of mogroside V and cisplatin at various concentrations. The results showed that, the Q value for each combination of mogroside V and cisplatin is greater than 1.15, indicating that the combined use of the two agents resulted in a synergistic effect and an increased effect of the drug. The combined administration of 5 gmol/L of mogroside V and 4 gmol/L of cisplatin resulted in an inhibition rate of SMMC-7721 of 51.79%, as compared to an inhibition rate of 13.19% when cisplatin was administrated alone, and a Q value of 2.17 which indicated a significant synergistic effect. In view of above, the combined use of mogroside V (even at a lower dose) and the chemotherapy drug may result in a synergistic effect, not only significantly inhibiting the proliferation of liver cancer cells, but also effectively decreasing the dose of the chemotherapy drug, and thus resulting in an increased therapeutic effect on patients and having a practical application value.
Example 3. Reversing effect of mogroside IV on the drug-resistance of leukemia.
1. The preparation of mogroside IV, comprising following steps:
1) The fruit of Grosvenor momordica was smashed and weighed. To the smashed fruit, water was added at a mass ratio of the smashed fruit to water of 1:6 and stirred at 80°C for 3 hours for extraction. The suspension was then centrifuged to collect supernatant, and the precipitate was subjected to the stirring for extraction and the centrifuging repeatedly for 3 times. The supernatants from each centrifugation were combined to give an extract solution.
2) To the extract solution, chitosan as a flocculant was added to remove tannins and soluble proteins from the extract solution, giving a clear aqueous
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2016204180 21 Jun 2016 solution.
3) The aqueous solution was absorbed on XAD-16 resin, which was then eluted with 30% ethanol, to give a water-ethanol mixed solution rich in mogrosides.
4) The water-ethanol mixed solution was concentrated under reduced pressure, during which ethanol was recovered, to give a paste-like extract which was then weighed. To the extract, deionized water was added at an amount which was 5-folds 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 was decolorized by using Diaion PA resin. Flow through was collected to give an enriched solution. Separation by using semi-preparative liquid chromatography was then performed to yield mogroside IV having a purity of greater than 98%. The semi-preparative liquid chromatography was performed under following conditions: reverse phase Cl8 column (a nonpolar column) as chromatography column, UV detection wavelength: 214nm; acetonitrile-water as mobile phase, elution with the gradient of 40% acetonitrile at 0-20 min, 50% acetonitrile at 20-40 min, and 60% acetonitrile at 40-60 min; flow rate: 1.0 mL/min; column temperature: 25°C. The eluent at a retention time of 35min was collected to provide mogroside IV.
2. The effect of mogroside IV on p-ERKl/2 in leukemia drug-resistant strain HL-60/ADR cells.
The effect of mogroside IV on p-ERKl/2 proteins in drug-resistant strain HL-60/ADR cells was determined by using western blotting. The tumor cells were seeded into a 6-well plate and treated with the agent at different concentrations for 24h. Then, the tumor cells were collected and lysed and the total proteins of the cells were extracted. The concentration of the proteins was measured by BCA method, using BSA as a standard. 50gg of each sample was loaded for electrophoresis separation. After the electrophoresis separation, the proteins were transfer onto PVDF membrane,
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2016204180 21 Jun 2016 which was then blocked with 5% skim milk at room temperature for lh, incubated with primary antibody at 4°C overnight, washed with TBST (3xl5min). Then, a corresponding secondary antibody was added and incubated at room temperature for 45min. The excessive secondary antibodies were washed with TBST (3xl5min). Subsequently, the PVDT membrane was placed on a preservative film, and covered with ECL working solution on its surface and incubated at room temperature for 5min. The expression levels of p-ERKl/2 and β-actin proteins were detected by sensitization and scanning.
It has been reported that inhibiting the overexpression of ERK signaling pathway may effectively increase the effect of chemotherapy on drug-resistant tumor cells. The effect of mogroside IV on the expression of p-ERK proteins in HL-60/ADR cells was determined by using western blotting.
Fig. 5 illustrates the results of the western blotting of Example 3 of the present invention, showing the regulation of p-ERKl/2 proteins in leukemia drug-resistant strain HL-60/ADR cells by mogroside IV. As can be seen from Fig. 5, mogroside IV may inhibit the phosphorylation of ERK1/2 proteins in HL-60/ADR cells. The expression of p-ERKl/2 proteins in HL-60/ADR cells decreased as the concentration of mogroside IV increased, suggesting that mogroside IV can inhibit ERK signaling pathway. The overexpression of ERK is closely related to the drug-resistance of tumors. Mogroside IV may increase the sensitivity of leukemia to drugs by inhibiting the phosphorylation of ERK. It has been shown by many studies that, the overexpression of ERK pathway is positively correlated with the chemotherapy drug-resistance of tumors. ERK is highly expressed in most of acute leukemia cells. By inhibiting ERK pathway, reducing the expression level of phosphorylated ERK1/2 proteins, and thus down-regulating the activity of telomerase, the sensitivity of various drug-resistant tumor cells such as leukemia and ovarian cancer to chemotherapy drugs can be increased.
3. The inhibition of tumor cells by drugs.
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Leukemia HL-60/ADR cells in log phase were adjusted to have a concentration of 1χ 106 cells/L, and seeded into a 96-well cell culture plate (100 pL/well) and pre-cultured for 24h. According to the treatment received, the cells were divided into following groups: blank control group, mogroside only group, drug only group, and combination group. The wells were added with lOOpL of cell culture containing the agents at different concentrations. Tumor cells cultured with DMSO were used as control group. After culturing the cells for 24h, 15pL of 5mg/mL MTT solution was added to each well. After continuing to culture for 4h, the culture was stopped. The culture medium was removed. 150 pL of DMSO was added to each well and shaken on a shaking table for lOrnin to dissolve crystals. The absorbance value of each well was detected at 490nm by using an ELISA reader. OD values were used to calculate inhibition rate.
The cells were divided into following groups: (1) blank control group, no agent added; (2) drug only group: cells treated with a single drug for 24h; and (3) combination group, cells treated with mogroside IV (lOpmol/L) in combination with doxorubicin (or daunorubicin) for 24h. As can be seen from the results of the effect on the drug-resistance of tumor cells (Table 3), as compared to the group without addition of mogroside IV, the combined use of a low dose of mogroside IV and a chemotherapy drug (doxorubicin or daunorubicin) increased the sensitivity of drug-resistant strain HL-60/ADR cells to the chemotherapy drugs, significantly reduced IC50 of the chemotherapy drugs (P<0.05), more significantly inhibit cell proliferation than the drug only group, and thus partially reversed the multidrug-resistance of HL-60/ADR cells. The combined use of mogroside IV and doxorubicin resulted in a reversing fold up to 5.31, and the combined use of mogroside IV and daunorubicin resulted in a reversing fold up to 2.84. As can be seen, mogroside IV, even at a lower dose, may increase the sensitivity of multidrug-resistant leukemia cells to chemotherapy drugs, and partially reverse the multidrug-resistance of HL-60/ADR cells, suggesting that
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2016204180 21 Jun 2016 mogroside IV may be used as a highly effective chemotherapy drug sensitizer and multidrug-resistance reversing agents for leukemia.
In view of above, the combination of mogroside IV and the chemotherapy drugs for acute leukemia provided in Example 3 of the present invention may be used as chemotherapy drug sensitizer and multidrug-resistance reversing agent for the treatment of acute leukemia. The combination of mogroside IV at a dose which has a low cytotoxicity (<IC20) and the chemotherapy drug doxorubicin (ADR) or daunorubicin (DNR) of Example 3 of the present invention had an inhibitory effect on the proliferation of multidrug-resistant HL-60/ADR cells. The down-regulation of p-ERK increased sensitivity to daunorubicin.
Table 3. The results of the effect of the combined administration of mogroside IV and chemotherapy drugs on the drug-resistance of HL-60/ADR cells.
| Drug | IC50 (pmol/L) | Reversing fold | |
| Drug only | Drug + Mogroside IV | ||
| Doxorubicin | 41.15 | 7.75 | 5.31 |
| Daunorubicin | 2.33 | 0.82 | 2.84 |
| Mogroside IV | 97 |
Example 4. Reversing effect of mogroside III on the drug-resistance of breast cancer drug-resistant strain MCF-7ADR.
1. The preparation of mogroside III, comprising following steps:
1) The fruit of Grosvenor momordica was smashed and weighed. To the smashed fruit, water was added at a mass ratio of the smashed fruit to water of 1:7 and stirred at 90°C for 1 hour for extraction. The suspension was then centrifuged to collect supernatant, and the precipitate was subjected to the stirring for extraction and the centrifuging repeatedly for 4 times. The supernatants from each centrifugation were combined to give an extract
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2016204180 21 Jun 2016 solution.
2) To the extract solution, chitosan as a flocculant was added to remove tannins and soluble proteins from the extract solution, giving a clear aqueous solution.
3) The aqueous solution was absorbed on XAD-16 resin, which was then eluted with 45% ethanol, to give a water-ethanol mixed solution rich in mogrosides.
4) The water-ethanol mixed solution was concentrated under reduced pressure, during which ethanol was recovered, to give a paste-like extract which was then weighed. To the extract, deionized water was added at an amount which was 6-fold 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 was decolorized by using Diaion PA resin. Flow through was collected to give an enriched solution. Separation by using semi-preparative liquid chromatography was then performed to yield mogroside III having a purity of greater than 98%. The semi-preparative liquid chromatography was performed under following conditions: reverse phase Cl8 column (a nonpolar column) as chromatography column, UV detection wavelength: 214nm; acetonitrile-water as mobile phase, elution with the gradient of 40% acetonitrile at 0-20 min, 50% acetonitrile at 20-40 min, and 60% acetonitrile at 40-60 min; flow rate: 1.0 mF/min; column temperature: 25°C. The eluent at a retention time of 41min was collected to provide mogroside III.
2. The effect of mogroside III on p-ERK in drug-resistant breast cancer MCF-7ADR cells.
The effect of mogroside III on the expression of p-ERKl/2 proteins in MCF-7ADR cells was determined by using western blotting. MCF-7ADR tumor cells were seeded into a 6-well plate and treated with mogroside III at different concentrations for 24h. Then, the tumor cells were collected and lysed and the total proteins of the cells were extracted. The concentration of
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2016204180 21 Jun 2016 the proteins was measured by BCA method, using BSA as a standard. 50gg of each sample was loaded for electrophoresis separation. After the electrophoresis separation, the proteins were transfer onto PVDF membrane, which was then blocked with 5% skim milk at room temperature for lh, incubated with primary antibody at 4°C overnight, washed with TBST (3><15min). Then, a corresponding secondary antibody was added and incubated at room temperature for 45min. The excessive secondary antibodies were washed with TBST (3xl5min). Subsequently, the PVDF membrane was placed on a preservative film, and covered with ECF working solution on its surface and incubated at room temperature for 5min. The expression levels of p-ERK and β-actin proteins were detected by sensitization and scanning.
It has been reported that inhibiting the overexpression of ERK signaling pathway may effectively increase the effect of chemotherapy on drug-resistant tumor cells. The effect of mogroside III on the expression of p-ERK proteins in MCF-7ADR cells was determined by using western blotting.
Fig. 6 illustrates the results of the western blotting of Example 4 of the present invention, showing the regulation of p-ERK proteins in breast cancer MCF-7ADR cells by mogroside III. As can be seen from Fig. 6, mogroside III may inhibit the phosphorylation of ERK in MCF-7ADR cells. The expression of p-ERKl/2 proteins in MCF-7ADR cells decreased as the concentration of mogroside III increased, suggesting that mogroside III can inhibit ERK signaling pathway. The overexpression of ERK is closely related to the drug-resistance of tumors. Mogroside III may increase the sensitivity of breast cancer cells to drugs by inhibiting the phosphorylation of ERK. It has been shown by many studies that, the overexpression of ERK pathway is positively correlated with the chemotherapy drug-resistance of tumors. ERK was highly expressed in most of acute leukemia cells. By inhibiting ERK pathway, reducing the expression level of phosphorylated ERK1/2 proteins, and thus down-regulating the activity of telomerase, the
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2016204180 21 Jun 2016 sensitivity of various drug-resistant tumor cells such as leukemia, breast cancer, and ovarian cancer to chemotherapy drugs can be increased.
3. The inhibition of tumor cells by drugs.
Breast cancer MCT-7ADR cells in log phase were adjusted to have a concentration of lx 106 cells/L, and seeded into a 96-well cell culture plate (100 pL/well) and pre-cultured for 24h. According to the treatment received, the cells were divided into following groups: blank control group, mogroside only group, drug only group, and combination group. The wells were added with lOOpL of cell culture containing the agents at different concentrations. Tumor cells cultured with DMSO were used as control group. After culturing the cells for 24h, 15pL of 5mg/mL MTT solution was added to each well. After continuing to culture for 4h, the culture was stopped. The culture medium was removed. 150 pL of DMSO was added to each well and shaken on a shaking table for lOmin to dissolve crystals. The absorbance value of each well was detected at 490nm by using an ELISA reader. OD values were used to calculate inhibition rate.
The cells were divided into following groups: (1) blank control group, no agent added; (2) drug only group: cells treated with a single drug for 24h; and (3) combination group, cells treated with mogroside III (10pmol/L) in combination with doxorubicin (or paclitaxel) for 24h. As can be seen from the results of the effect on the drug-resistance of tumor cells (Table 4), as compared to the group without addition of mogroside III, the combined use of a low dose of mogroside III and a chemotherapy drug (doxorubicin or paclitaxel) increased the sensitivity of drug-resistant strain MCT-7ADR cells to the chemotherapy drugs, significantly reduced IC50 of the chemotherapy drugs (P<0.05), more significantly inhibit the proliferation of tumor cells than the drug only group, and thus partially reversed the multidrug-resistance of MCF-7ADR cells. The combined use of mogroside III and doxorubicin resulted in a reversing fold up to 2.01, and the combined use of mogroside III and paclitaxel resulted in a reversing fold up to 1.69. As can be seen,
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2016204180 21 Jun 2016 mogroside III, even at a lower dose, may increase the sensitivity of multidrug-resistant breast cancer cells to chemotherapy drugs, and partially reverse the multidrug-resistance of MCF-7ADR cells, suggesting that mogroside III may be used as a highly effective chemotherapy drug sensitizer and multidrug-resistance reversing agents for breast cancer.
Table 4. The results of the effect of the combined administration of mogroside III and chemotherapy drugs on the drug-resistance of MCF-7ADR cells.
| Drug | IC50 (pmol/L) | Reversing fold | |
| Drug only | Drug + Mogroside III | ||
| Doxorubicin | 45.11 | 22.34 | 2.01 |
| Paclitaxel | 4.06 | 2.39 | 1.69 |
| Mogroside III | 121 |
Example 5. Reversing effect of mogroside salt A on lung cancer drug-resistant strain PC9.
Lung cancer drug-resistant strain PC9 cells in log phase were adjusted to have a concentration of lx 106 cells/L, and seeded into a 96-well cell culture plate (100 pL/well) and pre-cultured for 24h. According to the treatment received, the cells were divided into following groups: blank control group, mogroside only group, drug only group, and combination group. The wells were added with lOOpL of cell culture containing the agents at different concentrations. Tumor cells cultured with DMSO were used as control group. After culturing the cells for 24h, 15pL of 5mg/mL MTT solution was added to each well. After continuing to culture for 4h, the culture was stopped. The culture medium was removed. 150 pL of DMSO was added to each well and shaken on a shaking table for lOmin to dissolve crystals. The absorbance value of each well was detected at 490nm by using an ELISA reader. OD values were used to calculate inhibition rate.
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The cells were divided into following groups: (1) blank control group, no agent added; (2) drug only group: cells treated with a single drug for 24h; and (3) combination group, cells treated with mogroside salt A (10pmol/L) in combination with gefitinib for 24h. As can be seen from the results of the effect on the drug-resistance of tumor cells (Table 5), as compared to the group without addition of mogroside salt A, the combined use of a lower dose of mogroside salt A and the chemotherapy drug gefitinib increased the sensitivity of drug-resistant strain PC9 cells to the chemotherapy drugs, significantly reduced IC50 of the chemotherapy drug (P<0.05), more significantly inhibit the proliferation of tumor cells than the drug only group, and thus partially reversed the multidrug-resistance of PC9 cells. The combined use of mogroside salt A and gefitinib result in a reversing fold up to 2.12. As can be seen, mogroside salt A, even at a lower dose, may increase the sensitivity of drug-resistant lung cancer cells to chemotherapy drugs, and partially reverse the multidrug-resistance of drug-resistant strain PC9 cells, suggesting that mogroside salt A may be used as a highly effective chemotherapy drug sensitizer and multidrug-resistance reversing agents for lung cancer.
Table 5. The results of the effect of the combined administration of mogroside salt A and a chemotherapy drug on the drug-resistance of PC9 cells.
| Drug | IC50 (pmol/L) | Drug + Mogroside | |
| Drug only | Drug + Mogroside salt A | ||
| gefitinib | 7.11 | 3.34 | 2.12 |
| mogroside salt A | 96.33 |
In view of above, the mogrosides and the salts thereof of the present invention, as chemotherapy drug sensitizer and multidrug-resistance reversing agent, can be used in combination with chemotherapy drugs. The chemotherapy drugs which can be used are not limited to those disclosed in Examples, and the tumor cells which can be used are not limited to those
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2016204180 21 Jun 2016 disclosed in Examples. The mogrosides and the salts thereof of the present invention may act as sensitizer and drug-resistance reversing agent in, e.g., lung cancer, blood cancer, colon adenocarcinoma, lymphoma, laryngeal cancer, melanoma, colorectal cancer, skin cancer, renal cancer, and cervical cancer. Further, the mogrosides and the salts thereof of the present invention are not limited to the five ones disclosed in Examples. Other mogrosides and mogroside salts having the general formula of the mogrosides and the salts thereof of the present invention may have a similar effect.
The above examples only are preferred Examples of the present invention, and are not intended to limit the present invention. As will be appreciated by those skilled in the art, various modification and changes may be made to the present invention. Any modification, equivalent, and improvement within the spirits and principles of the present invention shall fall into the protection scope of the present invention.
2016204180 11 Dec 2017
Claims (5)
1. Use of the mogrosides and the salts thereof in the manufacture of a chemotherapy synergist for the treatment of tumors, wherein the mogrosides and the salts thereof are represented by the wherein, R is a H atom, metal ion, or n molecules of glucose, and Ri is a metal ion or n molecules of glucose, each n > 1, and wherein the mogrosides and the salts thereof are able to up-regulate the expression of Bax and down-regulate the expression of Bcl-2, or down-regulate the expression of p-ERK.
2. The use according to claim 1, wherein the mogrosides are represented by the following formulae:
mogroside VI
2016204180 11 Dec 2017 mogroside IV mogroside III.
3. The use according to claim 1 or 2, characterized in that, the chemotherapy synergist includes chemotherapy sensitizer and drug-resistance reversing agent.
4. The use according to claim 1 or 2, characterized in that, the
2016204180 11 Dec 2017 mogrosides and the salts thereof are able to regulate the expression of genes related to sensitivity of drugs and drug-resistance in tumor cells.
5. The use according to claim 1 or 2, characterized in that, the tumor is selected from leukemia, liver cancer, lung cancer, blood cancer, colon adenocarcinoma, lymphoma, breast cancer, pancreatic cancer, laryngeal cancer melanoma or cervical cancer.
2016204180 21 Jun 2016
Fig. 1 *H ©
SO
SO
SO
...^Gemcitabine 'WGemcitabine —Mogroside VI
Fig. 2
Drug Concentration (gmol/L)
Fig. 3
2016204180 21 Jun 2016
Fig. 4
P-ERK 1/2 {Thr202/Tyr204} ERK1/2 β-actin
Fig. 5
P-ERK 1/2 (Thr202/Tyr204) ERIC 1/2 β-actm
Fig. 6
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| CN201510497883.6 | 2015-08-13 | ||
| CN201510497883.6A CN105399794B (en) | 2015-08-13 | 2015-08-13 | Fructus momordicae triterpene saponin and salt thereof, preparation method and applications of fructus momordicae triterpene saponin and salt thereof, and pharmaceutical composition containing fructus momordicae triterpene saponin and salt thereof |
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| CN106176890A (en) * | 2016-06-30 | 2016-12-07 | 潜山县有余瓜蒌开发有限责任公司 | A kind of method extracting triterpene saponin from Fructus Trichosanthis seed |
| CN106432393A (en) * | 2016-09-23 | 2017-02-22 | 桂林电子科技大学 | Method of extracting and detecting mogrosides IV and V from momordica grosvenori of 55 days in fruiting age |
| CN106963801A (en) * | 2017-03-29 | 2017-07-21 | 桂林实力科技有限公司 | The preparation method of Fructus Monordicae extract |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101837001A (en) * | 2009-03-20 | 2010-09-22 | 帕特里克·T·普伦德加斯特 | Composition for treating infection and method |
| CN104042620A (en) * | 2013-03-12 | 2014-09-17 | 复旦大学 | Application of mogrol in preparation of tumor multi-medicine tolerant reversal agent |
| WO2016107471A1 (en) * | 2014-12-30 | 2016-07-07 | 北京农学院 | Stat3 and erk signal pathway inhibitor and use thereof |
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| CN104208073A (en) * | 2013-06-01 | 2014-12-17 | 复旦大学 | Application of protopanaxadiol to prepare tumor multidrug resistance reversers |
| CN104208074B (en) * | 2013-06-01 | 2017-10-10 | 复旦大学 | Purposes of the 2 α hydroxyl protopanoxadiols in tumor multi-drug resistance reversal agents are prepared |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101837001A (en) * | 2009-03-20 | 2010-09-22 | 帕特里克·T·普伦德加斯特 | Composition for treating infection and method |
| CN104042620A (en) * | 2013-03-12 | 2014-09-17 | 复旦大学 | Application of mogrol in preparation of tumor multi-medicine tolerant reversal agent |
| WO2016107471A1 (en) * | 2014-12-30 | 2016-07-07 | 北京农学院 | Stat3 and erk signal pathway inhibitor and use thereof |
Non-Patent Citations (3)
| Title |
|---|
| LIU, Can, et al., Chinese Pharmacological Bulletin, 2015 Sep, vol. 31(9), pages 1310-1314 (EPub 2015-8-10 at 14:37) * |
| TAKASAKI, M., et al., Cancer Letters, 2003, vol. 198, pages 37-42 * |
| ZHANG, H., et al., Journal of Food Composition and Analysis, 2012, vol. 25, pages 142–148 * |
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| AU2016204180A1 (en) | 2017-03-02 |
| CN105399794B (en) | 2017-04-12 |
| CN105399794A (en) | 2016-03-16 |
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