KR20220094941A - Pharmaceutical composition for prevention and treatment of pancreatic cancer comprising extracts of Tamarindus indica L. hull - Google Patents

Abstract

The present invention relates to an anti-cancer composition containing a Tamarindus indica bark extract as an active component. According to the present invention, the present invention relates to a compound having a remarkable cell proliferation inhibitory effect on human cancer cells, particularly human pancreatic cancer cells. More specifically, the Tamarindus indica bark extract is composed of sugars (32.5%), flavonoids (17.4%), phenolic compounds (20.32%), and proteins (11.48%), and the Tamarindus indica bark extract exhibits effects of inhibiting cell proliferation and killing cancer cells through the regulation of the expression and activation of proteins related to apoptosis in human pancreatic cancer cells, so that a pharmacological composition containing a natural material exhibits an anti-cancer effect is invented.

Description

Pharmaceutical composition for prevention and treatment of pancreatic cancer comprising extracts of tamarind bark {Pharmaceutical composition for prevention and treatment of pancreatic cancer comprising extracts of Tamarindus indica L. hull}

The present invention relates to a pharmaceutical composition for the prevention and treatment of pancreatic cancer comprising a tamarind bark extract.

According to Statistics Korea, as of 2017, the number of cancer cases was 109,963, and the age-standardized incidence rate per 100,000 people was 209.8, which showed a downward trend overall compared to the previous year. was seated Despite advances in cancer diagnosis and treatment technology according to scientific advances, cancer is still considered as one of the greatest challenges that modern medicine has to overcome. The International Agency for Research on Cancer under the World Health Organization (WHO) predicted that the number of cancer cases and deaths will increase by 1% every year, resulting in 25.4 million cancer cases and 16.4 million deaths in 2030.

Resection surgery and radiation therapy are representative cancer treatments, but it is difficult to precisely remove cancer cells and has limitations in metastasis to other tissues. The disadvantage is that it cannot be overlooked. The anticancer agent proposed as a solution to the above problem has been developed over the past several decades from the first generation of chemotherapy, the second generation of targeted anticancer agents, and even the third generation of immunotherapy. However, the first-generation anticancer drugs damage not only cancer cells but also normal cells, so side effects such as hair loss, vomiting, decreased physical strength, and fatigue occur. Although it has the advantage of killing cancer cells and reducing side effects on normal cells by inhibiting the growth factors of cancer cells, there are still problems to be solved in that the scope of application is limited to some carcinomas and drug resistance occurs. The third-generation immunotherapy shows a mechanism to kill cancer cells by activating the patient's immune system itself, which has been suppressed, and refers to an inhibitor of the defense mechanism that cancer cells have to avoid attack by immune cells. Because it strengthens and uses the patient's immune system, there are fewer side effects compared to the first or second generation anticancer drugs, and in particular, because it uses the memory and adaptability of the immune system, patients who are effective with immune anticancer drugs can see continuous anticancer effects. However, on the other hand, the possibility that it may cause side effects such as hyperimmune reaction is also suggested.

Pancreatic cancer is a cancer that has been steadily increasing in incidence since 1999. According to the national cancer registration statistics, as of 2017, pancreatic cancer accounted for 3% of all cancers, accounting for the 8th. ) and stomach cancer (14.9%), occupying fifth place. In addition, the incidence of gastric cancer, colorectal cancer, thyroid cancer, lung cancer (male), and liver cancer has recently decreased, whereas the incidence rate of pancreatic cancer has been increasing since 1999. This means that although the incidence of pancreatic cancer is not very high compared to other cancers, it has been steadily increasing for 10 years, and once it occurs, the mortality rate is very high.

Pancreatic cancer is almost impossible to operate due to its anatomical characteristics, and even with surgery, it has a high recurrence rate, so it is a cancer with a poor survival rate. In addition, early detection is not easy, and at the time of detection, it is often the case that the cancer has progressed considerably. The pancreas can be divided into head, trunk, and tail. Unlike other cancers, the prognosis of treatment depends on the location of the site of occurrence rather than the stage of progression. Even at a low stage, if the location of the cancer is not good, the prognosis after surgery is poor, and even after surgery, the biliary tract, liver, duodenum, stomach, and gallbladder must be excised at the same time, so postoperative sequelae can remain severe. In fact, only 20% of all pancreatic cancer patients can be operated on, and the remaining 80% undergo chemotherapy. In the past, there were only one or two pancreatic cancer treatments that could be applied to pancreatic cancer, and the response rate was less than 10% when anticancer monotherapy was administered. Most of the patients diagnosed with cancer were almost impossible to undergo surgery at stage 3 or 4, and the anticancer drug response rate was also very low. Recently, as various anticancer drugs have been developed, anticancer combination therapy, which administers 2 to 4 anticancer drugs at once, is being applied to the treatment of pancreatic cancer as a new treatment concept. However, although the anticancer combination therapy has the advantage of having a high therapeutic effect, it has a weakness in that the side effects are more severe than when the anticancer monotherapy is implemented. Therapeutic agents currently used for the treatment of pancreatic cancer are “Gemcitabine (cancer cell death and growth inhibition) and Abraxane (damage DNA required for cell division)”, “Folpirinox [5-FU (cancer cell death), oxaliplatin (in cancer cells)” DNA attack), irinotecan (inhibits growth by damaging cancer cells or DNA), and leucovorin (reduced toxicity by combination of three anticancer drugs)]”, etc. However, these drugs for pancreatic cancer may have side effects, which may significantly reduce the patient's quality of life, and in severe cases may even require discontinuation of the treatment itself. In addition, although most of pancreatic cancer patients receive Abraxane and gemcitabine anticancer combination therapy, some patients have fewer side effects and good treatment response rates. Therefore, there is a continuous need for research on discovering new therapeutic agents that can supplement the toxicity and side effects of existing therapeutic agents.

The fruits of Tamarindus indica L., a plant belonging to the legume family, are club-shaped, purple-brown, slightly curved, 7-20 cm long and 1.5 cm wide, and the skin of the fruit is thin and weak. , there is a soft large brown flesh inside, and brown, glossy seeds are contained in it. Seeds have a flat quadrilateral shape with a length of about 1-1.5 cm and a thickness of about 4 mm. Tamarind pulp has a sweet and sour taste, and is added to food as a spice other than raw food. Alternatively, it is used as a beverage by dissolving ground flesh in water and adding sugar. In addition, there is an endosperm in the seed, and the endosperm contains polysaccharides as a mass of cells with a size of about 40 to 80 μm. Tamarind seed sum is widely used in food preparation. Tamarind, the flesh of which is used as an anti-scurvy drug, an antipyretic drug, an analgesic agent, an antirheumatic drug, a hemorrhoid treatment drug, etc.; Seeds as a medicinal remedy; Although flowers and leaves have been used as folk medicines such as bath preparations, there have been no reports of cell proliferation inhibitory activity against pancreatic cancer cells. Therefore, the present inventors confirmed that the tamarind bark extract has excellent cell growth inhibition and apoptosis inducing ability for human pancreatic cancer cells, so that it can be utilized as an anticancer composition and completed the present invention.

The present inventors tried to develop a novel health functional material by identifying the new health functionality of the extract extracted from tamarind peel. As a result, the present invention was completed by elucidating the apoptosis mechanism in which the tamarind bark extract inhibits cancer cell proliferation in cancer, particularly pancreatic cancer. Accordingly, an object of the present invention is to provide a composition for anticancer. Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

The present invention may provide a pharmaceutical composition for the prevention and treatment of pancreatic cancer comprising a tamarind bark extract.

The present inventors obtained an extract by extracting the tamarind peel using alcohol. As a result of treating the tamarind bark extract on PANC-1 cells, a human pancreatic cancer cell line, a cell growth inhibitory effect of about 57% was exhibited at a concentration of 100 μg/ml ( FIG. 1 ). As a result of treatment with the tamarind bark extract, apoptosis forms such as cell number reduction, cell body condensation, neurite loss, segmentation, and cell membrane blistering were increased ( FIG. 2 ). The expression levels of cleaved-PARP (Poly ADP ribose polymerase), cleaved-caspase-3 and cleaved-caspase-9, known as apoptosis-related factors, increased in a concentration-dependent manner in the tamarind bark extract, while the expression level of procaspase-9 decreased. (Fig. 3). It plays a pivotal role in the apoptosis pathway, and its reduction increased as the concentration of tamarind bark extract increased, the expression level of Bax protein associated with high tumor malignancy and resistance to chemotherapy or radiotherapy ( FIG. 4 ). When the tamarind bark extract was treated, the mitochondrial membrane potential was reduced by 0.5 times, confirming that the mitochondrial membrane potential of pancreatic cancer cells was disrupted (FIG. 4).

As used herein, the term "extract" refers to an active ingredient separated from a natural product. The extract can be obtained by an extraction process using water, an organic solvent, or a mixed solvent thereof, and an extract, a dry powder thereof, or using the same Including all formulated forms.

The tamarind bark extract may be extracted with any one selected from the group consisting of alcohol, water, alcohols having 1 to 6 carbon atoms, and mixed solvents thereof, preferably extracted with alcohol, and the alcohol extract is purified water 50 % to 90% and a solvent composed of 10 to 50% alcohol, preferably 70% purified water and 30% alcohol.

According to a preferred embodiment of the present invention, the tamarind bark extract can inhibit the cell proliferation of human pancreatic cancer cells.

According to a preferred embodiment of the present invention, the tamarind bark extract may induce apoptosis against pancreatic cancer cells.

According to a preferred embodiment of the present invention, the tamarind bark extract can increase the expression level of the PARP protein in the cell.

According to a preferred embodiment of the present invention, the tamarind bark extract can increase the expression level of caspase protein in the cell.

According to a preferred embodiment of the present invention, the tamarind bark extract can increase the expression level of Bax protein, which is a mitochondria-dependent apoptosis-related protein, and increase the mitochondrial membrane potential collapse.

According to a preferred embodiment of the present invention, in the composition, the concentration of the tamarind bark extract may be 10 μg/ml to 100 μg/ml.

The composition may include a pharmaceutically acceptable carrier or diluent. "Pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable substance, such as a liquid or solid filler, diluent, excipient or solvent, which serves to transport the active ingredient from one organ or part of the body to another organ or part of the body; composition or vehicle.

The composition for treating pancreatic cancer of the present invention may be prepared as a pharmaceutical by adding one or more pharmaceutically acceptable carriers together with the active ingredient. The carrier includes, but is not limited to, saline, buffered saline, water, glycerol and ethanol, and any suitable formulation known in the art (Remingtons' Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA) can be used. .

The formulation for medicating the extract, which is a fragment of the present invention, can be administered orally during clinical administration and can be used in the form of general pharmaceutical formulations, and when formulated, commonly used fillers, extenders, binders, wetting agents, and disintegrants. , prepared by using a diluent or excipient such as a surfactant. Solid formulations for oral administration include tablets, pills, powders, granules, and capsules, and liquid formulations for oral administration include suspensions, internal solutions, emulsions, and syrups. In addition to liquid and paraffin, various excipients, such as wetting agents, sweetening agents, fragrances, preservatives, and the like may be included.

The composition of the present invention may be administered in various parenteral formulations during actual clinical administration. Solid formulations include tablets, pills, powders, granules, capsules, etc., and liquid formulations include suspensions, internal solutions, and emulsions. , syrup, etc., in addition to water, liquid, and paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. may be included. Specifically, preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, etc. may be used. In addition, calcium or vitamin D3 may be added to enhance the efficacy of the therapeutic agent. Such compositions may be presented in unit-dose (single-dose) or multi-dose (several-dose) containers, such as sealed ampoules and vials, and, immediately prior to use, in a sterile liquid carrier, e.g., veterinary for injection. It can be stored under freeze-drying conditions requiring only addition. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

The formulation of the present invention may be applied differently depending on the age, sex, condition of the subject, absorption of the active ingredient in the body, inactivation rate and excretion rate, and the drug used in combination. The present invention also includes dosage unit formulations. Formulations are presented in individual dosage forms, for example tablets, coated tablets, capsules, pills, suppositories and ampoules, wherein the content of the active compound in the medicament corresponds to a fraction or multiple of the individual dosage. Dosage units may contain, for example, one, two, three or four times, or one-half, one-third or one-quarter of the individual dosage. Individual dosages preferably contain an amount of the active compound administered in one dose, which is usually all, one-half, one-third or one-quarter times the daily dosage.

The present invention can provide a health functional food composition for the prevention and improvement of pancreatic cancer comprising a tamarind bark extract. The functional food of the present invention is not particularly limited in its formulation, for example, dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, and ice cream; There are various soups, beverages, teas, drinks, alcoholic beverages and vitamin complexes, and includes all fat foods in the ordinary sense.

In the present invention, "functional food" means a food manufactured and processed using raw materials or ingredients useful for the human body according to Health Functional Food Act No. 6727, and "functionality" means the structure of the human body. And it means ingestion for the purpose of obtaining useful effects for health purposes such as regulating nutrients for function or physiological action.

The tamarind bark extract of the present invention has an effect of inhibiting cell growth against pancreatic cancer cells, and increases apoptosis forms such as cell number reduction, cell body condensation, neurite loss, segmentation, and cell membrane blistering, and is an apoptosis-related factor. The expression levels of known cleaved-PARP (Poly ADP ribose polymerase), cleaved-caspase-3 and cleaved-caspase-9 increased, and the expression level of procaspase-9 decreased, and the Bax protein, which plays a central role in the apoptosis pathway, was When the tamarind bark extract is treated, the mitochondrial membrane potential is reduced by 0.5 times, and the mitochondrial membrane potential of pancreatic cancer cells is disrupted to induce apoptosis, thereby having an anticancer effect.

Figure 1A shows a graph confirming the anticancer effect using a CCK assay after processing and culturing tamarind bark extract in human pancreatic cancer cells (PANC-1 cells, human pancreatic cancer cells).
Figure 1B shows a graph confirming the cytotoxicity to normal cells using the CCK assay after processing and culturing tamarind bark extract in monkey kidney cells (Vero cells, Monkey kidney cells).
Figure 2 shows the morphological change of cells after tamarind bark extract is treated and cultured in human pancreatic cancer cells.
3 shows changes in the expression levels of cleaved-PARP, caspase-9 and caspase-3 in cancer cells using a Western blot method after tamarind bark extract was treated and cultured in human pancreatic cancer cells.
Figure 4A shows changes in the expression levels of Bax and Bcl-2 in cancer cells using a Western blot method after tamarind bark extract was treated and cultured in human pancreatic cancer cells.
Figure 4B shows a change in the mitochondrial membrane potential (Mitochondrial membrane potential) in cancer cells using the JC-1 staining method after treating and culturing human pancreatic cancer cells with tamarind bark extract.
5 shows the results of quantitative analysis of total sugar, total protein, total phenolic compounds, and total flavonoids for component analysis of TPE of tamarind peel extract.
6 is a graph showing the flavonoid composition of the tamarind bark extract.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and in accordance with the gist of the present invention, the scope of the present invention is not limited by these examples. It will be apparent to one of ordinary skill in the art.

Example 1: Extraction of active ingredients from tamarind

Buy tamarind from Vietnam ( Tamarindus indica ), separate only the tamarind peel, add 800 ml (10 times) of a solution of 70% purified water and 30% alcohol to 80 g of tamarind peel and add it at 60 ~ 80℃ Circulation extraction was carried out while stirring for 3 to 6 hours in a , and the extract was cooled to 30 to 35 °C. After filtering the extract using an extract cloth, the filtrate was centrifuged again (8,000 rpm) to remove the tamarind extract residue, and then the residual alcohol was removed through vacuum concentration under reduced pressure (60~70℃), and the obtained supernatant was 0.45. After final filtration using a μM syringe filter, the filtrate was concentrated and dried with a speed vacuum concentrator (Operon Korea, SVQ-120) to obtain 10 g of a solid extract, which was named “TPE”.

Example 2: Inhibitory effect on cancer cell growth

The effect of TPE on human pancreatic cancer cell lines (PANC-1 cells) and normal kidney cells (Vero cells) was tested according to the CCK assay (D-Plus™ CCK cell viability assay kit, Dongin LS). Pancreatic cancer cells (PANC-1) and normal kidney cells (Vero) were cultured at a density of 1x10 ⁴ cells/well in a 96-well culture plate for 24 hours at 37° C. and 5% CO ₂ condition. Thereafter, TPE was prepared at concentrations of 10, 25, 50 and 100 μg/ml and treated with cells. After incubation for 48 hours, 10 μl of CCK reagent was added and reacted for 30 minutes. After the reaction, the absorbance was measured at 450 nm using a microplate reader. As a positive control group, 5FU (5-Fluorouracil, Enzo Life Sciences, USA), which is currently used for pancreatic cancer, was treated at a concentration of 200 μM. When TPE was treated at concentrations of 10, 25, 50 and 100 μg/ml, it was confirmed that the cell viability decreased in a concentration-dependent manner in pancreatic cancer cells. At a concentration of 100 μg/ml, TPE inhibited cell growth by about 57%. effect was shown (FIG. 1A), and showed a lower cell growth inhibitory effect (20%) than in pancreatic cancer cells at the same concentration in normal cells (FIG. 1B).

Example 3: Changes in cell morphology

In order to confirm the morphological change of cancer cells by TPE, it was observed with an optical microscope. Pancreatic cancer cells (PANC-1) were cultured in a 6-well culture plate at a density of 2×10 ⁵ cells/well for 24 hours, and TPE was prepared at concentrations of 10, 25, 50 and 100 μg/ml and treated with the cells. . After culturing for 48 hours, the morphology of the cells was observed under an optical microscope (×40). After treatment with TPE, it was seen that the number of cells decreased, cell body condensation, neurite loss, segmentation, cell death patterns such as cell membrane blistering were increased when compared to the untreated control group (FIG. 2).

Example 4: Cell death analysis using Western blotting

Caspase, a key protein in apoptosis, is known to induce cell shape changes, DNA fragmentation and PARP activation by cleavage of the protein. To investigate the cancer cell death mechanism of TPE, the protein expression levels of Caspase and Cleaved-PARP in pancreatic cancer cells were investigated by Western blot method. Pancreatic cancer cells (PANC-1) were cultured in a 6-well plate for 24 hours, and TPE was prepared at concentrations of 10, 25, 50 and 100 μg/ml, and the cells were treated for 48 hours. After washing with PBS, each of 70 μl of lysis buffer was added, reacted on ice for 30 minutes, and centrifuged at 13,000 rpm for 15 minutes to harvest proteins. Proteins were quantified using the Bradford assay, and sample loading buffer was added, denatured at 95°C for 10 minutes, and then electrophoresed using 10% SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) gel. After transferring to a nitrocellulose (NC) membrane, blocking with 5% nonfat milk powder at room temperature for 1 hour 1,000), anti-caspase-3 antibody (1:1,000), and anti-beta actin antibody (1:1,000) were diluted in 5% skim milk powder at respective ratios and reacted at 4°C overnight. After washing 3 times every 10 minutes with TBST (TBS, 1% Tween 20), the HRP (horseradish peroxidase)-conjugated secondary antibody was diluted 1:2000 in 5% skim milk powder and reacted at room temperature for 2 hours. After washing with TBST three times in the same way as above, ECL (Enhanced Chemiluminescence) substrate solution was reacted with the membrane for 1 minute, and then developed on the X-ray film. As a result of the Western blot experiment, the expression levels of cleaved-PARP (Poly ADP ribose polymerase), cleaved-caspase-3 and cleaved-caspase-9, which are known as apoptosis-related factors, increased in a concentration-dependent manner according to the treatment of TPE, and also procaspase It was confirmed that the expression level of -9 was decreased (FIG. 3).

Example 5: Investigation of apoptosis mechanism

To determine whether TPE affects the expression of mitochondrial-dependent apoptotic proteins in cells, the expression level of Bax protein in pancreatic cancer cells was investigated by Western blot method. Pancreatic cancer cells (PANC-1) were cultured in a 6-well plate for 24 hours, and SHP was prepared at concentrations of 10, 25, 50 and 100 μg/ml, and the cells were treated for 48 hours. After washing with PBS, each of 70 μl of lysis buffer was added, reacted on ice for 30 minutes, and centrifuged at 13,000 rpm for 15 minutes to harvest proteins. Proteins were quantified using the Bradford assay, and sample loading buffer was added, denatured at 95°C for 10 minutes, and then electrophoresed using 10% SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) gel. After transferring to a nitrocellulose (NC) membrane, blocking with 5% nonfat dry milk for 1 hour at room temperature, primary anti-Bax antibody (1:1,000), anti-beta actin antibody (1:1,000) was diluted in 5% skim milk powder at each ratio and reacted overnight at 4°C. After washing 3 times every 10 minutes with TBST (TBS, 1% Tween 20), the HRP (horseradish peroxidase)-conjugated secondary antibody was diluted 1:2000 in 5% skim milk powder and reacted at room temperature for 2 hours. After washing with TBST three times in the same way as above, ECL (Enhanced Chemiluminescence) substrate solution was reacted with the membrane for 1 minute, and then developed on the X-ray film. Bax protein is mainly present in the cytoplasm, but when stress such as DNA damage is transmitted, it moves to the outer mitochondrial membrane and causes a conformational change of the outer mitochondrial membrane, thereby inducing apoptosis by releasing cytochrome C and other pre-suicide effectors. Therefore, it is reported that Bax protein plays a pivotal role in the apoptosis pathway, and its reduction is associated with high tumor malignancy and resistance to chemotherapy or radiation therapy. Accordingly, many studies have been conducted to show the anticancer effect by activating the action of the Bax protein. As a result of the Western blot experiment, it was confirmed that the expression level of the Bax protein increased as the concentration of TPE treated in the cells increased, indicating that TPE disrupts the mitochondrial function and induces apoptosis (apoptosis) of cancer cells. (Fig. 4A).

Example 6: Investigation of mitochondrial dysfunction

Mitochondrial dysfunction is caused by the loss of the potential difference of the mitochondrial membrane, which is known to cause apoptosis by releasing cytochrome C into the cytoplasm to form apoptosomes with Apaf1 and Caspase9. Mitochondrial dysfunction is caused by activation of Bcl-2 family proteins or by loss of membrane potential difference due to oxidation of mitochondrial membrane pores by ROS (Reactive Oxygen Species) released from within mitochondria. Therefore, to measure whether the mitochondrial membrane potential of pancreatic cancer cells (PANC-1) was lost by TPE, JC-1(5,5',6,6'-tetrachloroc-1,1',3,3' tetraethylbenzimidazolylcarbocyanine iodide) mitochondrial membrane potential measurement kit was used. Pancreatic cancer cells (PANC-1) were cultured at a density of 1×10 ⁵ cells/ml in a 96-well plate at 37° C. and 5% CO ₂ conditions for 24 hours. After culturing, cells were treated with TPE at a concentration of 100, 200, or 500 μg/ml and cultured for 24 hours. After incubation, 100 μl of JC-1 was treated in each well and incubated at 37° C. for 30 minutes. Using a fluorometer, fluorescence intensity was measured for red fluorescence at excitation 550 nm and emission 600 nm, and green fluorescence for fluorescence excitation. Fluorescence intensity was measured at 550 nm and emission 600 nm. Red fluorescence is measured in all living cells, and green fluorescence is measured in cells that have died due to apoptosis. Therefore, the degree of membrane potential collapse of mitochondria can be confirmed as the ratio of red fluorescence and green fluorescence. As shown in FIG. 4B, when 100 μg/ml of TPE was treated compared to the control group, it was confirmed that the mitochondrial membrane potential was reduced by 0.5 times. This suggests that the mitochondrial membrane potential of pancreatic cancer cells is disrupted by TPE.

Example 6: Quantitative analysis of tamarind bark extract (TPE)

For component analysis of TPE, quantitative experiments were performed for total sugar, total protein, total phenolic compounds, and total flavonoids.

(1) Quantitative analysis of total sugar in TPE

Quantitative analysis of total sugar in TPE was performed by the phenol-sulfuric acid method. Glocose (99.9%, Sigma) was prepared at concentrations of 0, 0.125, 0.25, 0.5, and 1 mg/ml and used as a standard, and TPE was prepared at concentrations of 0.5 and 1 mg/ml and measured. 100 μl of each standard material and sample was dispensed into a glass test tube, 100 μl of 5% phenol solution was added, and 500 μl of sulfuric acid stock solution was added each, followed by reaction for 30 minutes after blocking the light. After dispensing 100 μl of the reaction material into a 96-well plate, absorbance was measured at 490 nm using a spectrophotometer (Molecular Devices, USA) to quantify total carbohydrates. As a result, TPE was 32.5% (w/w, mass ratio) A carbohydrate component was detected.

(2) Total protein quantitative analysis of TPE

Total protein quantitative analysis of TPE was performed by Bradford assay. BSA (Bovine Serum Albumin 98%, Sigma) was prepared at concentrations of 0, 0.00625, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4 mg/mL and used as a standard, and TPE was used at concentrations of 0.5 and 1 mg/ml. prepared and measured. After preparing Bradford reagent (Bio-rad) by diluting 1:4 in distilled water, 10 μl of each standard material and sample were dispensed in a 96-well plate, and 200 μl of the diluted Bradford reagent was added. Then, it was reacted on a micro-plate shaker for 30 seconds. Absorbance was measured at 595 nm using a spectrophotometer and as a result of total protein quantification, 11.48% (w/w, mass ratio) of protein was detected in TPE.

(3) Quantitative analysis of total phenolic compounds in TPE

Quantitative analysis of total phenolic compounds in TPE was performed by the Folin-denis method. Gallic acid (97.5-102.5%, Sigma) was prepared at concentrations of 0, 0.25, 0.5, and 1 mg/ml and used as a standard, and TPE was prepared at a concentration of 1 mg/ml and measured. 200 μl of each standard material and sample was dispensed into a glass test tube, 2 ml of 2% Na ₂ CO ₃ solution was added, and reacted for 2 minutes. Then, 200 μl of Folin&Ciocalteu's phenol reagent was added, followed by reaction for 30 minutes. After dispensing 100 μl of the reactant into a 96-well plate, absorbance was measured at 750 nm using a spectrophotometer to quantify total phenolic compounds. As a result, 20.32% (w/w, mass ratio) of phenolic compounds in TPE This was detected.

(4) Quantitative analysis of total flavonoids in TPE

Quantitative analysis of total flavonoids in TPE was performed in a modified manner of the Davis method. Quercetin (95%, Sigma) was prepared at concentrations of 0, 0.25, 0.5, and 1 mg/ml and used as a standard, and S2 was prepared at a concentration of 1 mg/ml and measured. In a 96-well plate, 20 μl of each standard and sample were dispensed, and 180 μl of diethylene glycol was added. 20 μl of 1N NaOH was additionally added and reacted at 37° C. for 1 hour to block light. Total flavonoids were quantified by measuring at 420 nm absorbance using a spectrophotometer. As a result of measuring the total flavonoid content of S2, it was confirmed that TPE had a flavonoid component of 17.4% (w/w, mass ratio). As a result of comparative analysis of the composition, it was confirmed that TPE contained 20.32% of phenolic compounds, including flavonoids, which accounted for 17.4% of the total mass along with 32.5% of sugar, and contained 11.48% of protein (FIG. 5).

Example 7: Flavonoid composition analysis of TPE

For flavonoid composition analysis of TPE, it was analyzed using an HPLC system (Waters Alliance HPLC 2695, USA). For HPLC analysis, 10 μl of 0.5% S2 solution in HPLC water was injected into a Luna® 5 μm C18(2) 100 Å (250 × 4.5mm, phenomenex) column, and 2% (v/v) acetic acid was Acetonitrile was eluted at a rate of 0.7 ml per minute by gradient elution and was detected at 254 nm with a PDA (Photodiode array, Waters 2414, Waters) detector. As standards, galic acid, catechin, EGCG, folin, quercetin, naringenin, and kaempferol were used, and all were purchased from Sigma (USA). As a result of the experiment, TPE was galic acid: 0.08% (w/w), catechin: 0.02% (w/w), EGCG: 0.03% (w/w), folin: 0.01% (w/w), quercetin: 0.01% ( w/w) and kaempferol: 0.01% (w/w) was confirmed to be contained (FIG. 6).

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (9)

A pharmaceutical composition for the prevention and treatment of pancreatic cancer, comprising a tamarind bark extract. The pharmaceutical composition for preventing and treating pancreatic cancer according to claim 1, wherein the tamarind bark extract is extracted with any one selected from the group consisting of alcohol, water, alcohols having 1 to 6 carbon atoms, and mixed solvents thereof.
The pharmaceutical composition for preventing and treating pancreatic cancer according to claim 1, wherein the tamarind bark extract inhibits cell proliferation of human pancreatic cancer cells. The pharmaceutical composition for preventing and treating pancreatic cancer according to claim 1, wherein the composition is 10 μg/ml to 100 μg/ml of tamarind bark extract. The pharmaceutical composition for preventing and treating pancreatic cancer according to claim 1, wherein the composition comprises a pharmaceutically acceptable carrier or diluent. The pharmaceutical composition for preventing and treating pancreatic cancer according to claim 1, wherein the composition exhibits the expression of Bax protein and disruption of mitochondrial membrane potential in human pancreatic cancer cells. A health functional food composition for the prevention and improvement of pancreatic cancer, comprising a tamarind bark extract. The health functional food composition for preventing and improving pancreatic cancer according to claim 7, wherein the tamarind bark extract is extracted with any one selected from the group consisting of water, alcohols having 1 to 6 carbon atoms, and mixed solvents thereof. The health functional food composition for preventing and improving pancreatic cancer according to claim 7, wherein the composition is 10 μg/ml to 100 μg/ml of tamarind bark extract.

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