TWI840296B - Preparation method of oral dosage form of tea stalk for preventing oral cancer and lung cancer - Google Patents

Abstract

A preparation method of oral dosage form of tea stalk for preventing oral cancer and lung cancer is disclosed. The tea stalk comprises gallic acid and a Tannase-producing bacteria, which is Aspergillus, Mucor, Actinomucor elegans, or Rhizopus chinensis., wherein the Aspergillus is Aspergillus niger, Aspergillus japonicus, Aspergillus oryzae, Aspergillus sojae, or combination thereof, the Mucor is Mucor hiemalis, Mucor silvaticus, Mucor prainii, or Mucor subtilissimus.

Description

Preparation method of tea branches for preparing oral dosage forms for preventing oral cancer and lung cancer

This invention relates to a preparation method, and in particular to a preparation method of tea branches for preparing oral dosage forms for preventing oral cancer and lung cancer.

Gallic acid (chemical name: 3,4,5-trihydroxybenzoic acid) is an organic acid that can be found in plants such as gallnut, golden berry, sumac, oak bark, and tea leaves. It is also called gallic acid or gallic acid. Gallic acid has phenol and carboxylic acid and is easily soluble in water, alcohol, and ether. Gallic acid can be synthesized into gallic acid ester compounds, such as methyl ester, ethyl ester, propyl ester, octyl ester, lauryl ester, octadecyl alcohol ester, etc. These ester compounds are all excellent food antioxidants, especially propyl gallate as an antioxidant, which can be used in edible oils to prevent rancidity and deterioration. Many scientific research results indicate that gallic acid has a variety of biological effects such as anti-inflammatory, anti-mutation, antioxidant, anti-free radical, antibacterial, and antiviral. At the same time, gallic acid has anti-tumor effects and can inhibit the metastasis of mast cell tumors, thereby prolonging survival.

Park et al. found that the growth of Calu-6 and A549 lung cancer cells was weakened after 24 hours of treatment with gallic acid and/or protease inhibitors. Yoon et al. found that gallic acid has pro-apoptotic and anti-inflammatory activities on fibroblast-like synovial cells (FLS) of rheumatoid arthritis, and may be used to treat rheumatoid arthritis in the future. Wang et al. found that gallic acid may have a protective effect on liver damage. Chao et al. found that mice taking gallic acid orally can resist hepatic fatty lesions, obesity and hypercholesterolemia caused by a high-fat diet. The study by Shahrzad et al. used tannic acid tablets (10% gallic acid and 90% glucose) and brewed black tea (containing 10% gallic acid, of which 93% gallic acid exists in a free state) to determine the pharmacokinetics of gallic acid and the relative availability of healthy humans. The results showed that gallic acid is rapidly absorbed in the human body, with half-lives of 1.19±007h for tablets and 1.06±006h for tea bags, respectively, indicating that the availability of gallic acid in the two is similar. The results of the study by Su et al. showed that gallic acid significantly inhibited the synthesis of melanin and the activity of tyrosinase in a dose- and time-dependent manner, and reduced the expression of melanin synthesis-related protein genes.

Oolong tea is a popular drink in China and Taiwan. It contains various bioactive ingredients, including polyphenols and phenolic acid. According to reports, aged oolong tea has better taste and better health benefits than newly made or fresh oolong tea. Generally, if the tea leaves are of good quality, stored for more than five years and roasted every year, they can be called aged tea. Aged oolong tea is made by a series of roasting fresh Tieguanyin oolong tea at 120-140℃ for 72 hours, followed by long-term storage for 5, 10 and 20 years. Liquid chromatography ultraviolet visible detection-tandem mass spectrometry was used to analyze the tea soup of old oolong tea, which confirmed that the catechin content of old oolong tea was reduced, while the gallic acid content was relatively increased.

From the above research results, it can be seen that old tea containing gallic acid does have health benefits. However, the formation time of old tea is too long, and the gallic acid content of each tea variety is difficult to control. In addition, the complex polysaccharides in water-soluble tea polysaccharides can significantly reduce blood sugar in the human body, achieving the effect of preventing and treating diabetes without any side effects. Among them, the blood sugar lowering effect of sulfated tea polysaccharides is more obvious, and it is expected to replace or cooperate with other diabetes drugs, and has a good market prospect.

In view of the above-mentioned problems in the prior art, one purpose of this invention is to provide a method for preparing tea branches for oral dosage forms for preventing oral cancer and lung cancer.

To achieve the above-mentioned purpose, the present invention provides a use of tea branches in preparing an oral dosage form for preventing oral cancer and lung cancer, wherein the tea branches are inoculated with bacteria and then post-fermented, and contain gallic acid and a tanninase-producing fungus selected from the genus Aspergillus , the genus Mucor , Actinomucor elegans or Rhizopus chinensis , wherein the genus Aspergillus is selected from Aspergillus niger , Aspergillus japonicus , Aspergillus oryzae , Aspergillus sojae , or a combination thereof, and the genus Mucor is selected from Mucor hiemalis , Mucor silvaticus , Mucor prainii , or Mucor subtilissimus .

In one embodiment, 2 grams of the above-mentioned tea branches containing gallic acid were extracted with 15 ml of water, and the content of gallic acid in each ml of extract obtained was 322 to 527 micrograms/ml.

The present invention provides a method for preparing tea branches containing gallic acid for use in preparing oral dosage forms for preventing oral cancer and lung cancer, comprising: performing an inoculation step, inoculating a tanninase-producing bacterium selected from the genus Aspergillus , the genus Mucor , Actinomucor elegans or Rhizopus chinensis into tea branches that have been previously fermented, wherein the genus Aspergillus is selected from Aspergillus niger , Aspergillus japonicus , Aspergillus oryzae , Aspergillus sojae , or a combination thereof, and the genus Mucor is selected from Mucor hiemalis , Mucor silvaticus , Mucor prainii , or Mucor subtilissimus ; performing an aerobic post-fermentation treatment step, subjecting the tea branches inoculated with the tannase-producing bacteria to an aerobic post-fermentation treatment for 24 to 48 hours; and performing a baking step, baking the tea branches subjected to the aerobic post-fermentation treatment, so as to obtain the tea branches containing gallic acid.

In one embodiment, 2 grams of the above-mentioned tea branches containing gallic acid were extracted with 15 ml of water, and the content of gallic acid in each ml of extract obtained was 322 to 527 micrograms/ml.

In one embodiment, the step of inoculating the tanninase-producing bacteria into the tea branches comprises: screening the tanninase-producing bacteria with a plate culture medium; culturing the tanninase-producing bacteria with a liquid culture medium and a fermentation tank in sequence, and then taking out the tanninase-producing bacteria from the fermentation tank; and uniformly mixing the tanninase-producing bacteria and the tea branches in sterile water.

In one embodiment, the weight ratio of the sterile water to the tea branches is 0.5 to 1:3.

In one embodiment, the aerobic post-fermentation treatment step is to perform aerobic post-fermentation treatment on the tea branches inoculated with tannin-producing bacteria for 36 hours.

In one embodiment, the aerobic post-fermentation treatment step is to perform aerobic post-fermentation treatment on the tea branches inoculated with tannase-producing bacteria at 25 to 50°C for 24 to 48 hours.

In one embodiment, 2 grams of the above-mentioned tea branches containing gallic acid were extracted with 15 ml of water, and the content of gallic acid in each ml of extract obtained was 322 to 527 micrograms/ml.

In one embodiment, the tea branch is a tea branch of Camellia sinensis oolong tea, black tea or green tea.

This invention also provides an oral dosage form for preventing oral cancer and lung cancer, comprising an extract of tea branches containing gallic acid prepared by the above preparation method

In one embodiment, 2 grams of the above-mentioned tea branches containing gallic acid were extracted with 15 ml of water, and the content of gallic acid in each ml of extract obtained was 322 to 527 micrograms/ml.

In order to enable you to have a deeper understanding and knowledge of the technical features and technical effects of the present invention, we would like to provide a better implementation example and a detailed description as follows.

101, 102, 103: Steps

Figure 1 is a flow chart of the steps of the preparation method of the tea branches containing gallic acid in this invention.

Figure 2 shows the HPLC spectra of the extracts of processed and unprocessed oolong tea branches, as well as gallic acid standards.

Figure 3 is a graph showing the concentration of gallic acid produced by processed oolong tea branches and the post-fermentation time.

Figure 4 is a graph showing the concentration of gallic acid produced by processed oolong tea leaves versus post-fermentation time.

In order to facilitate understanding of the technical features, content and advantages of this creation and the effects it can achieve, this creation is described in detail as follows with diagrams and in the form of embodiments. The diagrams used are only for illustration and auxiliary instructions, and may not be the true proportions and precise configurations of this creation after implementation. Therefore, the proportions and configurations of the attached diagrams should not be interpreted to limit the scope of rights of this creation in actual implementation. In addition, for ease of understanding, the same elements in the following embodiments are indicated by the same symbols.

This invention discloses the use of a tea branch containing gallic acid in preventing oral cancer and lung cancer, and a method for preparing the tea branch containing gallic acid. This invention further applies the extract of the tea branch containing gallic acid to prepare a pharmaceutical composition for preventing oral cancer and lung cancer. The administration route of the pharmaceutical composition can be oral, sublingual, mucosal absorption, skin application, injection or spray inhalation or oral. The pharmaceutical composition is, for example, an oral dosage form, and is, for example, a powder, tablet, capsule, granule, powder, solution, syrup or concentrate. The above-mentioned pharmaceutical composition is, for example, an extract of the tea branch containing gallic acid and a pharmaceutically acceptable carrier. The carrier can be water, alcohol, glycerol, dimethyl sulfoxide, carboxymethyl cellulose, vegetable oil, organic ester, organic acid, saline solution, electrolyte solution or a combination thereof. The carrier is preferably water. In addition, the pharmaceutical composition may further contain an additive, for example. The additive may be a molding agent, a preservative, a diluent, a filler, a binder, a disintegrant, an absorption promoter, a sweetener, an emulsifier, a thickener, a lubricant, a fat or non-fat base, a surfactant, a suspending agent, a gelling agent, an adjuvant, an antioxidant, a stabilizer, a coloring agent, a fragrance or a combination thereof. The molding agent may be selected from sodium citrate, calcium carbonate, calcium phosphate or a combination thereof. The preservative may extend the shelf life of the pharmaceutical composition, such as benzyl alcohol, parabens. The diluent may be selected from water, ethanol, propylene glycol, glycerol or a combination thereof. The filler may be selected from lactose, galactose, high molecular weight polyethylene glycol or a combination thereof. The binder may be selected from sucrose, gelatin, gum arabic or a combination thereof. The disintegrant may be selected from potato starch, tapioca starch, silicate or a combination thereof. The absorption enhancer may be selected from dimethyl sulfoxide (DMSO), laurocapram, propylene glycol, glycerol, polyethylene glycol or a combination thereof. Sweeteners can be selected from Acesulfame K, aspartame, saccharin, sucralose, neotame or a combination thereof. In addition to the additives listed above, other suitable additives can be selected as needed without affecting the pharmaceutical effects of adenosine analogs. The pharmaceutical composition of the present invention can be used in the treatment of animals or humans, and can be made into any drug form and administered to the animal or human being treated by any route.

FIG1 is a flow chart of the steps of the preparation method of the gallic acid-containing tea branch of the present invention, wherein the gallic acid-containing tea branch is used to prepare an oral dosage form for preventing oral cancer and lung cancer. As shown in FIG1, the preparation method of the gallic acid-containing tea branch comprises: step 101, performing an inoculation step, inoculating tanninase-producing bacteria into the tea branch; step 102, performing an aerobic post-fermentation treatment step, performing a post-fermentation treatment on the tea branch inoculated with tanninase-producing bacteria; and step 103, performing a baking step, baking the tea branch treated with post-fermentation, so as to obtain the gallic acid-containing tea branch.

In detail, the above step 101 includes the sub-steps of strain culture and tea branch inoculation. The sub-steps of strain culture in step 101 can be further divided into stages such as strain screening, strain small-scale culture, strain shaking bottle culture and strain large-scale culture.

Bacteria screening: Tanninase-producing bacteria suitable for tea branch growth were screened using Potato Dextrose Agar (PDA) plate culture medium. Tanninase-producing bacteria can be selected from Aspergillus , Mucor , Actinomucor and Rhizopus . It is worth noting that the tanninase-producing bacteria screened are all strains that meet food safety standards. Specifically, the Aspergillus genus includes but is not limited to: Aspergillus niger , Aspergillus japonicus , Aspergillus oryzae , Aspergillus sojae , or a combination thereof; the Mucor genus includes but is not limited to: Mucor hiemalis , Mucor silvaticus , Mucor praini , Mucor subtilissimus ; the Actinomucor genus includes but is not limited to: Actinomucor elegans ; the Rhizopus genus includes but is not limited to: Rhizopus chinensis . These strains can be obtained from the American Type Culture Collection (ATCC) or the Hsinchu Food Industry Research Institute.

Small-scale culture of strains: In a sterile operating table, the selected tannin-producing bacteria, such as [ Aspergillus sojae] of the genus Aspergillus , are inoculated into several plates of PDA plate culture medium using aseptic operation techniques. The PDA plate culture medium is then placed in an incubator and cultured at an appropriate temperature for a period of time to allow the tannin-producing bacteria to grow to a basic number, for example, at 20 to 40°C for 1 to 3 days, preferably at 25 to 35°C for 1 to 3 days, and more preferably at 30°C for 3 days.

Strain shake flask culture: In a sterile operating table, use aseptic operation techniques to inoculate a small amount of tannin-producing bacteria into a 500 mL triangular shake flask containing 100 mL of potato dextrose broth (PDB) liquid culture medium. Place the triangular shake flask in an incubator and culture at an appropriate temperature for a period of time to allow the tannin-producing bacteria to grow to a basic number, such as 20 to 40°C for 1 to 3 days, preferably 25 to 35°C for 1 to 3 days, and more preferably 30°C for 3 days.

Large-scale culture of strains: Take 4 shake flasks/5L fermentation tank as the required bacterial volume ratio, use aseptic operation technology to centrifuge and remove the tanninase-producing bacteria from the triangular shake flask, and then inoculate the tanninase-producing bacteria into the fermentation tank for large-scale culture at an appropriate temperature, such as 20 to 40°C for 1 to 3 days, preferably 25 to 35°C for 1 to 3 days, and more preferably 33°C for 3 days.

The sub-step of tea branch inoculation in the aforementioned step 101 includes: centrifugally taking out the tanninase-producing bacteria cultured in large quantities of the aforementioned strain from the fermentation tank, and evenly mixing the tanninase-producing bacteria and tea branches in sterile water to inoculate the tanninase-producing bacteria into the tea branches. During inoculation, sterile water is added according to the weight of the tea branches, and the weight ratio of sterile water to tea branches is, for example, 0.5 to 1:3, preferably, 2 to 2.5 liters of sterile water is added for every 6,000 grams of tea branches. The amount of tanninase-producing bacteria is adjusted according to the amount of tea branches inoculated, based on the weight that can be evenly mixed with the tea branches. The inoculation amount of each batch of tea branches is, for example, about 3,000 to 6,000 grams, and the type of tea branches is, for example, oolong tea branches.

Secondly, step 102 includes: performing aerobic post-fermentation on the tea branches inoculated with tannase-producing bacteria at an appropriate temperature, the aerobic post-fermentation temperature is, for example, about 25 to 50°C, preferably, about 33°C; the aerobic post-fermentation time is, for example, about 24 hours to 48 hours, preferably, about 30 hours to 40 hours, and more preferably, about 36 hours.

Next, step 103 includes: baking the tea branches that have been post-fermented at an appropriate temperature to remove the moisture contained in the tea branches and increase the aroma components of the tea branches, so as to complete the preparation of the tea branches containing gallic acid of the present invention. In step 103, the baking temperature and time, taking oolong tea branches as an example, are, for example, about 70 to 150°C, preferably about 90 to 130°C, and more preferably about 110°C.

Please refer to Figure 2, which shows the HPLC spectra of tea branches that have not been processed by the above preparation process, tea branches that have been processed by the above preparation process, and gallic acid standards.

First, 2 grams of tea branches that have not been treated by the preparation method of the present invention (Comparative Example 1) and 2 grams of tea branches that have been treated by the preparation method of the present invention (Experimental Example 1) are taken, and the untreated and treated tea branches are extracted with 15 ml of hot water respectively, and then the amount of gallic acid contained in the extract (5 or 10 μl) of the untreated and treated tea branches is measured by high performance liquid chromatography (HPLC). The present invention takes oolong tea as an example, but is not limited to this. Therefore, the above-mentioned tea branches are, for example, oolong tea branches of the tea tree Camellia sinensis , and the tea tree variety is, for example, but not limited to, Sijichun. That is, the production method of the oolong tea branches used in this creation is, for example, to first ferment to make oolong tea and then pick the tea branches from the pile of tea leaves for subsequent inoculation (planting), post-fermentation and drying, so the tea branches and tea leaves have the same source. The oolong tea branches used in the data of this creation are, for example, but not limited to, purchased from Songfu Tea Factory in Mingjian Township, Nantou County, Taiwan. In addition, the tea branches used and made in this creation are not limited to oolong tea branches, but can also be tea branches with different degrees of post-fermentation such as green tea or black tea.

HPLC specifications: pump L-2130, Autosampler L-2200, UV Detector L-2400.

HPLC sample preparation

1. Weigh 2.0g of tea branch sample and place it in a 50mL centrifuge tube. Add 15mL of 100℃ hot water. Extract for 15 minutes and centrifuge for 10 minutes (speed 4,000rpm/min).

2. After centrifugation, take 1.2 mL of supernatant and place it in a 2.0 mL microcentrifuge tube, and centrifuge for another 10 minutes (speed 12,000 rpm/min).

3. After centrifugation, take 1 mL of supernatant and pass it through a 0.22 m filter membrane (nylon).

Chromatographic conditions

The column was Inertsil ^® ODS-2 C-18 (4.6 mm × 250 mm), and the mobile phase was (A) 0.026% phosphoric acid, (B) acetonitrile.

Gradient conditions: 0-40 minutes, the concentration of solution B increased from 10% to 30%; 40-40.1 minutes, the concentration of solution B decreased from 30% to 10%; after 40.1 minutes, the concentration of solution B was maintained at 10% until the end of 70 minutes.

Sample injection volume: 40μL of standard (gallic acid) (500μg/mL), 5 or 10μL of untreated tea branch sample, and 5 or 10μL of treated tea branch sample; flow rate: 1mL/min, absorbance: 254nm.

As shown in Figure 2, Spectrum A is the HPLC spectrum of untreated tea branches, Spectrum B is the HPLC spectrum of treated tea branches, and Spectrum C is the HPLC spectrum of gallic acid standard.

From Graph A and Graph B, it can be observed that compared with the tea branches that have not been treated by the preparation method of the present invention (Graph A), the GA content of the tea branches treated by the preparation method of the present invention (Graph B) has increased significantly, and the specific components in tannic acid, such as the EGCG content and the ECG content, have slightly decreased. The test results show that the ratio of gallic acid to tannic acid content in the untreated tea branches is lower than the ratio of gallic acid to tannic acid content in the treated tea branches. From this result, it can be inferred that the specific components of EGCG and tannic acid in the tea branches are the source pathways for producing gallic acid by the preparation method of the present invention.

This work further analyzes the effect of different post-fermentation times on the gallic acid content produced by tea branches. In this work, the gallic acid content is determined by using HPLC analytical grade standards, using the standard curve of gallic acid quantification with the standard, and then using the interpolation method to obtain the gallic acid content of each sample. Among them, this tea branch is the tea branch of oolong tea.

First, 2 grams of tea branches prepared according to the method of the present invention are taken, and the tea branches treated by the preparation method of the present invention are extracted with 15 ml of hot water, and then the amount of gallic acid contained in the extract (5 or 10 μl) of the treated tea branches is measured by high performance liquid chromatography (HPLC). The present invention repeats the measurement 3 times and then takes the average value. Figure 3 is a graph of the concentration of gallic acid produced by tea branches and time. It can be seen from Table 1 and Figure 3 that the gallic acid content of the untreated tea branches (post-fermentation time is 0) is only about 32 μg/ml. The gallic acid content of tea branches gradually increases with the post-fermentation time, and starts to rise rapidly at about the 24th hour of fermentation. When the post-fermentation time is about 36 hours, the gallic acid content of tea branches can reach the maximum value. After that, the gallic acid content of tea branches decreases instead of increasing, and gradually decreases with the post-fermentation time, and starts to drop rapidly at the 48th hour until it almost disappears. According to the preparation method of this creation, the gallic acid content in the extract obtained by extracting 2 grams of gallic acid-containing tea branches of this creation with 15 ml of water is about 322 to 527 micrograms/ml. For example, the weight of the gallic acid in each milliliter of extract obtained by extracting 2 grams of the roasted and post-fermented tea branches with 15 milliliters of water is preferably higher than 500 micrograms.

Figure 4 is a graph of the concentration of gallic acid produced by tea leaves versus time. The tea leaves were also processed using the preparation method of gallic acid-containing tea branches mentioned above in this work. The tea leaves are oolong tea leaves, and the raw material source is the same as the tea branches in Table 1. As can be seen from Table 2 and Figure 4, the gallic acid content produced by the tea leaves will also gradually increase with the post-fermentation time, but the increase is quite slow, and it will only start to rise rapidly at the 36th hour. When the post-fermentation time is about 72 hours, the gallic acid content can reach the maximum value. Afterwards, the gallic acid content of the tea branches will gradually decrease with the post-fermentation time, and will begin to drop rapidly at the 84th hour.

By comparing Table 1 and Table 2, it can be seen that according to the preparation method of this creation, the gallic acid content of the tea branches can reach the highest point after only 36 hours of fermentation time, and the required post-fermentation time is only about 0.5 times that of tea leaves. In other words, the gallic acid content of tea leaves needs 72 hours of fermentation time to reach the highest point. Although the gallic acid content of tea leaves can eventually reach 2.15 times the gallic acid content of oolong tea branches. However, in terms of raw material cost, taking oolong tea as an example, the price of tea leaves is 12 times that of tea branches. In comparison, the economic benefit ratio of tea branches prepared by the preparation method of this creation to tea leaves is 24:2.15.

In addition, in terms of the storage method of post-fermentation products, tea branches only need to be stored in plastic bags at room temperature, while tea leaves need to be vacuum-packed in aluminum foil (which must contain antioxidants and desiccant). In terms of product flavor and appearance, the appearance and flavor of tea branches change less, while the changes of tea leaves are greater (tea leaves are easy to unfold). In terms of the stability of gallic acid, tea branches have high reproducibility, but tea leaves have low stability. In addition, tea branches can reach the maximum gallic acid production in just 36 hours, which can reduce the chance of bacterial contamination. In contrast, it takes about 72 hours for tea leaves to reach the maximum gallic acid production, which increases the chance of bacterial contamination. In addition, because the catechin content of oolong tea branches is lower, it is less likely to inhibit the growth of tannin-producing bacteria, so the tannin-producing bacteria grow quickly and easily become the dominant strain. In contrast, the catechin content of oolong tea leaves is higher, which is easier to inhibit the growth of tannin-producing bacteria, so the tannin-producing bacteria grow slowly and are not easy to become the dominant strain.

Based on the above preparation method, the invention provides a tea branch containing gallic acid, comprising gallic acid and tannin producing bacteria, wherein the content of gallic acid in the extract obtained by extracting 2 grams of the tea branch of the invention with 15 ml of water is 322 to 527 micrograms/ml. In the tea branch of the invention, the tannin producing bacteria is selected from food-grade Aspergillus, Mucor, Actinomucor and Rhizopus, and the raw material of the tea branch can be selected from any tea tree (scientific name: Camellia sinensis ) oolong new tea, oolong old tea, black tea or green tea, and the tea tree stems are selected from them after the tea making process is completed, and the unwanted tea tree stems are discarded.

When the tea branches containing gallic acid obtained in this invention are applied to pharmaceutical compositions, the tea branches can be extracted first and then dried. The above-mentioned drying step is, for example, to obtain dry powder (without adding any molding agent) by using traditional freeze drying, wherein 1 ml of freeze-dried tea leaves and tea branch extract can obtain approximately 0.04 g and 0.028 g of dry powder respectively. Since the above-mentioned freeze drying method can be, for example, carried out by using traditional technology, it will not be described here separately.

In one embodiment, 1,800 grams of air-dried oolong tea branches and leaves are inoculated with tannin-producing bacteria selected from the genus Aspergillus at a temperature of 30 degrees Celsius, and then the tea branches and leaves are processed in the above manner to obtain tea branches and leaves containing gallic acid. Many scientific evidences show the importance of epigenetic and carcinogenic changes; almost all types of cancer have gene mutations regulating abnormalities in epigenome-encoded proteins. Targeting the epigenome and including the use of DNA methylation inhibitors and histone deacetylation are evolutionary strategies for cancer chemoprevention, and both methods have shown promise in cancer clinical trials. GA's ability to target abnormal epigenomes makes it a valuable chemopreventive agent at multiple cancer malignancy stages, in addition to its anti-cancer mechanism as previously explored. We developed a post-fermented oolong tea with high GA content through a two-week fermentation process using A. sojae. This tea retains GA's anti-DNA methylation function and has a better inhibitory effect on genomic 5mC content in lung cancer cells than oolong tea, and even higher than the well-known DNMT inhibitor 5azaC. The data of this work also showed that PFOTE (5μg/mL) treatment for 5 days resulted in a significant decrease in the cytoplasmic and nuclear content of all DNMTs in human lung cancer H1299 and A549 cells. These results clearly demonstrate the superior effect of PFOTE in inhibiting DNMT activity. Based on the widespread popularity of tea, this work proves that post-fermented oolong tea is a DNMT dietary inhibitor and can be used as a daily beverage to prevent cancer by reducing the burden of epigenomic abnormalities. This work further used oolong tea leaf extract (OTE) and processed oolong tea leaf extract (PFOTE) tea soup freeze-dried powder, gallic acid and EGCG standards as materials to analyze the survival rate of oral cancer cells OC3 and lung cancer cells H1299. The results showed that in the MTT test, oral cancer and lung cancer cells treated with PFOTE had a higher degree of reduced cell survival than OTE, and there was a statistically significant difference. When OC3 and H1299 cells were treated with PFOTE, the survival of both cells was lower than that of OTE. Time-dependent experiments were performed using 20μg/mL OTE or PFOTE tea to treat H1299 and OC3 cells. The results showed that the survival of oral cancer and lung cancer cells treated with PFOTE was lower than that of OTE after 24, 48, and 72 hours of culture. When OC3 and H1299 were treated with OTE or PFOTE tea and their IC ₅₀ values were compared, the results showed that the concentrations required for PFOTE were lower than those for OTE (235.35 and 199.79μg/mL for OC3, and 388.09 and 203.96μg/mL for H1299). Combining the above experimental results, it was found that PFOTE can more effectively reduce the survival rate of oral cancer and lung cancer cells than OTE, and in the antioxidant content and activity tests, it was also found that PFOTE is more effective than OTE.

As mentioned above, the pharmaceutical composition of the present invention can be tea branch powder and/or tea leaf powder. Because the freeze-dried post-fermented oolong tea (PFOTE) previously had an inhibitory effect on epigenetic abnormalities at a concentration of 5 micrograms per milliliter, and tea branches and tea leaves have the same source, it can be inferred that the freeze-dried powder of tea branches needs to have an inhibitory effect on epigenetic abnormalities at about 10 micrograms per milliliter. If you want to achieve a concentration that is effective at the cellular level, you need at least about 1,000 times, so each package of high gallic acid tea branch freeze-dried powder must contain at least 10 mg. Generally speaking, each package of tea branch powder contains about 2~3 grams, which is 200,000~300,000 times the effective concentration for cellular effects. The concentration of gallic acid in freeze-dried tea branch powder is about 81 times that of regular tea (3g/150mL).

In summary, this invention produces gallic acid by inoculating tannin-producing bacteria into tea branches, and then fermenting and baking the tea branches for 24-48 hours. The preparation method of this invention only takes half the time of tea leaves to obtain tea branches with high gallic acid content. Although the gallic acid content of tea branches is only half of that of tea leaves, because the price of tea branches is much lower than that of tea leaves, and the time required to reach the highest gallic acid content is only half of that of tea leaves, this invention can make the economic benefits of traditionally useless tea branches far higher than tea leaves.

The above description is for illustrative purposes only and is not intended to be limiting. Any equivalent modification or change made to the invention without departing from the spirit and scope of the invention shall be included in the scope of the patent application attached hereto.

101, 102, 103: Steps

Claims (6)

A method for preparing tea branches for oral dosage forms for preventing oral cancer and lung cancer comprises: performing an inoculation step, inoculating a tanninase-producing bacterium of the genus Aspergillus into a tea branch that has been previously fermented, wherein the genus Aspergillus is selected from the group consisting of Aspergillus niger , Aspergillus japonicus , Aspergillus oryzae , Aspergillus sojae, , or a combination thereof; performing an aerobic post-fermentation treatment step to treat the tea branches inoculated with the tannase-producing bacteria with aerobic post-fermentation for 24 to 48 hours; performing a baking step to bake the tea branches to obtain tea branches containing gallic acid; extracting the tea branches containing gallic acid to obtain an extract having a gallic acid content of 322 to 527 micrograms/ml; and drying the extract to obtain a dry powder. The preparation method as described in claim 1, wherein the inoculation step comprises: screening the tanninase-producing bacteria with a plate culture medium; culturing the tanninase-producing bacteria with a liquid culture medium and a fermentation tank in sequence, and then taking out the tanninase-producing bacteria from the fermentation tank; and uniformly mixing the tanninase-producing bacteria and the tea branches in sterile water. The preparation method as described in claim 2, wherein the weight ratio of the sterile water to the tea branches is 0.5 to 1:3. The preparation method as described in claim 1, wherein the aerobic post-fermentation treatment step is to subject the tea branches inoculated with the tannase-producing bacteria to aerobic post-fermentation treatment for 36 hours. The preparation method as described in claim 1, wherein the aerobic post-fermentation treatment step is to perform aerobic post-fermentation treatment on the tea branches inoculated with the tannase-producing bacteria at 25 to 50°C for 24 to 48 hours. The preparation method as described in claim 1, wherein the tea branch is a tea tree ( Camellia sinensis ) oolong tea branch.