TWI423809B - Pharmaceutical compositions for inhibiting cancer cell profileration induced by acetylcholine receptor activition - Google Patents

Description

Pharmaceutical composition for inhibiting proliferation of cancer cells by activation of acetylcholine receptor

The present invention relates to the use of a sesame extract, and more particularly to the use of a sesame extract for the preparation of a medicament which inhibits the activity of an acetylcholine receptor, such as a pharmaceutical composition comprising a sesame extract.

Sesame is the "medical material for food use" announced by the Department of Health. Sesame is a mature seed of Sesamum indicum L., which contains up to 60% oil. Sesame oil containing glycerol esters of oleic acid, linoleic acid, palmitic acid, arachidic acid, arachidonic acid, decadienoic acid, and the like, as well as sterol, sesamin, sesamolin, and sesamol. And vitamin E and so on. Traditional Chinese medicine says that the sesame is sweet and flat, and it enters the lungs, spleen, liver and kidney. Sesame is mainly used for laxative, nourishing liver and kidney, etc. It is usually taken in the form of decoction or frying.

The Republic of China Patent Publication No. 200618805 discloses that sesamin in sesame has a mechanism of activating the expression of the cancer suppressor gene P53 and inhibiting the pharmacological action of hepatoma cell proliferation. The Republic of China Patent Publication No. 200800165 discloses that sesamin in sesame can inhibit the proliferation and survival of breast cancer cells by inducing the production and activation of P21 protein, and thus can be used as a therapeutic agent for breast cancer. The Republic of China Patent Certificate No. I276439 discloses that sesamin and sesamin can inhibit the production of nitric oxide by LPS-stimulated primary glial cells, and thus can be used as a medicine for preventing stroke and protecting nerve degeneration. U.S. Patent No. 7,396,554 discloses the use of sesame extract containing sesame phenol as an antioxidant for the prevention of oxidation of edible oils and fats.

According to the above, the natural substances contained in sesame are used in traditional Chinese medicine or diet therapy for nourishing, intestine or preventing stroke; in recent years, it has been further explored that sesame extract can promote cancer cell apoptosis or resistance. Cell oxidation and other uses.

The inventors of the present invention have found that the sesame extract has an activity of inhibiting the acetylcholine receptor, and thus can be used as a medicine for treating or ameliorating a disease mediated by an acetylcholine receptor.

One aspect of the present invention provides a pharmaceutical composition for treating or ameliorating a disease condition by inhibiting an acetylcholine receptor comprising an effective amount of sesame extract and a pharmaceutically acceptable carrier therefor.

According to one or more embodiments of the present invention, the sesame extract refers to an extract extracted by a specific method, which comprises at least sesamin, sesamol, a pharmaceutically acceptable salt thereof, A pharmaceutically acceptable solvate thereof or any combination of the foregoing. The specific method comprises: mixing sesame seeds with a polar solvent in a ratio of about 1:3 by weight: and filtering to form a filtrate; and concentrating the filtrate under reduced pressure to obtain a sesame extract. According to an embodiment of the present invention, the polar solvent may be water, an alcohol solvent, a ketone solvent or a mixture thereof. The specific method further comprises: grinding the sesame seed to make the particle size uniform before mixing the sesame seed with the polar solvent. The specific method further comprises: subjecting the filtrate to a partition extraction with a non-polar solvent. Suitable non-polar solvents include, but are not limited to, n-hexane, ethyl acetate or a combination thereof. In one example, the non-polar solvent is n-hexane; in another example, the non-polar solvent is ethyl acetate. The specific method further comprises: further purifying the sesame extract using a high performance liquid chromatography column.

According to one or more embodiments of the invention, the disease treatable or ameliorated by the pharmaceutical composition of the invention may be cancer or overactive bladder (OAB). In one example, a disease suitable for treating or ameliorating a condition with a pharmaceutical composition of the invention is cancer, which may be selected from any of the following: human leukemia, muscle tumor, bone cancer, lymphoma, melanoma, ovarian cancer, skin cancer, Testicular cancer, gastric cancer, pancreatic cancer, kidney cancer, breast cancer, prostate cancer, rectal cancer, head and neck cancer, brain cancer, esophageal cancer, bladder cancer, adrenocortical carcinoma, lung cancer, bronchial cancer, endometrial cancer, cervical Cancer or liver cancer. In another example, a disease suitable for treating or ameliorating a condition with a pharmaceutical composition of the invention is OAB.

According to one or more embodiments of the present invention, the acetylcholine receptor inhibited by the pharmaceutical composition of the present invention may be a nicotine type acetylcholine receptor or a muscarinic acetylcholine receptor. In a specific example, the acetylcholine receptor is a muscarinic acetylcholine receptor.

According to one or more embodiments of the present invention, the pharmaceutical composition of the present invention may further comprise a chemotherapeutic agent. Suitable chemotherapeutic agents may be selected from paclitaxel, irinotecan HCl, docitaxel, doxorubicin, daunorubicin, aimycin ( Epirubicin), 5-fluorouracil, melphalan, cisplatin, carboplatin, cyclophosphamide, mitomycin, aminomethyl Fate (methotrexate), mitoxantrone, vinblastine, vincristine, indomethamine (ifosfamide), teniposide, etoposide, bleomycin, leucovorin, cytarabine, actinomycin D ( Dactinomycin), interferon alpha, streptozocin, prednisolone or procarbazine HCl. In one example, the chemotherapeutic agent is 5-fluorouracil; in another example, the chemotherapeutic agent is cisplatin.

Another aspect of the present invention provides a sesame extract for use in the preparation of a medicament for inhibiting the activity of an acetylcholine receptor. The sesame extract referred to above contains at least sesame phenol, sesamin or a combination thereof. From the above, it is understood that the present invention discloses an extract obtained by extracting sesame with a polar solvent, which has the effect of inhibiting the activity of the acetylcholine receptor, and can be applied to a new use of the function related to the acetylcholine receptor. For example, it is used as an adjuvant for cancer associated with acetylcholine receptors, and a drug related to the treatment or prevention of overactive bladder.

The inventors of the present invention unexpectedly found that the sesame extract has an activity of inhibiting the acetylcholine receptor, and thus can be used as a medicine for treating or ameliorating a disease mediated by an acetylcholine receptor.

Accordingly, one of the objects of the present invention is to provide a method for preparing a sesame extract comprising at least the following steps: first, mixing sesame seeds with a polar solvent in a weight: volume ratio of about 1:3; filtering to obtain a filtrate; The obtained filtrate was concentrated under reduced pressure to obtain a crude extract. The crude extract can be further subjected to a high performance liquid chromatography column (high performance liquid) Purification by chromatography, HPLC), thus confirming that the crude extract contains at least 11 compounds including at least sesame phenol and sesamin.

Sesame seeds suitable for extraction by the above method are not limited to sesame seeds obtained in a specific place or season, and the "sesame extract" described herein is covered by sesame seeds and/or seed coats unless the extraction site is specifically indicated. The resulting extract.

Polar solvents suitable for extraction may be water, alcohols, ketones or any combination thereof. The alcohols include ethanol, such as 80% or 95% alcohol or anhydrous alcohol. The ketone may be acetone.

According to one embodiment, the above method further comprises grinding the sesame seeds into uniform granules prior to extracting the sesame seeds with a polar solvent.

According to another embodiment, the above method further comprises subjecting the crude extract to a partition extraction with a non-polar solvent prior to purifying the crude extract by HPLC. Suitable non-polar solvents include, but are not limited to, n-hexane, ethyl acetate or a combination thereof. In one example, the non-polar solvent is n-hexane; in another example, the non-polar solvent is ethyl acetate.

According to a particular embodiment, the sesame extract obtained in the above manner contains at least 11 substances, at least comprising sesame phenol, sesamin or a combination thereof. According to another specific embodiment, the sesame extract has the function of inhibiting the calcium ion signal mediated by the acetylcholine receptor, and thus can be treated or improved by inhibiting the acetylcholine receptor function. The disease of the disease.

Accordingly, it is another object of the present invention to provide a pharmaceutical composition for treating or ameliorating a disease condition by inhibiting an acetylcholine receptor comprising an effective amount of a sesame extract, and a pharmaceutically acceptable carrier therefor .

According to a particular embodiment, the sesame extract comprises at least sesamin, sesaminol, a pharmaceutically acceptable salt or solvate thereof.

The disease that can be treated or ameliorated by the pharmaceutical composition of the invention can be cancer or overactive bladder (OAB). In one example, a disease suitable for treating or ameliorating a condition with a pharmaceutical composition of the invention is cancer, which may be selected from any of the following: human leukemia, muscle tumor, bone cancer, lymphoma, melanoma, ovarian cancer, skin cancer, Testicular cancer, gastric cancer, pancreatic cancer, kidney cancer, breast cancer, prostate cancer, rectal cancer, head and neck cancer, brain cancer, esophageal cancer, bladder cancer, adrenocortical carcinoma, lung cancer, bronchial cancer, endometrial cancer, cervical Cancer or liver cancer. In another example, a disease suitable for treating or ameliorating a condition with a pharmaceutical composition of the invention is OAB.

The term "an effective amount" means a dose of a therapeutic sesame extract administered to a subject. The measurement of efficacy can be objective (measured by some test or marker) or subjective (the subject gives a narrative of its effects and feelings). The effective dosage of the above sesame extract may range from about 1 mg/kg/day to about 500 mg/kg/day, preferably from 10 mg/kg/day to about 400 mg/kg/day, more preferably from 50 mg / kg / day to about 250 mg / kg / day. The effective dose will also vary with the route of administration and the other agents that may be used in combination.

The term "solvate" as used herein refers to a defect formed by the reaction of a compound (eg, sesamin or sesame phenol in the present disclosure) with its surrounding solvent molecules (eg, water, ethanol, etc.). Compound.

A pharmaceutically acceptable compound of the invention, for example, a sesamin or a sesame phenol or a salt of any of the active compounds in a sesame extract prepared according to the process of the invention, comprising a pharmaceutically acceptable inorganic acid base and an organic acid base . Suitable acid salts are, for example, acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate. ), butyrate, citrate, camphorate, camphorsulfonate, ethanesulfonate, formate, fumarate Fumarate, glycolate, heptanoate, hexanoate, hydrochloride, hydrobromide, 2-hydroxyB 2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, Nicotinate, nitrate, palmate, pectinate, 3-phenylpropionate, phosphate, picric acid Picrate, pivalate, propionate, salicylate, succinate, Sulfate, tartrate, thiocyanate, tosylate and undecanoate. Alternatively, pharmaceutically unacceptable acids, such as ethanedicarboxylic acid, can be used to prepare the salts of the intermediates to obtain the compounds of the invention and their pharmaceutically acceptable acid salts. The salts obtained from the appropriate bases include alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium ion and N(alkyl) ₄ ⁺ salts. The present invention also contemplates a nitrogen-containing quaternary ammonium salt of any of the compounds disclosed herein to provide a water soluble, oil soluble or dispersible product.

Further, the pharmaceutical composition of the present invention may further comprise a chemotherapeutic agent. Chemotherapeutic agents suitable for use with the pharmaceutical compositions of the invention include, but are not limited to, paclitaxel, irinotecan HCl, docitaxel, doxorubicin, dan Dounorubicin, epirubicin, 5-fluorouracil, melphalan, cisplatin, carboplatin, cyclophosphamide , mitomycin, methotrexate, mitoxantrone, vinblastine, vincristine, ifosfamide, podophyllotoxin Teniposide), etoposide, bleomycin, leucovorin, cytarabine, dactinomycin, alpha-interferon Interferon alpha), streptozocin, prednisolone or procarbazine HCl. In one example, the chemotherapeutic agent is 5-fluorouracil; in another example, the chemotherapeutic agent is cisplatin. The chemotherapeutic agent can be administered prior to, concurrently with, or subsequent to administration of the pharmaceutical composition of the invention to treat or ameliorate the disease condition thereof.

The dosage of the pharmaceutical composition disclosed herein may be affected by a number of factors, including the activity of the drug used, the age, weight, overall health, sex, diet, time of administration of the composition, route of administration, frequency of administration of the individual administered. The rate of drug excretion, whether there is a combination of drugs, the progress and severity of the disease, the location and condition of the disease, and the treatment and judgment of the doctor.

"Pharmaceutically acceptable carrier" means a carrier or adjuvant which can be administered to a body together with the sesame extract of the present invention, and which is not applied when a sufficient amount of a therapeutic dose of sesame extract is administered. The pharmaceutical activity of the sesame extract of the present invention is destroyed without causing any toxicity to the individual.

Pharmaceutically acceptable carriers, adjuvants or vehicles that can be used in the pharmaceutical compositions of the invention include, but are not limited to, ion exchangers, alumina, aluminum stearate , lecithin, self-emulsifying drug delivery systems (SEDDS) such as tocopherol polyethylene glycol 1000 succinate (d-α-tocopherol polyethylene glycol 1000 succinate), used in pharmaceutical Dosing surfactants, such as Tweens or similar polymeric delivery media, plasma proteins (eg, plasma albumin), buffers (eg, phosphate, glycine, sorbic acid, potassium sorbate) (potassium sorbate), partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (eg protamine sulfate, disodium hydrogen) Phosphate), potassium hydrogen phosphate, sodium chloride, zinc salt, colloidal silica, magnesium trisilicate, polyethylene Polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, paraffin ( Waxes), polyethylene-polyoxypropylene-block polymers, and wool fat. Cyclodextrins such as α-, β- and γ-cyclodextrin or Chemically modified derivatives, such as hydroxyalkylcyclodextrins, contain 2- and 3-hydroxyoxypropyl-β-cyclodextrin or other soluble derivatives.

The pharmaceutical composition of the present invention may be orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally. , vaginally, subdermally, transmucosally, or implanted reservoir into the body, preferably via the oral or parenteral route. The pharmaceutical compositions of the present invention may comprise any conventional non-toxic and pharmaceutically acceptable carrier, adjuvant or vehicle. In certain instances, a pharmaceutically acceptable acid, base or buffer may be used to adjust the pH of the formulation to increase the stability of the formulation compound or its delivery profile. The term "parenteral" as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular (intraarticular). ), intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, intraperitoneal, intracapsular (intraperitoneally) and intracranial (intracranial) Intracranial) injection or infusion technique.

The pharmaceutical compositions can be prepared in a sterile, injectable form, such as a sterile injectable aqueous solution or an oleaginous suspension. Suspensions can be formulated according to known techniques using conventional dispersing or wetting agents (such as Tween 80) and suspending agents. The sterile injectable preparation may also be in the form of a sterile injectable solution or suspension in a non-toxic non-orally acceptable diluent or solvent, such as 1,3-butanediol. Acceptable vehicles and solvents can be mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally used as a solvent or suspending medium. For this purpose, any type of fixed oil can be used to synthesize monoglyceride or diglyceride. Fatty acids such as oleic acid and its glyceride derivatives can be used in the preparation of injectables, as they are pharmaceutically acceptable natural oils such as olive oil or castor oil, especially its multiple ethoxylates. Polyoxyethylated form. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, or carboxymethyl cellulose and the like which are commonly used in pharmaceutical formulations such as emulsions and/or suspensions. Dispersant. Other commonly used surfactants such as Tween or Spans and/or other emulsifiers or bioavailability enhancers are commonly used to make pharmaceutically acceptable solids, liquids or other dosage forms that can be used as a formulation.

The pharmaceutical compositions of the present invention are preferably administered in any orally acceptable dosage, including, but not limited to, capsules, lozenges, suspensions of emulsions and waters, dispersions and solutions. In the case of oral lozenges, conventional carriers include lactose and corn starch. Lubricants such as magnesium stearate are also often added. In the case of oral capsules, useful diluents include lactose and dried corn starch. When aqueous suspensions and/or emulsions are administered orally, the active ingredient can be suspended or dissolved in the oil phase in association with the emulsifiers and/or dispersing agents. If necessary, add some sweeteners and/or spices.

The pharmaceutical compositions of the present invention may also be formulated as a suppository for rectal administration into an individual. This composition can be prepared by mixing the compound of the present invention with a suitable non-irritating excipient. The excipient must be solid at room temperature but liquid at the rectal temperature to facilitate dissolution of the active compound in the rectum. Excipients include, but are not limited to, cocoa butter, beeswax, and polyethylene glycols.

When the desired treatment involves topical topical application, it is preferred to administer the pharmaceutical composition of the present invention in a topical topical application. When the pharmaceutical composition of the present invention is applied to the skin by topical application, the pharmaceutical composition should be formulated to contain an active compound, such as sesamin or sesame, suspended or dissolved in a suitable carrier in a suitable ointment. Any of the active compounds of the phenol or the sesame extract made according to the process of the invention. Carriers suitable for topical surface application to apply the sesame extract of the present invention include, but are not limited to, mineral oil, liquid petroleum, white petroleum, polyethylene glycerin, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. In addition, the formulation of the pharmaceutical composition may also be in the form of a lotion or cream to contain the active compound (for example, sesamin or sesame phenol) or in suspension in a suitable carrier and a suitable emulsifier. Any of the active compounds in the sesame extract produced by the method of the invention). Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl alcohol wax, cetyl alcohol, 2-octyldodecanol , benzyl alcohol and water. The compositions of the invention may also be administered in the lower intestinal tract as an enteric plug or enemas. The topical surface application of the present invention also encompasses transdemal patches. The pharmaceutical composition formulations of the present invention also comprise a nasal aerosol or inhalation. The composition can be prepared into a saline solution according to known pharmaceutical formulations, using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance its bioavailability, halothanes, and/or other known solubilizers or dispersions. Agent.

It is to be understood that the singular forms "a", "a", "an" or "the"

The embodiments and the proper nouns are used to illustrate the invention and are not intended to limit the scope of the disclosure. The scope of the disclosure is also not specifically disclosed herein, but other embodiments that can be readily inferred by those skilled in the art after reading this disclosure.

Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described may be employed in the methods of the invention. The preferred embodiments and materials described herein are for illustrative purposes only. All references cited in this application are hereby incorporated by reference in their entirety to the extent of the disclosure of the disclosure of the disclosure. Moreover, the documents discussed herein merely disclose prior art techniques of the present application. And there is no literature showing that the present invention has been disclosed in the prior art. The actual data obtained in the context of the present invention may differ from the data disclosed in the present disclosure by the present invention.

The following examples are presented to illustrate the specific aspects of the disclosure and to assist those skilled in the art to understand and implement the present disclosure. However, the scope of the disclosure is not limited to these embodiments.

Example Experimental Example 1 Preparation and Analysis of Sesame Extract 1.1 Preparation of sesame extract

100 g of sesame seeds were taken at intervals of 10 seconds in a grinder, uniformly ground, placed in a clean and sterilized triangular flask, and poured into 300 ml of the above polar solvent, sealed with aluminum foil, and uniformly stirred for a while. The supernatant was poured into a 50 ml centrifuge tube, centrifuged at 15,000 rpm for 10 minutes, and the supernatant was filtered with a 0.22 μm filter. The filtered crude extract was concentrated under reduced pressure at 40 ° C for 10 hours to remove the polar solvent. The crude extract concentrated under reduced pressure was freeze-dried at -80 ° C for one week to obtain a dried crude extract. The sample was then stored at -20 ° C in the dark.

In the above embodiment, if the polar solvent used is water, it is maintained at a temperature of about 80 ° C, uniformly stirred for 40 minutes and allowed to stand overnight; if the polar solvent used is ethanol or acetone, the solution is 80% ethanol ( V/v, ethanol/water) or 80% propanol solution (v/v, ethanol/water) is preferred, and the crude extract concentrated under reduced pressure can be iced at -80 ° C to confirm whether it will freeze, if not When icing, indicating that the ethanol or acetone has not been completely removed, the depressurization concentration step is again performed to complete removal.

The dried crude extract can be further partitioned, and the substance in the crude extract is further separated into a substance dissolved in n-hexane, a substance dissolved in ethyl acetate, and insoluble in ethyl acetate and n-hexane. Substance.

The step of dispensing the extraction comprises dissolving the above extract in one-fifth dry weight of n-hexane, shaking uniformly, adding one-fifth dry weight of sterile tertiary water, shaking uniformly, and pouring into a separatory funnel. The extract is distributed, and the solution can be divided into three layers from top to bottom, respectively being dissolved in n-hexane layer, insoluble in n-hexane and water layer, and soluble in water. The top two layers were taken up in one tube, and the addition of n-hexane was carried out twice, and the lowermost layer was taken in one tube. The aqueous layer was then subjected to the same partition extraction with ethyl acetate three times. The obtained n-hexane layer, ethyl acetate layer, and the extract of ethyl acetate and n-hexane layer were placed in a fume hood and air-dried for two weeks, and then placed at -20 ° C for use.

1.2 High performance liquid chromatography analysis of sesame extract

In the extract obtained in the above-mentioned Embodiments 1 and 1, an ethanol extract is taken as an example, and a component in the solution is analyzed by High Performance Liquid Chromatography (HPLC). As a result of the analysis, it was found that the sesame extract contained 11 substances. Table 1 shows the results of high performance liquid chromatography analysis, please cooperate with the analysis map of Figure 1.

Figure 1 is an HPLC analysis of the ethanol extracted sesame extract. According to Table 1 and Figure 1, the ethanol extract of sesame was analyzed by HPLC, and there were 11 signal peaks, each of which was a substance. Among them, the 10th peak is sesame phenol, and the 11th peak is sesame.

Experimental Example 2 Functional analysis of sesame extract inhibiting acetylcholine receptor

Intracellular free calcium ions are known to be important secondary signals (calcium signals) and can be used to represent the function of the acetylcholine receptor. Since there are acetylcholine receptors on the nerve cells, including nicotine type and muscarinic type, the human neural embryonic tumor cell line SH-SY5Y can be used as a model system for detecting the function of acetylcholine receptors. The effect of a substance to be tested on the function of the acetylcholine receptor.

2.1 Analysis of the function of sesame extract to inhibit acetylcholine receptor

Treatment of cells with carbachol (CCh), a widely used stimulant of acetylcholine receptor, can cause an increase in intracellular free calcium concentration (calcium signal), and the calcium signal can represent acetylcholine. The function of the receiver.

Fig. 2 is an analysis of the sesame extract extracted by different polar solvents to inhibit the function of acetylcholine receptor; wherein the polar solvents used in Fig. 2 are (A) 80% acetone, (B) 80% ethanol, respectively. And (C) water.

The analysis of the function of acetylcholine receptor was carried out by using human embryonic tumor cell line SH-SY5Y cells loaded with calcium ion indicator (fura-2) as experimental materials, and classified into (a), (b), and (c). (d) Four groups were added with 4 different concentrations of sesame extract (abbreviated as E) at 50 seconds, and then 0.3 mM CCh was added as a stimulant at 100 seconds.

The concentration of sesame extract added in Fig. 2 (A) and (B) is the curves (a), (b), (c), and (d) are not added, 2 μg/ml, 20 μg/ml, and 200 μg, respectively. /ml; Figure 2 (C) added sesame extract concentration is the curve (a), (b), (c), (d) are not added, 5 μg / ml, 50 μg / ml and 500 μg / Ml.

The results of Fig. 2 (A), (B) and (C) show that the sesame extract extracted with acetone, ethanol or water can inhibit the calcium signal linked by the acetylcholine receptor and is dependent on the concentration. The (dose-dependent) method inhibits this calcium signal, that is, inhibits the function of the choline receptor.

Since the acetylcholine receptors include Nicotine acetylcholine receptors (nAChR) and muscarinic receptors. In order to understand which substances in sesame extract may have an inhibitory effect on nicotine-type or muscarinic acetylcholine receptors, the following will analyze individual substances in sesame extract for two different types of acetylcholine receptors. Impact.

2.2 Effect of sesame extract on nicotine-type acetylcholine receptor 2.2.1 Sesamin

Treatment of cells with nicotine-type acetylcholine receptor stimulating agent Epibatidine can cause an increase in intracellular free calcium concentration, and the calcium signal represents the function of the nicotine acetylcholine receptor.

Please refer to Figure 3A-3B. Figure 3A shows the analysis of the inhibition of nicotine-type acetylcholine receptor function by sesamin; and Figure 3B shows the statistical diagram of the calcium signal of Figure 3A.

The nicotine-type acetylcholine receptor function was analyzed by using fura-2-filled human neural embryonic tumor cell line SH-SY5Y as a material, and 10 μM of sesamin (SM in the figure) was added at 50 seconds, at 100 seconds. Add 10 μM of bombesin (referred to as Epi in the figure) as a stimulant. Sesamin inhibited the calcium signal linked by the nicotine-type acetylcholine receptor. The intracellular calcium ion concentration induced by 10 μM bombesin was 100%, and the ratio of 1-25 μM of sesamin was inhibited to inhibit the calcium signal induced by bombesin.

As can be seen from the results of Fig. 3A-3B, sesamin inhibits the calcium signal linked by the nicotine type acetylcholine receptor in a concentration-dependent manner, that is, inhibits the function of the nicotine acetylcholine receptor.

In addition, sesamin has no effect on the calcium signal caused by methotrexate methicillin stimulant methacholine, that is, sesamin does not affect the function of the muscarinic receptor.

2.2.2 Sesamol

Please refer to Fig. 4A-4B. Fig. 4A is a functional analysis of a nicotine type acetylcholine receptor for sesame phenol; and Fig. 4B is a calcium signal chart of Fig. 4A.

The analysis method of the effect of sesame phenol on the function of nicotine type acetylcholine receptor was carried out by adding 100 μM and 500 μM sesame phenol (abbreviated as SL) at 50 seconds in the same manner as shown in Fig. 3A-3B, and then at 100 10 μM of bombesin was added as a stimulant at the second. The inhibitory effect of sesamin on the calcium signal of nicotine-type acetylcholine receptor was 100%, and the ratio of 0, 100, 500 μM sesamin inhibited the calcium signal induced by bombesin. .

From the results of Fig. 4A-4B, it is known that sesame phenol inhibits the calcium signal linked by the nicotine type acetylcholine receptor in a concentration-dependent manner, that is, inhibits the function of the nicotine acetylcholine receptor.

In addition, sesame phenol has no effect on the calcium signal caused by the muscarinic acetylcholine receptor stimulant methyl acetylcholine.

Example 3 Effect of sesame extract on muscarinic acetylcholine receptor

From the results of the above Example 2, it was found that the sesame extract and the sesamin and the sesame phenol therein inhibited the function of the nicotine acetylcholine receptor, but did not affect the function of the muscarinic acetylcholine receptor.

Therefore, in order to understand whether the sesame extract containing 11 substances has an effect on the function of the muscarinic acetylcholine receptor, the muscarinic acetylcholine stimulant is treated with methyl acetylcholine. The cells were observed for the effect of sesame extract on the muscarinic acetylcholine receptor. Treatment of cells with methylcholine choline can cause an increase in intracellular free calcium ion concentration, and this calcium signal can represent the function of the acetylcholine receptor.

Referring to Fig. 5A, the sesame extract is used to inhibit the function of the muscarinic acetylcholine receptor; and Fig. 5B is the calcium signal chart of Fig. 5A.

The method for analyzing the function of the muscarinic acetylcholine receptor is to add the sesame extract (referred to as E) extracted with 80% ethanol at 50 seconds in the same manner as shown in Fig. 3A-3B. 0.3 mM methacholin (Meth for short) was added as a stimulant at 150 seconds.

In Fig. 5A, the concentration of the sesame extract extracted by ethanol is the curves (a), (b), (c) and (d), respectively, not added, 2 μg/ml, 20 μg/ml, 200 μg/ Ml. In Fig. 5B, the sesame extract inhibits the calcium signal linked to the muscarinic acetylcholine receptor by 300 μM methalin to induce a change in intracellular calcium concentration to 100%, respectively comparing 0.002- The 0.2 μM sesame extract inhibited the ratio of the calcium signal induced by bombesin.

From the results of the 5A-5B, it can be seen that the sesame extract can inhibit the calcium signal linked to the muscarinic acetylcholine receptor, that is, the function of inhibiting the muscarinic acetylcholine receptor, and the concentration dependence method Suppress this calcium signal.

From the above, it is understood that the action of the sesame extract having 11 substances is different from the action of sesamin and sesame phenol. Sesamin and sesame phenol only inhibit the action of nicotine acetylcholine receptor, and do not have the function of inhibiting muscarinic acetylcholine receptor, while sesame extract with 11 substances inhibits muscarinic bismuth The biological function of the alkali receptor. Therefore, the feasibility of applying sesame extract to diseases related to the function of muscarinic acetylcholine receptor can be further analyzed.

Example 4 Effect of sesame extract on the function of muscarinic receptor in lung tissue

Firstly, it was confirmed that the inhibitory effect of the substance in the sesame extract on the function of the muscarinic choline receptor was also present in the non-neurological acetylcholine receptor, and the human squamous lung cancer H520 filled with fura-2 was used as an experiment. For the material, sesame extract (E) was added at 50 seconds, and 0.3 mM of methyl choline (Meth) was added as a stimulating agent for 100 seconds.

Figure 6A is an analysis of the sesame extract extracted by different polar solvents to inhibit the muscarinic choline receptor in the lung tissue; wherein the polar solvent used in Figure 6 is (A) 80% acetone, (B ) 80% ethanol and (C) water. The concentration of sesame extract added in Fig. 6A (A) and (B) is curves (a), (b), (c) and (d), respectively, not added, 2 μg/ml, 20 μg/ml, 200 Gg/ml; Figure 6 (C) added sesame extract concentrations of (a), (b), (c) and (d), respectively, not added, 5 μg / ml, 50 μg / ml, 500 μg /ml.

The results of Fig. 6A (A), (B) and (C) show that the sesame extract extracted with acetone, ethanol or water can inhibit the calcium signal in a concentration-dependent manner, that is, the lung tissue chlorpyrifos. Choline receptor function.

Figure 6B is a graph showing the statistical changes in the inhibition of methotrexate-induced calcium signaling by extracts from different polar solvents. The calcium signal caused by 300 μM methyl acetylcholine was 100%. The results showed that the sesame extract extracted with ethanol inhibited the calcium signal most significantly.

The above results further demonstrate that sesame extract inhibits the function of muscarinic acetylcholine receptors, not only on nerve cells, but also in lung tissue cells.

Example 5 Sesame extract for prevention and treatment of overactive bladder

According to the above, it was confirmed that the sesame extract can inhibit the third type muscarinic acetylcholine receptor. Because human squamous lung cancer H520 cells only have a third type of muscarinic acetylcholine receptor, it can be used as a detection mode for the function of the third type of muscarinic acetylcholine receptor. The pharmacological effects associated with the function of the type III muscarinic acetylcholine receptor.

It is known that the overactive bladder is labeled as a third type muscarinic choline receptor, which can be treated with selective third-type muscarinic acetylcholine receptors with less clinical side effects. Darifenacin. According to the results of Fig. 6A-6B, it can be fully inferred that the sesame extract can completely inhibit the methacholine-stimulated sputum acetylcholine receptor on H520 cells, and thus has the potential to be used for the prevention and treatment of overactive bladder.

Example 6 Inhibition of sesame extract on acetylcholine receptors associated with canceration

The above results confirmed that the substance contained in the sesame extract has a function of inhibiting the activation of the muscarinic type and the nicotine type acetylcholine receptor. It is known that certain tumor cells having an acetylcholine receptor, such as lung cancer, bone cancer, rectal cancer, and liver cancer cells, are present in the acetylcholine receptor and have been shown to be associated with cancer.

For example, the Liu Peishan Laboratory of Soochow University published in 2010 the muscarinic acetylcholine receptor was present in osteosarcoma cell line, which can represent the proliferation of bone cancer. Stimulating the muscarinic acetylcholine receptor can promote the proliferation of this cell line, and inhibiting the muscarinic acetylcholine receptor can inhibit this proliferation.

Furthermore, according to the literature, acetylcholine receptors play an important role in the proliferation, survival and anti-apoptosis of lung cancer. For example, on lung cancer cell line H520, A549 or H1355, stimulation of choline receptors can cause cell proliferation and anti-apoptosis protection (Song et al., 2003; Xu and Deng, 2006; Song et al., 2008; Zhagn Et al., 2010). In addition, according to the literature, acetylcholine receptors also play an important role in the proliferation, survival and anti-apoptosis of rectal cancer (Xie et al., 2004; Ye et al., 2004; Xin and Deng, 2005; Arredondo et Al., 2007).

Therefore, in order to confirm whether the substance contained in the sesame extract of the embodiment of the present invention can inhibit the activation of the acetylcholine receptor, the effect of improving the efficacy of the cancer therapeutic drug can be improved. The substance contained in the extract has a function of inhibiting the growth of cancer cells.

Example 7 Inhibition of sesame extract on the proliferative benefit of muscarinic acetylcholine receptor in lung tissue

According to the results of Fig. 6A-6B, it has been confirmed that the sesame extract can inhibit the scorpion acetylcholine receptor signal, and the sesame extract can further investigate the inhibition of muscarinic acetylcholine by cell viability analysis. The mechanism of the link to the proliferation.

It is known that methionine choline receptor is stimulated by methotrexate to promote cell proliferation of lung cancer cells. To determine whether the substance in the sesame extract can inhibit the promotion of lung cell proliferation induced by methacholine (Meth), human squamous lung cancer H520 cells were used as experimental materials, and the cell survival rate analysis (Method of transcriptional) The MTT assay method detects the number of cells and compares the effect of adding sesame extract on cell growth.

Please refer to Figure 7A-7B for a comparison of the effect of adding sesame extract on cell growth after stimulation with methionine choline. Figure 7A shows the effect of cell hyperplasia after stimulating muscarinic choline receptor with methotrexate and inhibition of cell proliferation after the use of the inhibitor atropine. The cell viability analysis method was carried out by culturing a human squamous lung cancer H520 cell (5× ^{10 4} wells/plate) cultured in an 8-well plate in a 96-well plate. After culturing for 6 hours, the cells were adhered to a serum-free culture for 24 hours. In addition to serum, 1 μM of belladonna was added for 1 hour (the blank group was serum-free for 1 hour), and then cultured with different concentrations of methotrexate for 30 hours. Finally, cell viability was confirmed by MTT assay. The "*" in the figure indicates that the t-test analysis between groups is statistically significant, and the p value is less than 0.05. Fig. 7B is a view showing inhibition of cell proliferation by using the sesame extract of the present invention after stimulating the muscarinic choline receptor with methyl acetylcholine. Using the same cell culture method as in Fig. 7A, the effect on cell proliferation was observed after serum-free culture and addition of sesame extract for 72 hours, and the results of Figures 7A-7B were the sum of the results of the three batches of cells.

The results of Figure 7A illustrate the effect of treatment with methotrexate on cell proliferation, which can be inhibited by muscarinic acetylcholine receptor inhibitor belladonna; Figure 7B can be demonstrated by methyl acetamidine The cytostatic effect of choline-stimulated muscarinic choline receptor can be inhibited by the sesame extract of the examples of the present invention.

real Example 8 Effect of sesame extract on the efficacy of cancer medication

It is known that activation of the nicotine type acetylcholine receptor induces an anti-apoptotic pathway in cells, resulting in a decrease in the efficacy of cancer administration. Therefore, since the substance in the sesame extract can inhibit the activation of the nicotine type acetylcholine receptor, it is possible to further analyze whether the sesame extract can enhance or restore the efficacy of the cancer administration.

Please refer to Figure 8A-8B for the purpose of explaining that the human lung cancer cell line A549 treated with the apoptosis promoter camptothecin (CPT) causes a decrease in cell viability and is caused by CPT after being stimulated by bombesin. The inhibition of cell death. CPT is a cancer treatment drug that inhibits the activity of DNA topoisomerase I and causes apoptosis of cancer cells. Figure 8A is a cell viability analysis of CPT treatment to illustrate the effect of CPT on cell survival. The analysis method was to observe the cell viability by the same MTT assay as in Fig. 7A, and to treat DMSO (1%) cells as 100%. Figure 8B is a graph showing the inhibition of CPT-induced cell death by bombesin. The analysis method was to observe the cell survival rate by the same MTT analysis as in Fig. 8A; the cell death rate was 100% due to cell death caused by CPT. The results are the sum of the results of the three batches of cells. The "*" and "**" in the figure indicate that the t-test analysis between the groups is significant, and the p values are less than 0.05 and 0.01.

From the results of Fig. 8A, it can be seen that the cell survival rate of CPT-treated cells was reduced to about 60%. The results of Fig. 8B show that bombardin can significantly inhibit CPT-induced cell death at 1, 5 or 10 μM, indicating that stimulation of nicotine acetylcholine receptor can reduce apoptosis induced by CPT. It can be understood from Fig. 8A-8B that CPT can cause human lung cancer death by activating an apoptotic pathway, and that 20 μM CPT can cause significant cell death, while in the protection of 1 μM bombesin, a significant lethal loss of cellular CPT.

Therefore, if you want to know the substances in sesame extract, including sesamin, sesame phenol and sesame extract containing 11 substances, whether it can inhibit the acetylcholine receptor and the effect of the protection of the scutellarin can be lost. The cells were cultured in the same manner as in Fig. 8B, and the cells were treated with sesamin, sesame phenol and sesame extract containing 11 substances to confirm their individual effects.

Fig. 8C (A), (B), and (C) are the effects of sesame, sesame phenol, and sesame extract extracted with ethanol for 24 hours on the apoptosis of human lung cancer cell line A549 induced by bombardin. analysis. The results shown are the sum of the results of the three batches of cells. The "*" in the figure indicates that it is significant compared with the t-test analysis of the control group, p < 0.05. From the results of Fig. 8C (A), (B), (C), it can be seen that when the sesamin, sesame phenol or sesame extract is added, the inhibitory effect of the acetylcholine receptor can be produced to make the frog skin The protective effect on cells is lost.

Based on the above results, it can be seen that the substances in the sesame extract, including sesamin, sesame phenol and sesame extract containing 11 substances, can be promoted by the action of inhibiting the action of the acetylcholine receptor to assist the cancer drug camptothecin. The benefits of cancer cell apoptosis.

Example 9 Effect of Sesamin on the Chemotherapy of 5-Fluorouracil

5-Fluorouracil is a pyrimidine analog that can cause apoptosis by interfering with DNA synthesis and is a common cancer chemotherapy drug.

Fig. 9 is a graph showing the effect of 5-fluorouracil on cell survival, and the test results of 5-fluorouracil-assisted effect after co-setion treatment with sesamin and 5-fluorouracil. The results in Figure 9 illustrate the effect of 5-fluorouracil on cell survival. 10 nM 5-fluorouracil can cause a significant decrease in cell survival, but in the presence of nicotine-stimulated acetylcholine receptors, cells can be exposed. Protection so that the lethal effect is no longer significant. On the other hand, the results of Fig. 9 confirmed that sesamin can effectively reduce the cell viability by inhibiting the activity of the acetylcholine receptor when it is used together with 5-fluorouracil, and has an auxiliary effect on the therapeutic effect of 5-fluorouracil. Based on this result, it can be seen that sesamin can be used as an adjuvant cancer chemotherapy drug 5-fluorouracil.

Example 10 Effect of Sesamin on Cisplatin in Cancer Chemotherapy

Cisplatin is a common cancer chemotherapy drug that binds to the base of DNA to cause apoptosis. When cisplatin is stimulated by the bombesin, the effect of apoptosis is lost.

Figure 10 is the effect of cisplatin on cell survival, and the results of the test for the adjuvant effect of cisplatin for 48 hours after co-setion treatment with sesamin and cisplatin. Figure 10 illustrates that cisplatin can cause a decrease in cell survival, but in the presence of bombesin to stimulate the acetylcholine receptor, the cells can be protected and the lethal effect of cisplatin is lost. On the other hand, when sesamin is used together with cisplatin, it can effectively reduce cell viability by inhibiting the activity of acetylcholine receptor, and has an auxiliary effect on the therapeutic effect of cisplatin. That is, sesamin can be used as an auxiliary cisplatin.

Although the present invention has been disclosed in the above embodiments, it is not intended to limit the present invention, and the present invention can be modified and modified without departing from the spirit and scope of the present invention. The scope is subject to the definition of the scope of the patent application attached.

The above and other objects, features, advantages and embodiments of the present invention will become more apparent and understood.

Figure 1 is an HPLC analysis of the ethanol extracted sesame extract.

Figure 2 is an analysis of the sesame extract extracted by different polar solvents to inhibit the function of acetylcholine receptor.

Fig. 3A is an analysis of the inhibition of nicotine type acetylcholine receptor function by sesamin; and Fig. 3B is a statistical diagram of the calcium signal of the result of Fig. 3A.

Fig. 4A is an analysis of the function of the nicotine type acetylcholine receptor for sesame phenol; and Fig. 4B is a statistical diagram of the calcium signal of the result of Fig. 3A.

Fig. 5A is an analysis of the sesame extract inhibiting the function of the muscarinic acetylcholine receptor; Fig. 5B is a statistical diagram of the calcium signal of the result of Fig. 5A.

Figure 6A shows the analysis of the sesame extract extracted by different polar solvents to inhibit the muscarinic choline receptor in lung tissue; the 6B is the statistical change of the inhibition of methotrexate-induced calcium signaling by each polar solvent extract. Figure.

Fig. 7A is the effect of cell hyperplasia after stimulating muscarinic choline receptor with methotrexate and inhibiting cell proliferation after using the inhibitor belladonna; Figure 7B shows methyl acetylcholine The base inhibits the cytotoxic acetylcholine receptor and inhibits cell proliferation after treatment with sesame extract.

Figure 8A shows the cell viability analysis by CPT treatment to illustrate the effect of CPT inhibition on cell survival; Figure 8B shows the inhibition of CPT-induced cell death by bombesin; Figure 8C shows sesamin, Sesame phenol and sesame extract extracted with ethanol were treated for 24 hours, and the effect of bombesin on the apoptosis of human lung cancer strain A549 induced by CPT was inhibited.

Figure 9 shows the effect of 5-fluorouracil on cell survival and the adjuvant effect of co-serum treatment with 5-fluorouracil.

Figure 10 is the effect of cisplatin on cell survival, and the results of the test for the adjuvant effect of cisplatin for 48 hours after co-setion treatment with sesamin and cisplatin.

Claims (7)

A pharmaceutical composition for inhibiting proliferation of cancer cells caused by activation of an acetylcholine receptor, comprising an effective amount of sesame extract, a chemotherapeutic agent, and a pharmaceutically acceptable carrier thereof, wherein the chemotherapeutic agent is Camptothecin, 5-fluorouracil or cisplatin. The pharmaceutical composition according to claim 1, wherein the sesame extract comprises sesamin and sesamol. The pharmaceutical composition according to claim 1, wherein the cancer cell is derived from human leukemia, muscle tumor, bone cancer, lymphoma, melanoma, ovarian cancer, skin cancer, testicular cancer, gastric cancer, pancreatic cancer, renal cancer , breast cancer, prostate cancer, rectal cancer, cancer of the head and neck, brain cancer, esophageal cancer, bladder cancer, adrenocortical carcinoma, lung cancer, bronchial cancer, endometrial cancer, cervical cancer or liver cancer. The pharmaceutical composition according to claim 1, wherein the acetylcholine receptor is a nicotine type acetylcholine receptor or a muscarinic acetylcholine receptor. The pharmaceutical composition according to claim 1, wherein the sesame extract is obtained by a method comprising the steps of: mixing sesame seeds with a polar solvent in a ratio of about 1:3 by weight: and filtering to obtain a filtrate. And concentrating the filtrate under reduced pressure to remove the polar solvent, and obtaining the sesame extract Take things. The pharmaceutical composition according to claim 5, wherein the polar solvent is water, an alcohol solvent, a ketone solvent or a mixture thereof. A use of a sesame extract for the preparation of a medicament for inhibiting the proliferation of cancer cells caused by activation of an acetylcholine receptor.