AU2021310228A1

AU2021310228A1 - Pharmaceutical composition and use thereof

Info

Publication number: AU2021310228A1
Application number: AU2021310228A
Authority: AU
Inventors: Zhiguang DUAN; Daidi Fan; Jianya YAN; Shiyu YIN
Original assignee: Shaanxi Giant Biotechnology Co Ltd
Current assignee: Shaanxi Giant Biotechnology Co Ltd
Priority date: 2020-07-17
Filing date: 2021-07-15
Publication date: 2023-03-02
Anticipated expiration: 2041-07-15
Also published as: CN111759853B; AU2021310228B2; CN111759853A; WO2022012635A9; WO2022012635A1

Abstract

A pharmaceutical composition. The pharmaceutical composition comprises a ginsenoside monomer and bicyclol. The pharmaceutical composition has significant efficacy in chemoprevention and treatment of liver cancer, and has a significant synergistic effect compared with using a ginsenoside monomer alone or using bicyclol alone. Also provided are use of a ginsenoside monomer and bicyclol in preparation of a pharmaceutical composition for preventing and/or treating a tumor, and use of a ginsenoside monomer in promoting the effect of a pharmaceutical composition for preventing and/or treating a tumor.

Description

PHARMACEUTICAL COMPOSITION AND USE THEREOF FIELD OF THE INVENTION

The present application belongs to the field of pharmaceutical technology, and specifically, the

present application relates to a pharmaceutical composition and use thereof.

BACKGROUND OF THE INVENTION

Primary liver cancer, is one of the most common malignant tumors worldwide. The National

Cancer Center estimates that in 2015, there will be 466,000 liver cancer cases and 422,000 liver

cancer deaths in China, and liver cancer is currently the fourth most common malignant tumor and

the third cause of death in China, seriously threatening the lives and health of our people.

Patients with early stage liver cancer usually have no obvious symptoms, and , most patients are

already in the intermediate or advanced stage of liver cancer when they are diagnosed, and the

average survival time of untreated primary liver cancer after diagnosis is less than 4 months.

Currently, the best treatment methods for liver cancer are surgical resection and liver transplantation.

However, in the case of liver transplantation, it is difficult to find a donor; in the case of surgical

resection, only 20% of all patients can undergo surgery, so it is not suitable to be used as a treatment

for all liver cancer patients, and the recurrence rate of progressive liver cancer is high after either

surgical resection or orthotopic liver transplantation.

Other treatment methods include systemic anti-cancer therapy, hepatic artery chemical injection

therapy, hepatic artery chemoembolization and radiation therapy, etc., but these treatment methods

kill liver cancer cells while killing normal cells, which have fatal side effects. In addition, it has been

reported that sorafenib, a representative liver cancer target drug, has side effects such as shrinking

of the pancreas and drug resistance problems when taken for a long time. Multidrug resistance (MDR)

is a major obstacle to tumor treatment and its mechanism is relatively complex. Studies have found

that tumor cells have varying degrees of drug resistance to almost all individual drugs, but the

combination of multiple drugs can act on multiple target sites or signaling pathways of cancer cells at

the same time, which can effectively overcome the shortcomings of single drug resistance and

provide a new approach and hope for tumor prevention and/or treatment. It will also become the future trend of tumor prevention and/or treatment. Ginsenoside monomers are the main active ingredients of ginseng, American ginseng, pseudo-ginseng and other Chinese herbs. There are more than 30 ginsenoside monomers isolated from ginseng, and their functions such as antitumor activity, immune enhancing activity, hypoglycemia and memory improvement are becoming hot spots for development. Ginsenoside CK is a metabolite of natural diol-type ginsenoside monomers in the human intestine and is the entity that ginseng exerts pharmacological activity of ginseng in vivo. The ginsenoside CK is used for the treatment of rheumatoid arthritis and has entered the clinical trial stages. Antitumor-related studies have shown that the ginsenoside CK can significantly inhibit the proliferation of colon cancer HCT-116 cells, SW-480 cells and HT-29 cells in vitro, as well as the proliferation and invasion of liver cancer MHCC97-H cells, and can inhibit the growth of colon and liver cancers, etc. and hepatic metastasis in mice in vivo; the ginsenoside CK is associated with PI3K/Akt signaling pathway and promotes apoptosis of breast cancer cell MCF-7; the ginsenoside CK upregulates the expression of p53/p2l, Fox03a-p27/p15 and Smad3, downregulating the expression of cdc25A, CDK4/6 and cyclinD1/3, inducing the apoptosis and GIphase arrest in HCT-116 cells; the ginsenoside CK affects the interaction and cell nuclear colocalization of Annexin A2 protein and NF-KB p50 subunit in liver cancer HepG2 cells, inhibiting the transcriptional activation of NF-KB and the expression of downstream anti-apoptotic genes X-IAP, c-IAP1, c-IAP2 and Survivin, promoting the activation of Caspase 9 and the occurrence of apoptosis in HepG2 cells, and suppressing the downstream MMP-2/9 expression levels, thereby inhibiting tumor cell metastasis. Bicyclol (trade name: Bicyclol) is the first anti-hepatitis new drug with independent intellectual property rights in China. Clinical trial results have shown that it can significantly reduce the serum and levels of chronic hepatitis B and C patients, and can make some patients' hepatitis B virus indexes turn negative. The results of pharmacological studies show that bicyclol has the effects of anti- liver damage caused by chemical poisons, anti-liver fibrosis, and inhibition of apoptosis of hepatocytes caused by immune damage. According to literatures, Bicyclol can significantly inhibit the damage to the cell genome caused by chemical mutagens and restore the inhibition of gap junction intercellular communication function in hepatic epithelial cells caused by carcinogenic agents. The relationship between gap junction intercellular communication (GJIC) function and tumorigenesis is gaining more and more attention, and a variety of evidence shows that GJIC function has a close relationship with the occurrence and development of tumors, suggesting that it may have a certain blocking effect on the occurrence and development of tumors. Bicyclol inhibits the malignant transformation of rat liver epithelial WB-F344 cells co-induced by 3MC/TPA in vitro and prevents the occurrence of liver cancer. Bicyclol has significant inhibitory effects on the adhesion of highly metastatic human liver cancer cell line MHCC97-H to LN and FN, reducing

VEGF mRNA expression in MHCC97-H cells, and inhibiting angiogenesis, thereby inhibiting tumor

invasion and metastasis, suggesting that it may have certain potential value in the aspect of

preventing and inhibiting the occurrence and development of liver cancer.

Patent Document 1 discloses a liver cancer targeted ginsenoside CK chitosan polymeric micelle

delivery system, characterized by comprising: a targeting headgroup, a basic carrier, and an

anticancer drug; wherein the targeting headgroup is an A54 polypeptide, the basic carrier is an

amphiphilic polymer, and the anticancer drug is ginsenoside CK; the targeting headgroup is

connected to the hydrophobic end of the basic carrier via a linker group to form a drug delivery

carrier; the drug delivery carrier is encapsulated with ginsenoside CK, forming a liver cancer targeted

ginsenoside CK chitosan polymeric micelle delivery system.

Patent Document 2 discloses a composition comprising the following components: ginsenoside

Rkl, ginsenoside Rg5, ginsenoside CK, total ginsenoside extract, wherein the composition is an

interleukin (IL)-6 modulator.

Patent Document 3 discloses the use of bicyclol and their pharmaceutically acceptable

derivatives for the preparation of drugs for preventing or treating drug-induced liver injury.

The prior art either uses ginsenoside monomers or bicyclol, however, their effectiveness in

preventing and treating tumors is limited, and the prior art does not disclose that ginsenoside

monomers can promote the effect of pharmaceutical compositions for preventing and/or treating

tumors, nor does it disclose the combined use of ginsenoside monomers and bicyclol to synergize

and improve the effectiveness of preventing and/or treating tumors.

Prior art documents

Patent document 1 CN109793711A disclosure

Patent document 2 CN110090236A disclosure

Patent document 3 CN109106711A disclosure

SUMMARY OF THE INVENTION

In order to solve the problem of limited effectiveness of ginsenoside monomers used alone or

bicyclol used alone in the preventing and treating tumors in the prior art, the present application

provides a synergistic anti-tumor drug composition which can be safer and more potent in the

preventing and treating tumors, especially liver cancer.

The specific technical solutions of the present application are as follows:

1. A pharmaceutical composition, wherein the pharmaceutical composition comprises:

ginsenoside monomers and bicyclol.

2. The pharmaceutical composition according to item 1, wherein the weight ratio of the

ginsenoside monomers and the bicyclol is 1:0.3 to 3.

3. The pharmaceutical composition according to item 2, wherein the weight ratio of the

ginsenoside monomers and the bicyclol is 1:1.

4. The pharmaceutical composition according to any one of items 1-3, wherein the ginsenoside

monomers are one or two or more selected from: ginsenoside CK, ginsenoside 20-(S, R)-Rg3,

ginsenoside 20-(S, R)-Rh2, ginsenoside 20-(S, R)-PPD, ginsenoside Rkl, ginsenoside Rg5,

ginsenoside Rk3 or ginsenoside Rh4;

preferably, the ginsenoside monomers comprise ginsenoside CK.

5. Use of ginsenoside monomers and bicyclol in the preparation of pharmaceutical composition

for preventing and/or treating tumors.

6. The use according to item 5, wherein the weight ratio of the ginsenoside monomers and

bicyclol is 1:0.3 to 3.

7. The use according to item 6, wherein the weight ratio of the ginsenoside monomers and

bicyclol is 1:1.

8. The use according to any one of items 5-7, wherein the ginsenoside monomers are one or two

or more selected from: ginsenoside CK, ginsenoside 20-(S, R)-Rg3, ginsenoside 20-(S, R)-Rh2,

ginsenoside 20-(S, R)-PPD, ginsenoside Rkl, ginsenoside Rg5, ginsenoside Rk3, and ginsenoside

Rh4;

preferably, the ginsenoside monomers comprise ginsenoside CK.

9. The use according to any one of items 5-8, wherein the tumor is liver cancer.

10. Use of ginsenoside monomers in promoting the effect of a pharmaceutical composition for preventing and/or treating tumors.

11. The use according to claim 10, wherein the ginsenoside monomers are one or two or more

selected from: ginsenoside CK, ginsenoside 20-(S, R)-Rg3, ginsenoside 20-(S, R)-Rh2, ginsenoside

-(S, R)-PPD, ginsenoside Rk1, ginsenoside Rg5, ginsenoside Rk3 or ginsenoside Rh4;

preferably, the ginsenoside monomers comprise ginsenoside CK.

12. The use according to claim 10 or 11, wherein the pharmaceutical composition for preventing

and/or treating tumors comprises bicyclol.

13. The use according to any one of claims 10-12, wherein the effect is a synergistic effect.

Effect of the application

The pharmaceutical composition of the present application comprising ginsenoside monomers

and bicyclol, in particular the pharmaceutical composition comprising ginsenoside CK and bicyclol,

is capable of inhibiting the proliferation of tumor cells, in particular liver cancer cells; and inhibiting

the malignant transformation of rat liver epithelial-like stem cells to liver cancer cells induced by

chemical mutagens; has a significant inhibitory effect on the growth of liver cancer in nude mice;

and is capable of preventing carcinogen-induced liver cancer. The pharmaceutical composition

conprising ginsenoside CK and bicyclol has significant synergistic and beneficiate effects compared

with single drug thereof, and is a kind of highly effective and low-toxic antitumor pharmaceutical

composition. Molecular biology studies related to this application also suggest that this kind of

pharmaceutical composition has a molecular mechanism and theoretical basis for the significant

synergistic effect. Bicyclol can restore the inhibition of gap junction intercellular communication

(GJIC) function in hepatic epithelial cells induced by chemical carcinogens, protect cellular genomic

DNA, and inhibit the malignant transformation of hepatic epithelial cells and the development of

liver tumors induced by chemical carcinogens. Ginsenoside CK is related to several signaling

pathways including PI3K/Akt signaling pathway, NF-KB signaling pathway and cell cycle regulation

signaling pathway, and can inhibit tumor cell growth and metastasis. Ginsenoside CK and bicyclol

act on different stages of hepatocarcinogenesis, different targets and signaling pathways, respectively,

resulting in stronger efficacy in the prevention and/or treatment of liver cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 (a)-(b) show the cell morphology of WB-F344 after 3MC/TPA co-induction in the

blank control group and the model group, respectively.

Fig. 2 (a)-(e) show the H&E staining pathological observations of the livers of SD rats for liver

cancer prevention experiment in the blank control group, model group, ginsenoside CK group,

bicyclol group and ginsenoside CK/bicyclol combination group, respectively.

DETAILED DESCRIPTION OF THE INVENTION

The following is a detailed description of this application.

It should be noted that the term "comprise" or "include" throughout the specification and claims

is referred to an open-ended term and should be interpreted as "include but are not limited to". The

specification subsequently description is preferred embodiments to implement this application, but

the description is intended to be a general principle of the specification and is not intended to limit

the scope of this application. The scope of protection of this application shall be as defined in the

appended claims.

The present application provides a pharmaceutical composition wherein the pharmaceutical

composition comprises: ginsenoside monomers and bicyclol.

The ginsenoside monomers of this application may be commercially available ginsenoside

monomers, or ginsenoside monomers isolated from ginseng cultivated or collected in nature, or other

ginsenoside monomers converted from isolated ginsenoside monomers, or ginsenoside monomers

synthesized by synthetic methods, however, any ginsenoside monomers demonstrating the preventive

or therapeutic effects of liver cancer of this application may be used without limitation. The

ginsenoside monomers can be one or two or more selected from: ginsenoside CK, ginsenoside 20-(S,

R)-Rg3, ginsenoside 20-(S, R)-Rh2, ginsenoside 20-(S, R)-PPD, ginsenoside Rkl, ginsenoside Rg5,

ginsenoside Rk3, ginsenoside Rh4, and other monomers, preferably comprise ginsenoside CK.

"Ginsenoside CK", with the chemical name 20-0-p-D-glucopyranosyl-20-protopanaxadiol

(ginsenoside CK), molecular formula: C36H620, molecular weight: 622.87, odorless, white powder.

It is soluble in methanol, ethanol, slightly soluble in ethyl acetate, poorly soluble in water, insoluble

in trichloromethane and ether, CAS No. 39262-14-1. It is one of the ginsenosides serving as a main

active ingredient of ginsengs such as panax ginseng or red ginseng, referring to the ginsenoside monomers belonging to the protopanaxadiol (PPT) class. The ginsenoside CK used for the experiments in this application is a highly active and rare ginsenoside CK with a purity of 98% or more obtained from the common ginsenoside monomer extracted from panax ginseng by directed biotransformation technology and the following isolation and purification.The structural formula of ginsenoside CK is.

H HH

HH H Ha

HOO

"Ginsenoside 20-(S, R)-Rg3", molecular formula: C 42 H 70 0 12 , molecular weight 767.0, off-white

powder, soluble in methanol, CAS No.: 14197-60-5, the structural formula is:

HO HQH H OH OH OH

"Ginsenoside 20-(S, R)-Rh2", molecular formula: C 3 6H 62 0 8, molecular weight 622.87, colorless

needle crystal, soluble in methanol, CAS No.: 78214-33-2, the structural formula is:

-6HH "00

"Ginsenoside 20-(S, R)-PPD", molecular formula: C3 0 H 52 0 3 , molecular weight 460.74, white

crystal, soluble in acetone, DMSO and other solvents. CAS No.: 30636-90-9, the structural formula is:

O

"Ginsenoside Rkl", molecular formula: C4 2 H7 0 0 12 , molecular weight 767.0, odorless, white

powder, soluble in methanol and ethanol, slightly soluble in ethyl acetate, poorly soluble in water,

insoluble in trichloromethane and ether, CAS No.: 494753-69-4, the structural formula is:

soH HO O

0

OO HO L °

"Ginsenoside Rg5", molecular formula: C 4 2 H 7 0 0 12 , molecular weight: 767.0, odorless, white

insoluble in trichloromethane and ether, CAS No.: 74964-14-0, the structural formula is:

HO HO O HO H H HO HO

"Ginsenoside Rk3", molecular formula: C36H600, molecular weight: 620.9, odorless, white

powder, soluble in water, methanol and ethanol, CAS No.: 364779-15-7, the structural formula is:

OHH HH HO .

H OH 'O HO OH OH "Ginsenoside Rh4", molecular formula: C 3 6 H 6 0 0; molecular weight: 620.4; scientific name:

6-0-p-D-glucopyranosyl-20(-HO)-trans-protopanaxatriol; odorless, white powder, soluble in

methanol and ethanol, slightly soluble in ethyl acetate, poorly soluble in water, insoluble in

trichloromethane and ether; CAS No.: 174721-08-5; the structural formula is:

HO 20

3I OH O-GIc

"Bicyclol", whose chemical name is

4,4'-dimethoxy-5,6,5',6'-bis(methylenedioxy)-2-hydroxymethyl-2'-methoxycarbonybiphenyl, with

the molecular formula C 19H 8 0 9, can be clinically used to treat aminotransferase elevation caused by

chronic hepatitis. Bicyclol is a structural analogue of bifendatatum, having effects on both

anti-hepatitis virus and anti-hepatocyte damage.

The pharmaceutical composition of the present application comprises effective doses of

ginsenoside monomers and bicyclol as active ingredients. The effective dose is the dose to be used

when the pharmaceutical composition exerts its pharmacological function.

In a specific embodiment, the ginsenoside monomers and bicyclol are combined in the ratio range limited by the present application, exhibiting synergistic effects in terms of inhibition of proliferation of liver cancer cells in vitro, inhibition of malignant transformation of liver cancer precursor cells in vitro, treatment of liver cancer in vivo, and prevention of liver cancer in rats. The inventors also found that ginsenoside monomers and bicyclol were also low in toxicity within the above-mentioned ratio ranges. Considering the significant synergistic effect, the weight ratio of ginsenoside monomers and bicyclol in the pharmaceutical composition is preferably 1:0.3 to 3, specifically 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5 , 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, preferably 1:1. In a specific embodiment, in the process of the pharmaceutical composition used in vitro to inhibit the proliferation of cancer cells, or in vitro to inhibit the malignant transformation of normal cells to cancer cells, the concentration of ginsenoside monomers can be 3 to 30pM, specifically 3pM, 4gM, 5gM, 6gM, 7gM, 8gM, 9M, 10gM, 11gM, 12gM, 13gM, 14p M, 15M, 16M, 17gM, 18gM, 19gM, 20gM, 21gM, 22gM, 23gM, 24gM, 25gM, 26gM, 27M, 28M, 29M, gM, preferably 5M to 20gM; and the concentration of bicyclol can be 1 to 20M, specifically 1gM, 2gM, 3gM, 4gM, 5g M, 6M, 7M, 8M, 9M, 10M, 11gM, 12M, 13M, 14M, 15M, 16gM, 17gM, 18gM, 19M, 20M, preferably 3gM to 15gM. In a specific embodiment, the pharmaceutical composition of the present application, can be administered orally or non-orally. The administration amount varies depending on the degree of symptoms, the patient's age, gender, body weight, differences in sensitivity, the method of administration, the period of administration, the interval of administration, the nature of the pharmaceutical formulation, the type of active ingredient, etc. There are no special limitations, but usually 10mg~1.5g, preferably 50mg900mg, more preferably 100mg600mg, more preferably 300mg-500mg (by total weight of ginsenoside CK) per day for adults (body weight 60kg), and the above administration amount can usually be administered by dividing into 1 to 3 times daily. In a specific embodiment, the ginsenoside monomers in the pharmaceutical composition of the present application are one or two or three or four or more than five selected from: ginsenoside CK, ginsenoside 20-(S, R)-Rg3, ginsenoside 20-(S, R)-Rh2, ginsenoside 20-(S, R)-PPD, ginsenoside Rkl, ginsenoside Rg5, ginsenoside Rk3, and ginsenoside Rh4. In a specific embodiment, in the pharmaceutical composition of the present application, the ginsenoside monomers comprise ginsenoside CK. In addition to ginsenoside CK, it may comprise one or two or more other ginsenoside monomers: ginsenoside 20-(S, R)-Rg3, ginsenoside 20-(S,

R)-Rh2, ginsenoside 20-(S, R)-PPD, ginsenoside Rkl, ginsenoside Rg5, ginsenoside Rk3, and

ginsenoside Rh4, or may not comprise other ginsenoside monomers. In the case that the

pharmaceutical composition of the present application comprises other ginsenoside monomers, and

from the viewpoint of better exerting synergistic effect, based on the total weight of total ginsenoside

monomers comprised in the pharmaceutical composition being 100 parts by weight, the content of

the ginsenoside CK is preferably 50 parts by weight or more, more preferably 60 parts by weight or

more, more preferably 70 parts by weight or more, more preferably 80 parts by weight or more, more

preferably 90 parts by weight or more, more preferably 100Parts by weight (i.e., the ginsenoside

monomer comprised in the pharmaceutical composition is only ginsenoside CK). The purity of the

ginsenoside monomers used in the pharmaceutical compositions of the present application can be

98% or more.

In a specific embodiment, the pharmaceutical composition of the present application may

comprise a pharmaceutically acceptable carrier, such as an excipient. There are no special restrictions

on the excipient in the pharmaceutical composition of the present application, for example, the

excipient commonly used in medicines or health products in the art can be used. Specifically, the

excipient is starch, dextrin, lactose, mannitol, sodium hypromellose, xanthan gum, protein sugar and

the like. In a specific embodiment, there are no special restrictions on the dosage forms of the

pharmaceutical composition of the present application, for example, they may be oral dosage forms

or injection dosage forms. The oral dosage forms can be liquid dosage forms or solid dosage forms.

The oral dosage forms can be, for example, hard capsules, soft capsules, sustained and controlled

release capsules, compressed tablets, sugar-coated tablets, powders, granules, dropping pills,

water-honeyed pills, syrups or oral liquids; the injection dosage forms can be, for example, solution

forms, suspension forms, emulsion forms or lyophilized powders. The administration mode of the

pharmaceutical composition for improving sleep can be, for example, oral administration, drip or

injection.

In a specific embodiment, when preparing solid preparations for oral administration, after

adding excipients and optionally binders, disintegrating agents, lubricating agents, coloring agents, flavoring agents and the like to the main drugs, it can be prepared into tablets, coated tablets, granules, fine granules, powders, capsules, and the like according to a conventional method. In the specific embodiments above, as excipients, for example, lactose, corn starch, white sugar, glucose, sorbitol, crystalline cellulose, silicon dioxide and the like can be used; as binders, for example, polyvinyl alcohol, ethyl cellulose, methyl cellulose, gum arabic, hydroxypropyl cellulose, hydroxypropyl methyl cellulose and the like can be used; as disintegrant agents, dry starch, sodium carboxymethyl starch, polyvinyl pyrrolidone, cross-linked polymer of sodium carboxymethyl cellulose and the like can be used; as lubricating agents, for example, magnesium stearate, talc, silicon dioxide, and the like can be used; as coloring agents, those allowed to be added to medicines can be used; as flavoring agents, cocoa powder, menthol, aromatic acid, peppermint oil, borneol, and cinnamon powder can be used. Of course, the above-mentioned tablets and granules can also be coated with a sugar coating, a gelatin coating, and other necessary outer coatings. In a specific embodiment, when preparing injections, pH regulators, buffers, suspending agents, solubilizers, stabilizers, isotonic agents, preservatives and the like can be added to the main drug as required, and then intravenous, subcutaneous, and intramuscular injections can be prepared according to conventional methods. At this time, if necessary, a freeze-drying product can be prepared with a conventional method. In the specific embodiments above, examples of the suspending agents include methyl cellulose, Tween 80, hydroxyethyl cellulose, gum arabic, tragacanth gum powder, sodium carboxymethyl cellulose, polyoxyethylene sorbitan monolaurate, and the like. Examples of the solubilizers include polyoxyethylene hydrogenated castor oil, Tween 80, niacinamide, polyoxyethylene sorbitan monolaurate, polyethylene glycol, castor oil fatty acid ethyl ester, and the like. Examples of the stabilizers include sodium sulfite, sodium metasulfite, and the like; examples of the preservatives include methylparaben, ethylparaben, sorbic acid, phenol, cresol, chlorocresol, and the like. The pharmaceutical composition of the present application can be administered to a subject to prevent and/or treat liver cancer. The subject may be a mammal, such as a human, a rat, a rabbit, a sheep, a pig, a cow, a cat, a dog, a monkey, etc., and preferably a human. On the other hand, the inventors have found that the pharmaceutical composition of the present application has a significant effect in the prevention and/or treatment of tumors and in particular in the prevention and/or treatment of liver cancer. Therefore, the present application also provides the application of the pharmaceutical composition of the present application in the preparation of pharmaceutical compositions for preventing and/or treating tumors. Further, the tumor is preferably liver cancer.

The present application also provides the use of ginsenoside monomers in promoting the effect

of pharmaceutical compositions for preventing and/or treating tumors.

The pharmaceutical composition of the present application exhibits synergistic, even significant

synergistic effects in terms of inhibition of proliferation of liver cancer cells in vitro, inhibition of

malignant transformation of liver cancer precursor cells in vitro, treatment of liver cancer in vivo, and

prevention of liver cancer. Specifically, the interaction index CI value of ginsenoside monomers and

bicyclol is less than 1, preferably less than or equal to 0.7. Further, ginsenoside monomers and

bicyclol are combined in the ratio defined in this application so that the pharmaceutical composition

with superior effects in the prevention and/or treatment of liver cancer can be obtained.

EXAMPLE

The content of the present application is further described below by examples. If not specifically

indicated, the technical means used in the embodiments are conventional means known to those

skilled in the art and commercially available and commonly used instruments.

Example 1 Inhibition of proliferation of liver cancer HepG2 cells in vitro by the combination of ginsenoside CK and bicyclol

In this example, MTT was used to detect the inhibitory effect of ginsenoside CK on the

proliferation of liver cancer HepG2 cells. The chemical name of "MTT" is

3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide, which is a yellow dye. "The MTT

method", also known as the "MTT colorimetric method", is a method for detecting cell survival and

growth. The principle of the assay is that succinate dehydrogenase in the mitochondria of living cells

can reduce exogenous MTT to water-insoluble blue-purple crystalline formazan and deposit in the

cells, while dead cells do not have this function. Dimethyl sulfoxide (DMSO) can dissolve the

formazan in the cells, and its absorbance value was measured at 490 nm with an microplate reader.

Within a certain cell number range, the amount of MTT crystals formed was proportional to the

number of cells. According to the measured absorbance value (OD value), the number of living cells is judged, and the larger the OD value, the stronger the cell activity (if the drug toxicity is measured, the less toxic the drug is indicated). This method has been widely used for the activity assay of some bioactive factors, large-scale anti-tumor drug screening, cytotoxicity test and tumor radiosensitivity determination. It is characterized by high sensitivity and economy.

First, HepG2 cells in good growth state with cell density of 80% or more were taken, digested

by adding trypsin, and the cell density was adjusted to 1x105 cells/mL, added to a 96-well plate with

a volume of 100 pL per well, and incubated at 37°C in a 5% incubator. 24 h later, the supernatant

was discarded, and the ginsenoside CK group, bicyclol group and the ginsenoside CK/bicyclol

combination group were set up, 200 pL of different concentrations of ginsenoside CK, bicyclol and

ginsenoside CK/Bicyclol were added to each dosing group as shown in Table 1. Correspondingly,

200pL of 1640 culture medium was added to the blank control group and 6 replicate wells were set

for each concentration. 48h later, 150pL of a mixture of MTT and cell culture medium with a

corresponding volume ratio of 1:2 was added to each well and the incubation was continued for 4h.

The medium was carefully removed and 150pL of DMSO was added and slowly shaken for 10min to

dissolve the generated blue-purple crystalline Formazan. Finally, the absorbance OD value

corresponding to each well was measured at 490 nm and the following formula was used to calculate

the inhibition ratio of cells: inhibition ratio of cells (%)= (1 - average OD value of the dosing group/

average OD of the control group) x 100%.

The interaction index CI of the two monomers was used to determine the effect of the

combination, CI = AB/(AxB), T is the OD value of the cells in the dosing group, C is the OD value

of the cells in the control group, AB is the T/C value of the combination group of the two monomers ,

A and B are the T/C values of the single drug acting groups. CI <1 indicates a synergistic effect of

the two monomers, and CI < 0.7 indicates a very significant synergistic effect.

The experimental data were expressed as x s and analyzed by SPSS17.0 statistical software.

Pairwise comparison between groups was performed by LSD method. p < 0.05 indicates significant

difference, andp < 0.01 indicates extremely significant difference.

The results of the inhibitory effects of ginsenoside CK and bicyclol on the proliferation of liver

cancer HepG2 cells in vitro measured by MTT method are shown in Table 1. Ginsenoside CK alone

with different concentrations and bicyclol alone with different concentrations could show certain

degrees of inhibition of tumor cell growth with a concentration-dependent effect. Compared with the single drugs, the combination of ginsenoside CK and bicyclol has a stronger inhibitory effect on

HepG2. Compared with the single drugs, the combination of the two monomers has a significant

difference (P < 0.05), and the CI index also shows that the combination of the two monomers has a

significant synergistic effect. Moreover, Under the condition that the total concentration of

ginsenoside CK and bicyclol is fixed, the CI index is low when the ratio of the two is within the

range of 1:0.3 to 3 as described in this application, and when the ratio is 1:1, the CI index is the

smallest and the synergistic effect is the most obvious.

Table 1 Inhibitory effects of ginsenoside CK and bicyclol on the proliferation of liver cancer

HepG2 cells in vitro (x s)

Bicyclol Ginsenoside CK/Bicyclol CI No. Blank control Ginsenoside CK group group group combination group INDEX

1 Drug concentration OtM 5M 5pM 5pM/5pM Inhibition ratio (%) 0.25±0.32 10.32±1.12 7.43±1.67 27.90±3.58 0.87 2 Drug concentration OtM 10pM 10pM 10tM/10tM Inhibition ratio (%) 0.25±0.32 19.68±1.54 16.60±0.76 52.27±4.36 0.71 Drug concentration OtM 8pM 12pM 8pM/12pM Inhibition ratio (%) 0.25±0.32 11.97±1.54 22.26±2.13 45.10±3.15 0.80 Drug concentration OtM 15.4pM 4.6pM 15.4pM/4.6pM Inhibition ratio (%) 0.25±0.32 28.13±2.25 6.93±2.10 45.56±3.07 0.81 Drug concentration OtM 5pM 15pM 5pM/15pM Inhibition ratio (%) 0.25±0.32 10.32±0.96 22.81±2.13 43.40±3.86 0.82 6 Drug concentration OtM 16pM 4pM 16pM/4pM Inhibition ratio (%) 0.25±0.32 30.06±1.01 3.77±0.69 42.71±1.13 0.85 Drug concentration OtM 4pM 16pM 4pM/16pM Inhibition ratio (%) 0.25±0.32 8.32±3.31 23.81±0.69 37.15±3.21 0.90

Example 2 Combination of ginsenoside CK and bicyclol inhibits the malignant transformation of WB-F344 cells co-induced by 3MC/TPA to cancer cells in vitro

The models for studying tumor chemical preventive drugs are mainly in vitro systems and in

vivo systems. The in vitro system uses various carcinogens to directly or indirectly induce malignant

transformation of normal cells, and its effective index is the inhibition of malignant transformation of

cells. It has the advantages that the multi-stage process of tumorigenesis in vivo is able to be well

simulated and the experimental conditions are easily controlled. Rat liver epithelial-like stem cells

(WB-F344 cells) were obtained from normal adult isogenic male rats. They are a kind of liver

immature epithelial cells with the properties of liver stem cells, relating to the occurrence of liver cancer and are precursor cells of liver cancer. Model group: 1500 WB-F344 cells/well were seeded in 6-well plates with 2mL culture medium per well, respectively. 24h after cell seeding, the carcinogen 3-methylcholanthrene (3MC) was added to the culture medium, making its final concentration 2 g/mL, and after 96h of culture, 3MC was removed and replaced with fresh medium, and the culture was continued for 3 days, then phorbol myristate acetate (TPA) was added, and on day 8 to 12, TPA concentration was 50ng/mL for induction, followed by TPA concentration of 80ng/mL for induction, on day 25, TPA was removed, and on day 30, the experiment was completed, cells were rinsed with PBS solution, Wright-Giemsa staining was performed, and the number of transforming foci was observed microscopically and counted. Blank control group: On the basis of the model group, 0.2% DMSO was used to replace 3MC and TPA. Dosing group: On the basis of the model group, the cells were treated by adding drugs at 24 h after seeding, and the ginsenoside CK single administration group (concentrations of 5 M, 7.8 [M, 2.5 [M, 2 M), bicyclol single administration group (concentrations of 5 M, 2.2 jM, 7.5 jM, 8 paM), ginsenoside CK/bicyclol combination group 1 (concentrations of 5 M/5 M), ginsenoside CK /bicyclol combination group 2 (concentration of 7.8 jaM/2.2 jaM), ginsenoside CK/bicyclol combination group 3 (concentration of 2.5 M/7.5 M), ginsenoside CK/bicyclol combination group 4 (concentration of 2 M/8 jM) were set, until the end of the experiment. Other treatments were the same as the model group. For the cells, the medium were changed every 1 to 3 days after adding TPA depending on the cell status. The following formula was used to calculate the inhibition ratio of liver cancer cells: inhibition ratio (%) = (1 - number of transforming foci in the dosing group/number of transforming foci in the blank control group) x 100%. The interaction index CI of the two monomers was used to determine the effect of the combination, CI= AB/(AxB), T is the number of transforming foci in the cells of the dosing group, C is the number of transforming foci in the cells of the blank control group, AB is the T/C value of the combination group of the two monomers, and A and B are the T/C values of the groups where the two monomers acting alone. CI < 1 indicates that the two monomers have a synergistic effect, and CI < 0.7 indicates that the synergistic effect is very significant. After the experiments, the cell morphology observation results of the blank control and model groups were as shown in Figure 1. Figure 1(a) is the cell morphology of the blank control group. It can be seen from the figure that the normally grown WB-F344 cells have regular morphology and polygonal shape, and spread well on the substrate, rich in cytoplasm and with contact inhibition.

Figure 1(b) is the cell morphology of the model group. It can be seen from the figure that the

transformed WB-F344 cells were irregular morphology, shuttle-shaped or polygonal shaped, with

unequal cell size, loss of polarity of growth, loss of contact inhibition, often overlapping growth, and

scattered transforming foci were visible. The results of transforming foci counting were shown in

Table 2 below. At the end of the experiment, the model group formed obvious transforming foci, and

the number of transforming foci was 58.6±7.2. In the blank control group, cells were cultured for 30

days, and there were fewer transforming foci formed. There was a extremely significant difference

compared with the model group (P<0.05). The ginsenoside CK group, bicyclol group and

ginsenoside CK/bicyclol combination group 1, ginsenoside CK/bicyclol combination group 2,

ginsenoside CK/bicyclol combination group 3, ginsenoside CK/bicyclol combination group 4 have

inhibitory effects on the formation of transforming foci (see Table 2 for specific results), and the

interaction index CI values of the two monomers were both less than 1, which has a synergistic effect

on inhibiting the in vitro malignant transformation of WB-F344 cells.

Table 2 Effect of ginsenoside CK and bicyclol on the in vitro malignant transformation of

WB-F344 cells co-induced by 3MC/TPA(x s) Transforming Inhibition ratio CI focus (%) INDEX Blank control group 4.1+2.7 Model group + 58.6±5.7## Ginsenoside CK 5 M + 38.6±3.6** 34.13 Bicyclol 5 M + 40.8±4.8** 30.38 Ginsenoside CK/bicyclol combination + 17.2±1.9*** 70.65 0.64 group Ginsenoside CK 7.8[tM + 30.6+3.3*** 47.78 Bicyclol 2.2[tM + 49.2+2.7* 16.04 Ginsenoside CK/bicyclol combination + 21.5±2.0*** 63.31 0.84 group 2 Ginsenoside CK 2.5[tM + 45.9±4.3** 21.67 Bicyclol 7.5[tM + 35.9+3.1*** 38.74 Ginsenoside CK/bicyclol combination + 24.2±2.9 58.70 0.86 group 3 Ginsenoside CK 2[M + 47.7±3.3* 18.60

Bicyclol 8[M + 33.1±1.7** 43.52 Ginsenoside CK/bicyclol combination + 2 26.4±2.*- *** 54.95 0.98 group 4 gop4+ Compared with the blank group, #P<0.05, ##P<0.01; compared with the model group, *P<0.05,

**P<0.01, ***P<0.01.

Example 3 In vivo efficacy evaluation of the combination of ginsenoside CK and bicyclol in the treatment of liver cancer

Establishment of human liver cancer xenograft tumor model in nude mice: 4 to 5 weeks old

female BALB/c nude mice were subcutaneously inoculated with Hep-3B cells in the left axilla, and

each nude mouse was injected with 4x106 cells. When the tumor volume reached 100-300 mm 3, the

nude mice were randomly divided into 16 groups with 10 animals in each group.

The 16 groups were 1 model group and 15 dosing groups. The 15 dosing groups were (1) 5

ginsenoside CK groups, administered intragastrically, at doses of 60 mg/kg, 30 mg/kg, 90 mg/kg, 100

mg/kg, and 24 mg/kg, respectively; (2) 5 bicyclol groups, administered intragastrically, at doses of

60 mg/kg, 90 mg/ kg, 30mg/kg, 20mg/kg, and 96mg/kg, respectively; (3) 5 ginsenoside CK/bicyclol

combination groups, administered intragastrically, with ginsenoside CK/bicyclol administered at

doses of 60mg/kg/60mg/kg, 30mg/kg/90mg/kg, 90mg/kg/30mg/kg, 100mg/kg/20mg/kg, and

24mg/kg/96mg/kg, respectively. The administration volume of each dosing group was 0.2 mL,

administrated once a day for 21 days. The animals were observed once a day and their clinical status

was recorded, including whether they were dead, dying, appearance, mental status and activity

status. The tumor length and tumor width were measured twice a week, and the tumor volume and

tumor inhibition ratio (%) were calculated according to the following formulas. Tumor volume

V=0.5xaxbxb, in which, a is the length of the tumor and b is the width of the tumor. Tumor

inhibition ratio (%)=(model group V-dosing group V)/model group Vx100%.

The interaction index CI of the two monomers was used to determine the effect of combination,

CI=AB/(AxB), T is the tumor volume of the dosing group, C is the tumor volume of the model group,

AB is the T/C value of the combination group of the two monomers, A and B are the T/C values of

the groups where the two monomers acting alone. CI<1 indicates that the two monomers have a

synergistic effect, CIG0.7 indicates that the synergistic effect is very significant.

After continuous administration for 21 days, the animals showed no abnormal clinical symptoms. The tumor volume test results were shown in Table 3, and the CI index of the combination of the two monomers was 0.68 (CI < 0.7), with a significant synergistic effect.

Moreover, with the total concentration of ginsenoside CK and bicyclol unchanged, the CI index was

lower when the ratio of the two was within the range of 1:0.3 to 3 as described in this application

than that when the ratio of the two was outside the above range, and the CI index was the smallest

and the synergistic effect was the most obvious when the ratio was 1:1.

Table 3 Effect of ginsenoside CK and bicyclol on tumor volume in human liver cancer

xenograft tumor model in nude mice Model Ginsenoside CK Bicyclol Ginsenoside CK/Bicyclol CI group group group combination group INDEX Administration dosage - 60 60 60/60 (mg/kg) Tumor volume (mm3 ) 1097.41 612.05 838.89 302.87

tumor inhibition ratio( - 44.23% 23.56% 72.40% 0.65

Administration dosage - 90 30 90/30 (mg/kg) Tumor volume (mm3 ) 1097.41 541.08 936.34 380.22

tumor inhibition ratio( - 50.69% 14.68% 65.35% 0.82

Administration dosage - 30 90 30/90 (mg/kg) Tumor volume (mm3 ) 1097.41 793.32 659.67 402.23

tumor inhibition ratio( - 27.71% 39.89% 63.35% 0.84

Administration dosage - 100 20 100/20 (mg/kg) Tumor volume (mm3 ) 1097.41 502.45 988.89 441.56

tumor inhibition ratio( 54.21% 9.89% 59.76% 0.98

Administration dosage - 24 94 24/94 (mg/kg) Tumor volume (mm3 ) 1097.41 817.77 639.26 456.98

tumor inhibition ratio( - 25.48% 41.75% 58.36% 0.96

Compared with the model group, *P<0.05, **P<0.01.

Example 4 Combination of ginsenoside CK and bicyclol to prevent DEN-induced liver cancer in rats

Model establishment and administration: 100 SD rats were purchased, and after one week of

adaptation in the animal room, they were randomly divided into 5 groups, namely, blank control

group, model group, ginsenoside CK group, bicyclol group, and ginsenoside CK/bicyclol

combination group. The ginsenoside CK group and bicyclol group were administered intragastrically,

the ginsenoside CK group was administered at a dose of 60 mg/kg, the bicyclol group was

administered at a dose of 60 mg/kg, the ginsenoside CK/bicyclol combination group was

administered at a dose of 60 mg/kg, and the blank control group and the model group were given an

equal volume of solvent and administered by intragastric administration for 20 weeks. One week

after intragastric administration, except for the blank control group, the animals in each group were

intraperitoneally injected with the chemical mutagen diethylnitrosamine (DEN) at 20 mg/kg for 2

weeks, 40 mg/kg for 2 weeks, 60 mg/kg for 2 weeks and 80 mg/kg for 2 weeks.

Detection of index: After 20 weeks of intragastric administration, the animals were sacrificed.

The rats were weighed before the animals were sacrificed. The rats were anesthetized by

intraperitoneal injection of sodium pentobarbital solution, the abdominal wall was dissected to

expose the liver, and the size, shape, color, and the presence of nodules of the liver were observed.

Blood was collected from the abdominal aorta for the detection of alanine aminotransferase (ALT)

and aspartate aminotransferase (AST). The liver was taken out completely, the liver weight was

measured, and the liver coefficient was calculated, the liver coefficient = liver weight/body weight *

100%. Some liver samples were taken, fixed with 4% formaldehyde, and H&E stained pathological

sections were made. H&E staining is a commonly used staining method for pathological sections and

it is a Hematoxylin-Eosin staining method. This method can be used for tissues fixed in any fixative

solution and for sections with various embedding methods. Hematoxylin is an alkaline dye that can

stain the basophilic material in the tissue blue, such as chromatin in the nucleus, and eosin is an

acidic dye that can stain the eosinophilic material in the tissue red, such as the cytoplasm and

nucleolus of most cells which are red in H&E stained sections.

Experimental results.

(1) Serum ALT, AST and ALP levels in liver cancer prevention model rats

The results are shown in Table 4. Compared with the blank control group, the serum ALT, AST

and ALP levels in the model group rats increased significantly (P<0.05), indicating that the chemical

mutagen DEN was accompanied by changes of serum liver function indexes ALT, AST and ALP

levels in the process of inducing liver cancer in SD rats. Compared with the model group, the serum

ALT, AST and ALP levels were significantly lower in the ginsenoside CK and bicyclol groups

(P<0.05), while the effect of the ginsenoside CK/bicyclol combination group was significantly better

than that of the groups using single drugs, and the serum ALT, AST and ALP levels were close to

those of the blank control group.

Table 4 Effects of ginsenoside CK and bicyclol on serum ALT, AST and ALP levels in liver

cancer prevention model rats (x s)

Dosage ALT AST ALP Group (mg/kg) U/L U/L IU/L Blank group 142.35+8.37 75.55+4.84 348.59+36.85 Model group - 181.45±15.08" 86.40±3.88" 410.27±35.34" Ginsenoside CK group 60 162.79+14.85* 81.54+6.00* 350.59+27.35* Bicyclol group 60 167.83+6.16* 80.18+4.42* 367.53+30.68* Ginsenoside CK/bicyclol 60/60 149.83+9.81* 76.32+3.19* 337.53+30.68* combination group Compared with the blank group, #P<0.05; compared with the model group, *P<0.05.

(2) Body weight, liver weight and liver index of liver cancer prevention model rats

After 20 weeks of intragastric administration, as shown in Table 5, the body weight of rats in the

model group was significantly lower than that of the blank control group, and the liver weight and

liver coefficient were significantly higher than those of the blank control group (P<0.05). Changes in

organ coefficients generally reflect the enlargement, congestion and hyperplasia and hypertrophy of

organs, which are indexes of liver injury. Intraperitoneal injection of DEN can increase the liver

coefficients of rats, and it is presumed that liver injury may occur in rats. The body weight of rats in

ginsenoside CK group, bicyclol group and ginsenoside CK/bicyclol combination group was

significantly higher than that of the model group (P < 0.05), and the liver coefficient was

significantly lower than that of the model group (P < 0.05), and the effect of ginsenoside CK/bicyclol

combination group was significantly better than that of the single drug dosing group.

Table 5 Effects of ginsenoside CK and bicyclol on body weight, liver weight and liver coefficient of rats (x s)

Dosage Body weight Liver weight Liver coefficient Group (mg/kg) (g) (g) (%)

Blank group - 470.33+55.06 12.308+0.113 0.0262+0.0021

Model group - 398.33±39.43# 15.407±0.204# 0.0387±0.0052#

Ginsenoside CK group 60 422.43+26.53* 13.528+0.058* 0.0320+0.0022*

Bicyclol group 60 440.38+49.51* 13.843+0.089* 0.0314+0.0018* Ginsenoside CK/bicyclol 60/60 411.25+20.68* 11.643+0.089* 0.0283+0.0036* combination group Compared with the blank control group, #P<0.05; compared with the model group, *P<0.05.

(3) liver H&E-stained pathological sections

All rats were sacrificed after 20 weeks of intragastric administration, as shown in Figure 2(a)-(e),

the liver tissues of the rats in the blank control group were dark red, normal in size, soft and fragile,

with smooth liver surface without abnormalities, and regular hepatic lobules and hepatic cord

structures were visible in H&E stained sections; while the livers of the animals in the model group

showed severe lesions, with hardened liver texture and cancer nodules of different sizes scattered in

each lobe, some of which were in the form of giant masses, and H&E staining confirmed liver cancer

or moderate to severe liver cirrhosis; in the ginsenoside CK and bicyclol groups,there were some

gray-white nodules in livers of some animals, and the number of cases of liver cancer and the

degree of liver lesions were lighter than those in the model group; whereas only a few gray-white

nodules existed in the livers of only a few animals in the ginsenoside CK/bicyclol combination group,

and the number of cases of liver cancer and the degree of liver lesions were significantly lower than

those in the single drug dosing group.

It is shown by this example that the combination of the two monomers, ginsenoside CK and

bicyclol, disclosed in the present application exhibited excellent efficacy when applied to prevent

liver cancer model induced by carcinogenic agent DEN, which was significantly better than that of

the single drugs, and no obvious adverse effects were found, and it had good safety.

The above mentioned is only preferred embodiments of the present application, but not a

limitation of the present application in other forms, and any person skilled in the art may use the

technical content revealed above to change or remodle to equivalent embodiments with equivalent

changes. However, without departing from the content of the technical solution of this application and based on the technical essence of this application, any simple modifications, equivalent changes and remodlings made to the above embodiments still fall within the protection scope of the technical solution of this application.

Claims

ginsenoside monomers and bicyclol.

2. The pharmaceutical composition according to claim 1, wherein the weight ratio of the

ginsenoside monomers and the bicyclol is 1:0.3 to 3.

3. The pharmaceutical composition according to claim 2, wherein the weight ratio of the

ginsenoside monomers and the bicyclol is 1:1.

4. The pharmaceutical composition according to any one of claims 1-3, wherein the ginsenoside

ginsenoside Rk3 and ginsenoside Rh4;

preferably, the ginsenoside monomers comprise ginsenoside CK.

5. Use of ginsenoside monomers and bicyclol in the preparation of a pharmaceutical

composition for preventing and/or treating tumors.

6. The use according to claim 5, wherein the weight ratio of the ginsenoside monomers and

bicyclol is 1:0.3 to 3.

7. The use according to claim 6, wherein the weight ratio of the ginsenoside monomers and

bicyclol is 1:1.

8. The use according to any one of claims 5-7, wherein the ginsenoside monomers are one or

two or more selected from: ginsenoside CK, ginsenoside 20-(S, R)-Rg3, ginsenoside 20-(S, R)-Rh2,

ginsenoside 20-(S, R)-PPD, ginsenoside Rkl, ginsenoside Rg5, and ginsenoside Rh4;

preferably, the ginsenoside monomers comprise ginsenoside CK.

9. The use according to any one of claims 5-8, wherein the tumor is liver cancer.

10. Use of ginsenoside monomers in promoting the effect of a pharmaceutical composition for

preventing and/or treating tumors.

-(S, R)-PPD, ginsenoside Rkl, ginsenoside Rg5, ginsenoside Rk3 or ginsenoside Rh4; preferably, the ginsenoside monomers comprise ginsenoside CK.

and/or treating tumors comprises bicyclol.