JP2009509960A - Black tea polyphenol and its use - Google Patents

Abstract

  Contemplated compositions and methods include combinations of tea polyphenol compositions and lactoferrin to prevent and / or treat oral cancer with significantly higher efficiency than treatment with either tea polyphenol compositions or lactoferrin alone. It is effective. Such improved efficiency is also reflected in various clinical and systemic parameters, which are regulated activity of phase 1 / phase 2 enzymes, cytokeratin, NF-κB, Bcl− 2, including expression of PCNA, and alteration of cell proliferation and induction of apoptosis.

Description

  This application claims priority to our co-pending US provisional patent application having serial number 60/720007, filed September 22, 2005.

(Field of the Invention)
The field of the present invention is the treatment and / or chemoprevention of oral cancer and in particular the treatment and / or chemoprevention of oral cancer by administration of a combination of tea extract and lactoferrin.

  Oral cancer is the world's fifth most common malignancy, with approximately 500,000 new cases diagnosed annually. Unfortunately, about 75% of them occur in developing countries, and no effective treatment has been found that is widely available and available in such countries.

  For example, Tanaka et al. Reported the chemopreventive effect of at least some bovine lactoferrin on 4-nitroquinoline 1-oxide-induced tongue carcinogenesis in male F344 rats, and the chemopreventive effect on tongue tumor formation is It was suggested that this may have been mediated through modification of activity and / or activity of detoxifying enzymes (Jpn J Cancer Res. 2000 Jan; 91 (1): 25-33). In other examples, various tea polyphenols have been reported in various publications as having some degree of antineoplastic effect. Hsu et al. Show that p21WAF1 can be induced by green tea polyphenol EGCG in several cancer cell types and that p21WAF1 is involved in EGCG-induced growth inhibition of OSC2 cells, which can promote caspase-3 mediated apoptosis. Observed. Based on these observations, Hsu hypothesized that the expression of functional p21WAF1 could promote phytochemical-mediated growth arrest and apoptosis in oral cancer cells (Anticancer Res. 2005 Jan-Feb; 25 ( 1A): 63-7). In further experiments, Hsu demonstrated that green tea and selected constituents of green tea selectively induced apoptosis to at least some extent only in oral cancer cells, while EGCG was able to inhibit oral cancer cell growth and invasion. Further discovered (Gen Dent. 2002 Mar-Apr; 50 (2): 140-6).

  EGCG has also been reported to play an important role in promoting oxidative stress, as demonstrated by Weisburg et al. Here, the authors reported that EGCG acts as a prooxidant and that cancer cells are more sensitive to oxidative stress than normal cells (Basic Clin Pharmacol Toxicol. 2004 Oct; 95 (4): 191-200). .

  Green tea has also been demonstrated to reduce to some extent the average tumor burden and the incidence of dysplasia and oral cancer (Nutr Cancer. 1999; 35 (1): 73-9), Li et al. Presented data that showed a significant reduction in oral visible tumor incidence, squamous cell carcinoma incidence, tumor volume and the number of dysplastic lesions and papillomas (Wei Sheng Yan Jiu. 2002 Oct; 31 (5): 354-7).

  Unfortunately, while most of the reported compositions and methods exert at least some chemopreventive or therapeutic effect, a variety of difficulties still remain. In particular, the concentration of polyphenols required to sustain the effect is relatively high. Furthermore, due to the relatively rapid metabolic conversion of polyphenols, serum concentrations are rapidly reduced in animals and humans, and efficacy is lower than desired.

  Thus, while many compositions and methods for the treatment or prevention of oral cancer are known in the art, all or nearly all of them have one or more disadvantages. Thus, there is still a need for improved compositions and methods for chemoprevention and / or treatment of neoplastic diseases, and particularly oral cancer.

(Summary of Invention)
The present invention is directed to compositions and methods for chemoprevention and / or treatment of oral cancer in which tea polyphenols and lactoferrin are combined to exert a significant anti-malignant effect, and in such combinations The compounds can be administered alone or together.

  In one aspect of the present inventive subject matter, the nutritional or pharmaceutical product reduces oral cancer with an efficiency greater than that achieved by oral administration of the product with either the tea polyphenol composition alone or lactoferrin alone. A combination of a tea polyphenol composition and lactoferrin in an effective amount. Most preferably, the polyphenol composition comprises a mixture of a plurality of catechins obtained from black tea and / or green tea, and may also comprise one or more synthetic catechins. In a further preferred embodiment, the polyphenol composition is present in an amount between 100 mg and 600 mg per dosage unit and the lactoferrin is present in an amount between 200 mg and 1200 mg per dosage unit and the combination is synergistic. It is particularly preferred. Contemplated nutritional products specifically include solid formulations (eg, capsules, tablets, snack bars, etc.), but if desired, the polyphenol composition and / or lactoferrin may also be present in a liquid carrier.

  Therefore, methods for improving oral cancer treatment or chemoprevention using either oral administration of tea polyphenol compositions or oral administration of lactoferrin generally include oral administration of tea polyphenol compositions or oral administration of lactoferrin. Including ensuring that oral cancer is effectively reduced. In the second stage, information is provided that oral administration of the tea polyphenol composition and lactoferrin combination reduces oral cancer with a second efficiency greater than the first efficiency.

  In a particularly preferred method, the tea polyphenol composition comprises a mixture of catechins obtained from black tea and / or green tea and the lactoferrin is bovine lactoferrin. Typically, the weight ratio between tea polyphenol composition and lactoferrin in combined oral administration is between 1: 1 and 1: 3, the most typically synergistic ratio. Even more preferably, the oral administration is co-administration in a single dosage unit form.

  In summary, and from another perspective, we use the combination of tea polyphenol composition and lactoferrin in the manufacture of a nutritional or pharmaceutical product for the treatment or chemoprevention of oral cancer. Contemplate. In such use, the tea polyphenol composition and lactoferrin are present in a weight ratio between 1: 1 and 1: 3, and most typically in a synergistic weight ratio. Further, the tea polyphenol composition comprises a mixture of catechins (or at least one synthetic catechin) obtained from at least one of black tea and green tea, and the tea polyphenol composition and lactoferrin are in a single dosage unit. It is generally preferred to be formulated (eg, in a nutritional or pharmaceutical product).

  Various objects, features, aspects and advantages of the present invention will become more apparent from the following detailed description of preferred embodiments of the invention.

(Detailed explanation)
The inventors have unexpectedly discovered that the chemopreventive and / or therapeutic effects of tea polyphenols can be significantly improved by the combination of such compounds and compositions and lactoferrin. Among other notable results (discussed below), the inventors have found that the improved effect is often synergistic, selective induction of (mitochondrial mediated) apoptosis in cancer cells, reduction in proliferation of cancer cells Has been found to be driven by a number of intrinsic effects, including regulation of phase I / phase II enzymes, and enhancement of antioxidant effects.

  Thus, based on the results presented below and further considerations, it is contemplated that the nutritional or pharmaceutical product now includes a combination of tea polyphenol composition and lactoferrin in an effective amount to reduce oral cancer. Most typically, such combinations are provided such that the combination has greater efficiency than oral administration of a product having a tea polyphenol composition or lactoferrin alone. Such increased efficiency is most preferably synergistic. From a different perspective, it is therefore contemplated that the combination of tea polyphenol composition and lactoferrin is used in the manufacture of nutritional or pharmaceutical products for the treatment or chemoprevention of oral cancer.

  In a further particularly preferred embodiment of the present inventive subject matter, we confirm in one step that oral administration of tea polyphenol composition or oral administration of lactoferrin reduces oral cancer with first efficiency. Contemplates a method of improving the treatment or chemoprevention of (such as using either a tea polyphenol composition or lactoferrin alone). In one further step of the preferred method, information that oral administration of a combination of tea polyphenol composition and lactoferrin reduces oral cancer with an efficiency greater than the first efficiency (eg, printed, displayed, audible Sex) is provided.

  Ensure that oral administration of tea polyphenol composition or oral lactoferrin reduces oral cancer (eg, incidence, multiplicity, severity and / or rate of progression to more malignant forms) with first efficiency It should be understood that there are numerous methods for such validation known in the art. Most typically, however, such confirmation steps are accomplished using experimental determinations performed as described below or in a similar manner. For example, a control study using animals or statistical analysis of oral cancer data in humans can be used. Alternatively or additionally, the confirmation stage may also include published results (for humans or animals; for example, J Pharmacol Sci. 2005 May; 98 (1): 41-8, or Nutrition. 2006 Sep; 22 (9): 940-6; Toxicol In Vitro. 2005 Mar; 19 (2): 231-42, or Clin Biochem. 2005 Oct; 38 (10): 879-86) and publication thereof. The data obtained is preferably identical, but not necessarily identical to the data ascertained with respect to chemical composition, dose, dosing schedule and / or route of administration.

  Similarly, with respect to providing information that oral administration of a combination of tea polyphenol composition and lactoferrin reduces oral cancer with greater efficiency than primary efficiency, all known methods for providing information are here. It is intended to be suitable for use. For example, such information may be provided in printed form, displayed form, and / or audible form. For example, if the intended method is used to obtain regulatory approval (eg, for pharmaceutical and / or nutritional use, or to embody efficiency requirements), the stage of confirmation is animal And / or can be performed using human trials, whereas the step of providing information can be performed in printed (written and drawing) form in the application for authorization. On the other hand, if the contemplated method is used to market a combination product comprising tea polyphenols and lactoferrin, the steps of confirmation and information provision will be described in publications detailed with respect to first efficiency and first This can be done by providing commercial materials that reference (together or separately) publications detailing efficiency that is greater than efficiency.

Contemplated compounds With respect to contemplated polyphenol compositions, it is generally preferred that a suitable polyphenol composition be a mixture of plant-derived catechins. For example, plants that are particularly suitable for the isolation of polyphenols include Chinese tea plants (Camellia sinensis, especially its leaves) or grapes (Vitis vinifera, especially its seeds). Particularly preferred polyphenol compositions include those marketed under the names of polyphenone E (green tea polyphenol formulation) and polyphenone B (black tea polyphenol formulation) from Mitsui Norin Japan. However, it should be understood that numerous other compositions can be obtained from various plants as long as such plants provide significant amounts of polyphenols.

  Among other suitable ingredients, plant polyphenol compositions include epigallocatechin-3-gallate (EGCG), epigallocatechin (EGC), epicatechin-3-gallate (ECG), epicatechin (EC), gallocatechin -3-gallate (GCG), gallocatechin (GC), theaflavin, theaflavin monogallate-A, theaflavin monogallate-B, theaflavin digallate, tannin etc., each of which can be stereochemically / optically pure or At least 25%, more typically at least 50%, and even more typically at least 75% by weight of one or more (generally between 2 and 7). And most generally at least 85% by weight. It is further preferred that the plant polyphenol composition has a reduced caffeine content at a concentration of generally less than 5 wt%, more typically less than 3 wt%, and most typically less than 1 wt% Generally preferred. Note that depending on the plant, the weight ratio of a particular polyphenol to each other can vary considerably. However, particularly preferred plant polyphenol compositions include those generally derived from green tea extract, black tea extract, and those in which EGCG is the major component (generally at a concentration of at least 30 wt%, more typically at least 40 wt%). . Black tea extract can also be characterized as containing catechin polymerized to polymerized catechin. Most commonly, polymerized catechins contain theaflavins and theearugabins in various weight ratios and average molecular weights.

  In further contemplated embodiments, plant polyphenol compositions can be prepared such that the composition comprises two or even polyphenols. In such a formulation, it may be advantageous to replace the isolated plant polyphenol with one or more synthetic polyphenols, and in particular with EGCG. If desired, contemplated polyphenol compositions can be enriched in galloylated catechins. The catechins contemplated here (galloylated and non-galloylated) contain optical isomers, chiral centers and / or stereoisomers, and all such forms (and mixtures thereof) Note that is contemplated herein. Still further contemplated polyphenols are also represented by formula 1:

［式中、Ｒ_１、Ｒ_２、Ｒ_３、Ｒ_４、Ｒ_３’、Ｒ_４’及びＲ_５’は、独立してＨ、ＯＨ又はＭ（ここで、Ｍは、ＯＣ（Ｏ）Ｒ、ＯＣ（Ｓ）Ｒ、ＯＣ（ＮＨ）Ｒ、ＯＲ、又はＲ（ここで、Ｒは場合により置換されているアルキル、アルケニル、アルキニル、アルカリル、又はアリールである。）である。）であり；及びＲ_３”は任意の没食子酸エステル基又はＨである。］
で示される構造を有する（例えば、合成の、非天然型の）ポリフェノールも含み得る。

[Wherein R ₁ , R ₂ , R ₃ , R ₄ , R ₃ ′, R ₄ ′ and R ₅ ′ are independently H, OH or M (where M is OC (O) R, OC (S) R, OC (NH) R, OR, or R, where R is an optionally substituted alkyl, alkenyl, alkynyl, alkaryl, or aryl. R ₃ ″ is any gallate group or H.]
(Eg, synthetic, non-naturally occurring) polyphenols having the structure of

  With regard to suitable lactoferrin compositions, it is generally preferred that the lactoferrin is bovine lactoferrin, which can be isolated from milk or can be a recombinant product. Thus, it should be noted that the iron load of lactoferrin can vary considerably and that all known iron loads are considered here. For example, lactoferrin loading can be between 0% and 20% iron saturation, between 20% and 40% iron saturation, between 4% and 60% iron saturation, or between 60% and 100%. The iron saturation between. Alternatively, lactoferrin can also be isolated from human or colostrum or other mammalian milk sources. Again, it should be noted that lactoferrin from such alternative sources can be recombinant.

  In still further contemplated embodiments, it should be understood that various other iron and especially trivalent iron ion complexing agents are also suitable. Particularly suitable compounds include such chelating and non-protein molecules. For example, desferrioxamine can be employed when the compound is a non-protein, and ferritin or hemosiderin can be used when the compound is a protein. Furthermore, if the protein is relatively large and the iron binding domain is characterized, a suitable iron chelator can be an iron binding fragment.

Contemplated nutritional compositions In general, all combinations of polyphenols and lactoferrin for chemoprevention and / or treatment of oral cancer are in an edible carrier, and in particular both polyphenols and lactoferrin are the same edible item ( That is, it is contemplated that it may be included in an edible carrier such as present in a nutritional food or food).

  For example, suitable foods include plant materials (eg, cereals, fruits, vegetables, berries, etc.) and / or animal materials (eg, beef, pork, lamb, chicken, fish, crustaceans, milk, dairy products) Etc.), or prepared from them, such materials can be raw or at least partially processed. Thus, in further contemplated embodiments of the present inventive subject matter, the food is solid or liquid and can be orally administered with at least one nutrient (eg, carbohydrates, proteins, lipids, minerals, vitamins, etc.), fiber and / or water. Provided in various forms. The contemplated combination of polyphenol composition and lactoferrin can be added to the food in solid or liquid form, or the polyphenol composition is in the first edible carrier and the lactoferrin is in the second edible carrier. Can be provided in various ways. Most preferably, the food is a food for human consumption and is formulated as a liquid, liquid nutrient or formulated in a capsule, tablet or powder (most preferably a single dosage unit of catechin and lactoferrin in combination Formulated as).

  For example, where the food product is in a solid form, contemplated forms include, in particular, dietary supplements formulated as capsules, tablets or other nutritionally known solid forms (eg, as a powder). However, many alternative solid formulations would also be suitable, including snack bars, chewing gums, lozenges to which the contemplated compound was added (or incorporated abundantly in the contemplated compound) Such as products that can be consumed as they are, fermented milk products, and the like. Similarly, when the food is in liquid form, contemplated food products are in particular tea or grape juice, dairy products, carbonated beverages, fruit juice beverages (eg, beverages containing fresh fruit juices, juices from juices or fruit juices) Sports drinks, alcoholic beverages, enriched water and the like.

  In another example, it is anticipated that the degree of processing of the contemplated food product can vary considerably, and all degrees of food processing are considered suitable for use in the present invention. For example, if the food is not processed (eg, harvested fruits or vegetables), the compounds according to the present subject matter can be added as a coating, mixture, solution, injection, or otherwise combined with the food Intended. On the other hand, if the food is processed to at least some degree (eg, the physical form changes (eg, pressed oats) or the chemical composition changes (eg, in combination with a fruit extract or food)), it is contemplated. The compound can be added as a coating, as a mixed component, or can be increased by processing.

  Thus, it should be understood that depending on the particular type of food and / or processing, the contemplated polyphenol composition and lactoferrin can be added as isolated individual compounds and / or as a mixture of individual compounds. Thus, such compounds can be relatively pure or can exist as enriched fractions in one or more of such compounds. Alternatively or additionally, the concentration of the contemplated polyphenol composition and / or lactoferrin in the food product can also be increased by processing steps (eg, processing that increases the concentration of the contemplated compound). It is further understood that mixing of the contemplated compounds into the food product can be carried out in a number of ways, and it is contemplated that all known methods are suitable for use in combination with the teachings presented herein. .

  With respect to the amount of polyphenol composition and lactoferrin in food products contemplated, the amount of such compound is such that the combination has greater efficiency than oral administration of the product with tea polyphenol composition or lactoferrin alone. It is generally preferred. Thus, polyphenol compositions are generally present in a range between about 200 mg and 1200 mg per daily dose, and more generally between 400 mg and 800 mg (these amounts can be administered in 1 to several units). To do. However, in alternative embodiments, smaller amounts of polyphenol compositions are also considered suitable (eg, between 100 mg and 200 mg, or even less, especially in the form of a troche or chewing gum). Similarly, higher amounts are considered, and such amounts are generally in the range between about 1200 mg and 2000 mg (and higher amounts). The term “about” as used herein with a number refers to the range +/− 10% of that number inclusive.

  Similarly, the amount of lactoferrin can vary considerably and generally ranges between about 400 mg and 3000 mg and more generally between 800 mg and 2000 mg per daily dose (these amounts can range from 1 unit to several Can be administered in units). However, in alternative embodiments, smaller amounts of the polyphenol composition would be suitable (eg, between 200 and 400 mg or even less, especially in the form of a troche or chewing gum). Similarly, higher amounts are considered (eg, in beverages), and such amounts generally range between about 3000 mg and 5000 mg (and even higher). Accordingly, it should be understood that a particularly preferred ratio of polyphenol to lactoferrin is in the range of about 1: 1 to about 1: 3, and even more preferably about 1: 2. Although lactoferrin can be administered with polyphenols, it is also considered suitable herein to administer lactoferrin in liquid form and polyphenols in solid form. In most cases it is preferred that the weight ratio of polyphenol to lactoferrin is a synergistic weight ratio (see experiment below). Thus, in a particularly preferred food product, the polyphenol composition is present in an amount between 100 mg and 600 mg per dosage unit and lactoferrin is present in an amount between 200 mg and 1200 mg per dosage unit. It will be appreciated that in yet a further aspect of the present inventive subject matter, the contemplated food product may comprise additional ingredients that have at least a recognized or demonstrated nutritional value. For example, particularly preferred additional components include those known or stated to reduce oxidative stress, improve immune status, and the like.

Contemplated pharmaceutical compositions It is generally contemplated that all combinations of polyphenols and lactoferrin can also be administered as a pharmaceutical formulation, wherein the administration of polyphenols and lactoferrin can be performed together or separately in a single dosage unit. . Regardless of the particular formulation, it is preferred that the polyphenol and / or lactoferrin be mixed with a pharmaceutically acceptable carrier. It will be appreciated that depending on the particular application, the formulation, route and / or dosage regimen can vary considerably, and it is generally contemplated that the subject matter of the invention is not limited to a particular formulation, route and / or administration. .

  Accordingly, suitable formulations include those for oral, parenteral and / or topical administration (including nasal, buccal and sublingual administration), where the contemplated formulation is in unit dosage form. More preferably. It is even more preferred that the amount of polyphenol and / or lactoferrin contemplated in combination with the carrier to form a unit dosage form is an amount that produces a chemopreventive and / or therapeutic effect. Accordingly, the percentage (% by weight) of active ingredient is generally from about 10% to about 99% of the total weight, preferably from about 20% to about 95%, more preferably from 50% to about 90%. And most preferably in the range of about 70% to about 90%.

  However, the dose administered of the pharmaceutical composition varies considerably, and the specific dose is at least partly (a) the amount of polyphenols and lactoferrin effective to achieve the desired therapeutic response, (b) the contemplated compound It should be understood that it depends on other factors, including: (c) the route of administration, and (d) age, gender, weight, general health, history of the patient being treated. One skilled in the art can readily determine and formulate an effective amount of the pharmaceutical composition required.

  It is generally preferred that the daily dose of the contemplated compound generally corresponds to the amount of the compound that is the lowest dose effective to produce the desired therapeutic effect. Such an effective dose generally depends on the factors described above. Thus, the dose of the compound according to the present inventive subject matter is from about 0.5 mg to about 100 mg per kg body weight per day, more preferably from about 1.0 mg to 50 mg per kg per day, and even more preferably The range is from about 2.5 mg to about 25 mg per kg per day. Thus, the unit dose of polyphenol generally ranges from about 100 mg to about 5000 mg, more preferably from about 200 mg to about 1200 mg, and most preferably from about 200 mg to about 800 mg. Similarly, the unit dose of lactoferrin generally ranges between about 400 mg and about 3000 mg and more generally between 800 mg and 2000 mg per day. If desired, the effective daily dose of the active compound may be administered in unit dosage forms, optionally in 2, 3, 4, 5, 6 or more divided doses administered separately at appropriate intervals throughout the day. Can be done.

Exemplary Oral Formulations It is generally preferred that pharmaceutical combinations according to the present inventive subject matter be administered orally (individually or separately), and all known forms of oral administration, including solid and liquid forms, are described herein. It is considered suitable for use in writing. For example, solid oral forms include capsules, tablets, troches, powders, while preferred liquid oral forms include solutions or suspensions in suitable media (generally aqueous solutions).

  Typical suitable pharmaceutically acceptable carriers are fillers or bulking agents (eg starch, lactose, sucrose, glucose, mannitol and / or silicic acid), binders (eg alginate, gelatin, carboxy Methylcellulose or polyvinylpyrrolidone), wetting agents (eg glycerol), disintegrants (eg agar, calcium carbonate or potato or tapioca starch), solution retarders (eg paraffin), absorption enhancers (eg quaternary ammonium salts) ), Wetting agents (eg cetyl alcohol, glycerol monostearate), absorbents (eg kaolin, bentonite clay), lubricants (eg talc, calcium stearate, magnesium stearate, solid polyethylene glycol), colorants, buffers Including agents. Contemplated oral solid doses can also be formulated to provide slow or controlled release of the active ingredient (eg, hydroxypropyl methylcellulose, other polymer matrices, liposomes in various ratios that provide desirable release characteristics) And / or with fine particles). It is to be understood that the preparation of contemplated oral solid dosage forms is known in the art and that all known methods are considered suitable for use with the teachings presented herein.

  Liquid dosage forms for oral administration of the contemplated compounds can be prepared as pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. Thus, and depending on the particular formulation, liquid dosage forms may also contain inert diluents, solubilizers and emulsifiers including water or other aqueous and non-aqueous solvents (eg, ethyl alcohol, isopropyl alcohol, ethyl carbonate, Ethyl acetate, benzyl alcohol, etc.), suspending agents (eg, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol), oils (eg, cottonseed, corn, germ, olive, etc.), glycerol, tetrahydrofuryl alcohol, polyethylene glycol and Additional known pharmaceutically acceptable liquid ingredients may also be included.

Exemplary Parenteral Formulations It is generally contemplated that combinations according to the present inventive subject matter can be prepared in a formulation for parenteral use, and specifically contemplated parenteral formulations are liquid formulations for injection. It is a thing. Accordingly, suitable formulations generally include pharmaceutically acceptable solvents (eg, sterile isotonic aqueous or non-aqueous solutions) and / or can be prepared as dispersions, suspensions or emulsions. Alternatively, parenteral formulations can also be provided as a kit containing the contemplated compound and other ingredients that can be reconstituted into a liquid product prior to use.

  Examples of suitable aqueous and non-aqueous carriers that may be employed in pharmaceutical compositions include water, ethanol, polyols (eg, glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils and ethyl oleate And injectable organic esters. The proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

Contemplated uses It is generally preferred that the compositions and methods according to the present inventive subject matter be employed for chemoprevention and / or treatment of oral cancer. Thus, it should be understood that suitable conditions include pre-tumor conditions as well as tumor conditions that may or may not be metastatic. For example, oral pre-tumor conditions include pain (from viruses and non-viruses) and pre-tumor lesions. Typical oral and / or pharyngeal cancers include, among others, squamous cell carcinoma that is common to the bottom of the oral cavity, the inside of the cheek, the gingiva and / or palate.

  It should be noted that administration of contemplated compounds and compositions can be purely prophylactic or can begin in the first diagnosis of pre-tumor lesions or even cancer. Thus, general administration is preferably long-term. For example, daily administration over at least 30 days, more typically at least 60 days, and most typically at least 180 days is particularly preferred. However, in alternative embodiments, administration need not be daily, but can be between once a week (or even less frequently) and more than three times a week.

Experiment (I) Effect of black tea polyphenol alone We have chemopreventive effect of black tea polyphenol (polyphenone B) administration during the pre-onset stage of 7,12 dimethylbenz [a] anthracene (DMBA) induced hamster cheek sac (HBP) carcinogenesis Evaluated. Expression of proliferating cell nuclear antigen (PCNA) in the buccal sac and lipid peroxide, protein carbonyl levels, and antioxidant status in the buccal sac, liver and erythrocytes were used as biomarkers for chemoprevention.

Chemicals Bovine serum albumin, 2-thiobarbituric acid, 2,4-dinitrophenylhydrazine, GSH, 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB8), DMBA and 3,3'-diaminobenzine Purchased from Chemical Company (St. Louis, Mo, USA). PCNA mouse monoclonal antibody and anti-mouse biotin-labeled secondary antibody were purchased from Dako, Carprinteria, CA, USA. Mitsui Norin Co. , Ltd., courtesy of Japan, Polyphenon B is epicatechin (0.4%), epigallocatechin-3-gallate (1.4%), epicatechin-3-gallate (0.1% ), Gallocatechin-3-gallate (0.2%), free theaflavin (0.32%), theaflavin monogallate-A (0.14%), theaflavin monogallate-B (0.15%), theaflavin digallate (0.21%), tannin (35.6%) and caffeine (4.9%). All other reagents used were analytical grade.

Animals and diet All experiments were performed using 6 to 10 week old male Syrian hamsters weighing between 90 and 110 g, obtained from the Central Animal House Annalabi University, India. Five animals were housed in one polypropylene cage and were allowed free access to food and water. The animals were maintained in a controlled environment under standard conditions of temperature and humidity with a 12 hour alternating light / dark cycle. The animals were maintained according to the guidelines of the National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, India, and approved by the Annamali University Ethics Committee. The experimental diet was prepared daily by mixing Polyphenon B into a weighed standard solid meal (Mysore Snack Feed, Mysore, India) at a concentration of 0.05%. The dose for polyphenone B used in this study corresponds to a daily intake of 4 cups of tea (30 to 40 mg tea polyphenol per kg body weight in humans). Diet was replenished daily and diet consumption was recorded.

Treatment plan Hamsters were randomly divided into experimental and control groups and divided into 4 groups of 10 animals in each group. Group 2 animals were fed a diet containing polyphenone B 4 weeks prior to carcinogen administration at 6 weeks of age and continued until final exposure to the carcinogen. At 10 weeks of age, Group 1 and Group 2 hamsters were coated with a solution of DMBA in 0.5% liquid paraffin on the right cheek pouch three times a week for 14 weeks using a # 4 brush. Each application leaves about 0.4 mg DMBA. Hamsters in group 1 received no further treatment. Animals in group 3 were administered polyphenone B alone as in group 2. Group 4 animals received a basal diet and served as a control.

  The experiment was terminated at the end of 18 weeks and all animals were sacrificed by cervical dislocation after an overnight fast. Prior to sacrificing the animals, the right pouch was examined visually to assess the development of precancerous lesions or tumors and photographed. Tumor volume was calculated by multiplying the average tumor volume (4 / 3πr3) (r = 1/2 tumor diameter (mm)) by the average number of tumors. The buccal sac and liver tissue were subdivided and variously processed for distribution to each experiment.

Histopathology Sac tissue was fixed in 10% formalin, embedded in paraffin, mounted on a polylysine-coated glass slide and stained with hematoxylin and eosin. Basal cell hyperplasia, dysplasia and squamous cell carcinoma (SCC9) were diagnosed. Hyperplasia of the buccal sac, indicated by an increase in the number of basal cells, was subjectively graded as mild, moderate and severe based on the thickness of the inner epithelium. Dysplasia was characterized by irregular epithelial stratification, an increase in the number of fission images, an increase in the ratio of nuclei to cytoplasm, and a loss of basal cell polarity. Histological grading of dysplasia as mild, moderate and severe was based on 1/3, 1/2 or all involvement of the epithelium, respectively. SCC was diagnosed by underlying tissue invasion, nuclear polymorphism and increased mitosis. Tumors that closely resemble the tissue of their origin were graded as well-differentiated SCC.

Immunohistochemistry Tissue sections were deparaffinized by heating at 60 ° C. for 10 minutes and then washed three times in xylene. After gradual hydration with special alcohol, the slides were incubated in citrate buffer (pH 6.0) in a microwave for 2/5 cycles for antigen activation. The sections were allowed to cool for 20 minutes and then rinsed with Tris buffered saline (TBS10). Sections were treated with 3% H ₂ O ₂ in distilled water for 15 minutes to inhibit endogenous peroxidase activity. Nonspecific antibody binding was reduced by incubating sections with normal goat serum for 25 minutes. The sections were then incubated overnight at 4 ° C. with PCNA mouse monoclonal antibody. Slides were washed with TBS and then incubated with anti-mouse biotin-labeled secondary antibody followed by streptavidin-biotin-peroxidase for 30 minutes each at room temperature. Immunoprecipitation was visualized by treatment with 3,3′-diaminobenzidine and counterstaining with hematoxylin. For negative controls, the primary antibody was replaced with TBS. Positive controls for each antibody were also processed simultaneously. The percentage of positive tumor cells expressing these proteins is graded as 0 = less than 5%, 1 = 5% to 25%, 2 = 26% to 50%, 3 = 51% to 75% and 4 = 75% over The immunoreactivity intensity was graded as-= none, + = weak staining, ++ = moderate staining, and +++ = strong staining. A tumor was considered positive when more than 10% of the tumor cells were stained.

Preparation of tissue homogenate and erythrocyte lysate Biochemical evaluation was performed using fresh tissue. The buccal sac tissue and liver tissue after weighing were homogenized in a total glass homogenizer equipped with a Teflon pestle and stored in ice until use. Blood samples were collected in heparinized tubes and plasma was separated by centrifugation at 1000 g for 15 minutes at 4 ° C. After centrifugation, the buffy coat was removed, and blood cells were washed 3 times with physiological saline. Erythrocyte samples (0.5 ml) were solubilized with 4.5 mL of 0.2 M phosphate buffer containing 0.1 M NaCl solution (pH 7.4). The hemolyzed blood was separated by centrifugation at 2500 g, 2 ° C. for 15 minutes. Biochemical assays were performed immediately.

Biochemical assay Lipid peroxidation was assessed as evidenced by the formation of thiobarbituric acid reactive substance (TBARS11), lipid hydroperoxide (LOOH12) and conjugated diene (CD13). TBARS was assayed by the method of Ohkawa et al. In sac and liver tissues and by the method described by Buge and Aust in erythrocytes. Lipid hydroperoxides are prepared according to Jiang et al. Conjugated dienes were evaluated by the method of Rao & Recnagel. Protein oxidation was measured by the method of Levine et al. Based on a reaction in which a carbonyl group reacts with 2,4-dinitrophenylhydrazine to form 2,4-dinitrophenylhydrazone. Total SOD activity and Mn-SOD activity were assayed as described by Oberley et al. Based on 50% inhibition of nitroblue tetrazolium (NBT14) reduction. Cu-Zn SOD activity was calculated by subtracting the activity of Mn-SOD from the total SOD activity. The activity of CAT was assayed by Sinha's method based on the use of hydrogen peroxide by the enzyme. GSH was measured by the method of Anderson by measuring the yellow color that develops when DTNB is added to a sulfhydryl group-containing compound. Oxidized glutathione (GSSG15) was evaluated after oxidation of NADH with glutathione reductase (GR16, EC 1.6.4.2) at 340 nm according to Anderson's method. Selenium-dependent glutathione peroxidase (GPx17, EC1.11.1.9) activity was assayed by following the use of hydrogen peroxide according to the method of Rotruck et al. Independent GPx activity was assayed according to the method described by Lawrence and Burk using cumene peroxide as a substrate. The activity of γ-glutamyl transpeptidase (GGT18) was assayed by the method of Fiala et al. using gamma glutamyl p-nitroanilide as a substrate. GR activity was assessed by the method of Carlberg and Mannervic using GSSG as a substrate and FAD as a cofactor. Protein content was assessed by the method of Lowry et al. Using bovine serum albumin as a standard.

Statistical analysis Data are expressed as mean ± standard deviation. Body weight was analyzed by Student's t test. Tumor incidence was analyzed using χ2 test combined with Yate correction. Statistical analysis on tumor volume, immunohistochemical and biochemical assay data was performed using analysis of variance (ANOVA19) and group means were compared by least significant difference test (LSD20). Results were considered statistically significant when the p-value was less than 0.05.

Results Overall Observation Results Table 1 of FIG. 1 shows the average body weight, food consumption, tumor incidence, tumor multiplicity and average tumor volume in various groups. The hamsters in group 1 showed a tendency to lose weight during the experiment, and the average final weight was significantly (p <0.01) lower compared to the control (group 4). No significant difference in body weight was observed in Groups 2-4. There was no significant difference in the amount of food consumed in groups 1 to 4. At the end of the experimental period, the tumor incidence in group 1 was 100% with a multiplicity of 1.8 per hamster and a tumor volume of 346 mm ³ . Administration of polyphenone B reduced tumor incidence to 20% with a multiplicity of 0.3 per hamster. Furthermore, the tumor was significantly smaller compared to group 1 (mean tumor volume 17.2 mm ³ ). Tumors were not observed in groups 3 and 4.

Histopathological Observations Table 2 in FIG. 1 summarizes histopathological changes in the hamster cheek pouch in the experimental and control groups. All Group 1 hamsters exhibited severe keratosis, hyperplasia, dysplasia, and well-differentiated SCC. Dietary administration of polyphenone B to hamsters (Group 2) coated with DMBA significantly reduced the incidence of SCC and hyperplastic and dysplastic lesions. Of the 10 animals, only 2 developed SCC, 4 animals showed mild to moderate dysplasia, and the remaining 4 hamsters showed only moderate hyperplasia. Mild hyperplastic changes were observed in 2 of Group 3 hamsters. In the control group of animals, the epithelium was normal, intact and continuous. Representative micrographs of histopathological changes in each group are shown in FIG. FIG. 2 shows photomicrographs of H and E stained areas of the buccal pouch mucosa of control and experimental animals. A. Well-differentiated SCC with extensive infiltration into the connective tissue of Group 1 animals 14 weeks after DMBA treatment (H and E staining, 10 fold). B. A photomicrograph of buccal sac epithelium from group 2 hamsters administered DMBA and polyphenone B exhibiting moderate dysplasia (H and E staining, 40x). C. Photomicrograph of buccal sac epithelium from group 3 hamsters administered with polyphenone B alone exhibiting mild hyperplasia (H and E staining, 40x). D. Photomicrograph showing normal buccal histology of control animals (Group 4, H and E staining, 40x).

Immunohistochemical findings FIG. 3 shows the effect of polyphenone B on PCNA expression in the buccal pouch mucosa of control and experimental animals. In hamsters coated with DMBA, the average protein expression of PCNA (81%) was significantly higher compared to control animals (Group 4). Administration of polyphenone B (group 2) significantly reduced PCNA expression (52%) compared to group 1. No significant change in PCNA expression was observed in group 3 animals compared to the control group. A representative photomicrograph of immunostaining is shown in FIG. Here, photomicrographs (10X) of immunohistochemical staining of PCNA expression in control and experimental animals are Overexpression of PCNA protein in group 1 animals; Reduced expression of PCNA protein in group 2 animals, C.I. Expression of PCNA protein in group 3 animals, D. PCNA expression in control animals is shown.

Biochemical findings Table 3 in FIG. 5 shows the effect of pretreatment with polyphenone B on lipid peroxidation and protein oxidation. The degree of lipid peroxidation and protein carbonyl formation was significantly lower in the cheek sac of DMBA-coated hamsters (Group 1) and higher in the liver compared to the control group. Dietary administration of polyphenone B (Group 2) significantly mitigated DMBA-induced changes in lipid and protein oxidation as reflected by a significant increase in the buccal sac with a decrease in the liver compared to Group 1. Administration of polyphenone B alone significantly reduced the degree of tissue lipid peroxidation and protein oxidation in group 3 animals compared to the control group.

  FIG. 6 shows the activities of the antioxidant enzymes SOD (total SOD, Mn-SOD, Cu-Zn SOD) and CAT in the buccal sac, liver and erythrocytes of the animals of the control group and the experimental group. In animals applied with DMBA (Group 1), the activity of SOD and CAT was significantly lower compared to the control group. Dietary administration of polyphenone B significantly increased antioxidant enzyme activity in group 2 animals compared to group 1. Administration of polyphenone B alone significantly increased the activity of SOD and CAT in group 3 animals compared to the control group (group 4). Changes in GSH concentration and activity of GSH-dependent enzymes in the buccal sac, liver and erythrocytes are presented in FIGS. In animals applied with DMBA (Group 1), GSH concentration, GSSG concentration and GSH / GSSG ratio and GPx (Se dependent and independent), GR and GGT activities were significantly increased in the cheek pouch In liver and erythrocytes, these antioxidants were significantly reduced compared to controls. Dietary administration of polyphenone B significantly increased all antioxidants and detoxifying enzymes in the buccal sac, liver and red blood cells of group 2 animals compared to group 1. Treatment with polyphenone B alone significantly enhanced GSH and GSH-dependent enzymes in group 3 animals compared to the control group.

  All hamsters applied for 14 weeks with DMBA alone developed buccal sac cancer associated with increased PCNA expression, decreased lipid and protein oxidation, and enhanced antioxidant status. In the liver and erythrocytes of tumor-bearing animals, enhanced lipid and protein oxidation was accompanied by an infectious antioxidant defense. Dietary administration of polyphenone B effectively suppressed DMBA-induced HBP carcinogenesis, as evidenced by the reduced incidence of tumor and PCNA expression. Polyphenone B also moderated lipid and protein oxidation and enhanced antioxidant status in sac, liver and erythrocytes. We suggest that polyphenone B exerts its chemopreventive effect by inhibiting cell growth in the target tissue and modulating the oxidation-antioxidant state in the target tissue as well as the host tissue.

  Thus, it should be understood that the administration of black tea catechin, and in particular polyphenone B, regulated the redox state in the target organ and host tissue and significantly suppressed PCNA expression in the buccal sac. HCPC-hamster buccal sac cancer cell line polyphenone B reverses the sensitivity to lipid and protein oxidation while simultaneously increasing the antioxidant status in the buccal sac, while the extent of lipid and protein oxidation in the liver and erythrocytes Decreased with increasing antioxidants. Thus, the differential oxidative events induced in tumor and host tissues by polyphenone B reflect its inhibitory role on cell proliferation, as evidenced by downregulation of PCNA expression and suppression of tumor development. ing. These results demonstrate that dietary administration of polyphenone B exerts significant protection against HBP carcinogenesis by downregulating PCNA expression, modulating lipid and protein oxidation, and enhancing antioxidant status. ing.

  We further describe the expression of cytokeratin and the proliferation and apoptosis related proteins of black tea polyphenol (polyphenone B (P-B)) during 7,12-dimethylbenz [a] anthracene-induced hamster buccal sac (HBP) carcinogenesis. The effect on expression was evaluated. As a marker for carcinogenesis, we used the development of buccal sac carcinoma with increased expression of cytokeratin, PCNA, NF-κB, and Bcl-2 and decreased expression of cytochrome C and caspase-3.

Chemicals DMBA and 3′-diaminobenzidine are available from Sigma Chemical Company, St. Purchased from Louis, MO, USA. All other reagents used were analytical grade.

Animal experiments were performed using 6 to 10 week old male Syrian hamsters weighing between 90 and 110 g obtained from the Central Animal House, Annamali University, India. Five animals were housed in one polypropylene cage, provided food and water ad libitum, and maintained in a controlled environment under standard temperature and humidity conditions with a 12 hour alternating light / dark cycle. The animals were maintained according to the guidelines of the National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, India, and approved by the Annamali University Ethics Committee. The experimental diet was prepared daily by mixing P-B at a concentration of 0.05% into a pre-measured standard solid meal (Mysore Snack Feed, Mysore, India). The dose for P-B used in this study corresponds to a daily intake of 4 cups of tea (30 to 40 mg tea polyphenol per kg body weight in humans). Diet was replenished daily and diet consumption was recorded.

Treatment plan Hamsters were randomly divided into experimental and control groups and divided into 4 groups of 10 animals in each group. Group 2 animals were fed a diet containing P-B 4 weeks prior to carcinogen administration at 6 weeks of age and continued until final exposure to the carcinogen. At 10 weeks of age, 0.5% liquid paraffin solution of DMBA was applied to group 1 and 2 hamsters on the right cheek pouch three times a week for 14 weeks using a No. 4 brush. Each application leaves about 0.4 mg DMBA. Two hamsters in group 1 received no further treatment. Group 3 animals received P-B alone as in Group 2. Group 4 animals received a basal diet and served as a control. The experiment was terminated at the end of 18 weeks and all animals were sacrificed by cervical dislocation after an overnight fast. Prior to sacrificing the animals, the right pouch was examined visually to assess the development of precancerous lesions or tumors and photographed. Tumor volume was calculated by multiplying the average tumor volume (4 / 3πr ³ ) (r = 1/2 tumor diameter (mm)) by the average number of tumors. The buccal sac tissue was subdivided and processed variously for distribution to each experiment. Tissues were fixed in 10% formalin, embedded in paraffin and mounted on polylysine-coated glass slides. One section from each specimen was stained with hematoxylin and eosin. Residual sections were used for immunohistochemical staining.

Immunohistochemistry Tissue sections were deparaffinized by heating at 60 ° C. for 10 minutes and then washed three times in xylene. After gradual hydration with special alcohol, the slides were incubated in citrate buffer (pH 6.0) in a microwave for 2/5 cycles for antigen activation. The sections were allowed to cool for 20 minutes before rinsing with Tris buffered saline (TBS) and treated with 3% H ₂ O ₂ in distilled water for 15 minutes to inhibit endogenous peroxidase activity. Nonspecific antibody binding was reduced by incubating sections with normal goat serum for 25 minutes. The sections were then cytokeratin, PCNA and Bcl-2 (DAKO, Carprinteria, CA, USA) mouse monoclonal antibody and NF-κB and caspase-3 rabbit polyclonal antibody and cytochrome C monoclonal antibody (all Neo Markers, USA). And incubated at 4 ° C. overnight. The slides were washed with TBS and then incubated with anti-rabbit and anti-mouse biotin labeled secondary antibodies (both DAKO, Carprinteria, CA, USA) followed by streptavidin-biotin-peroxidase for 30 minutes each at room temperature. did. Immunoprecipitation was visualized by treatment with 3,3′-diaminobenzidine and counterstaining with hematoxylin. For negative controls, the primary antibody was replaced with TBS. Positive controls for each antibody were processed simultaneously. The labeling index for PCNA was calculated as the number of cells with positive staining per 100 counted cells in 3 high power fields. The expression of NF-κB, Bcl-2, cytochrome C and caspase 3 was determined according to the number of positively stained cells per 100 counted cells, I = 5% to 25%, II = 26% to 50 %, III = 51% to 75% and IV = 75% over. Cytokeratin expression is graded as 0 = no detectable keratin, I = stain limited to some evidence of basal or suprabasal area staining, II = positive staining through basal and / or suprabasal area Divided.

Colorimetric evaluation of caspase 3 activity DEVD-specific caspase 3 activity was assayed using the CASP-3-C colorimetric kit (Sigma, St. Louis Mo, USA) according to the manufacturer's instructions. The cytoplasmic extract was prepared by homogenizing the tissue in solubilization buffer containing 50 mM HEPES (pH 7.4), 5 mM CHAPS and 5 mM DTT. The supernatant was collected as an enzyme source. The caspase 3 colorimetric assay is the release of the p-nitroaniline (pNA) moiety as a result of hydrolysis by the caspase 3 of the peptide substrate acetyl-Asp-Glu-Val-Asp-nitroanilide (Ac-DEVD-pNA). Is based on what happens. The concentration of pNA released from the substrate is calculated from the absorbance value at 405 nm or from a calibration curve created with a pNA defined solution.

Statistical analysis Tumor incidence and NF-κB, cytokeratin, Bcl-2, cytochrome C and caspase 3 grading were statistically compared using the X2 test. Statistical analysis of data on tumor burden was performed using Student's t-test. PCNA labeling index and colorimetric assay data for caspase 3 were analyzed using LNO after using ANOVA. Results were considered statistically significant when the p-value was less than 0.05.

Results Table 4 in FIG. 9 shows tumor incidence, average tumor volume and histopathological changes in control and experimental animals. The tumor incidence in group 1 was 100% and the average tumor volume was 346 mm ³ . Histologically, HBP tumors induced by DMBA were invasive squamous cell carcinomas with squamous papillary processes to connective tissue. Two of the 10 hamsters treated with DMBA and P-B developed SCC, while 4 hamsters showed mild to moderate dysplasia and the remaining 4 hamsters had moderate overload. Showed formation. Mild hyperplastic changes were observed in 2 of the hamsters in group 3. In control animals, the epithelium was normal, intact and continuous.

  Table 5 in FIG. 10 shows the effect of P-B on the PCNA labeling index and the expression of NF-κB, cytokeratin, Bcl-2, cytochrome C and caspase 3 in the buccal sac of control and experimental animals. In animals applied with DMBA (Group 1), the expression of PCNA, NF-κB, cytokeratin and Bcl-2 is significantly higher compared to control animals (Group 4), and the expression of cytochrome C and caspase 3 is significant. Was lower. Administration of P-B (group 2) significantly reduced the expression of PCNA, NF-κB, cytokeratin and Bcl-2 and significantly increased the expression of cytochrome C and caspase 3 compared to group 1. It was. No significant changes in the expression of PCNA, NF-κB, cytokeratin, Bcl-2, cytochrome C and caspase 3 were observed in group 3 animals. PCNA immunostaining showed nuclear localization, whereas NF-κB, cytokeratin, Bcl-2, cytochrome C and caspase 3 were found in the cytoplasmic region. Representative micrographs of immunostaining are shown in FIGS.

  FIG. 13 shows the activity of DEVD-specific caspase 3 in the buccal sac in control and experimental hamsters. In animals that received DMBA (Group 1), caspase 3 activity was significantly reduced compared to controls (Group 4). Treatment with P-B significantly increased enzyme activity in group 2 animals compared to group 1. In animals treated with P-B alone (Group 3), the activity of caspase 3 was not significantly different from that of the control.

  Thus, it should be noted that dietary administration of black tea polyphenols, and in particular P-B, reduced the incidence of DMBA-induced HBP cancer and lesions prior to neoplasia. P-B also significantly downregulated the expression of PCNA, NF-κB and Bcl-2 in the buccal sac and up-regulated cytochrome C, caspase 3 and cytokeratin. These data indicate that P-B acts as an inhibitor and its anti-malignant tumor properties are reduced by downregulating PCNA, NF-κB and Bcl-2 and upregulating cytochrome C, caspase 3 and cytokeratin. It strongly suggests that it demonstrates. Interestingly, P-B induced apoptosis in tumor cells but not in normal cells. Hsu et al. Found that EGCG induces growth inhibition of transformed cells by activating the apoptotic pathway without affecting normal growth, and the protective effect on normal cells is closely related to cell differentiation. It was suggested that it might be caused by the related molecule p57. These findings support the hypothesis that dietary drugs that cause tumor cells to undergo apoptosis and lead normal cells to the survival pathway are ideal chemopreventive agents.

(II) Effect of Black Tea Polyphenol-Lactoferrin Combination We have reported that bovine lactoferrin (bLF) and black tea polyphenol (Polyphenone B: The antiproliferative and apoptosis-inducing effects of the combination with P-B) were evaluated. Expression of cytokeratin and other markers including proliferating cell nuclear antigen (PCNA), NF-κB, p53 mutant, Bcl-2, Bax, Fas and caspase 3 was used as a marker for chemoprevention of oral cancer .

Chemicals DMBA was purchased from Sigma Chemical Company, St. Purchased from Louis, MO, USA. BLF (lot number 020119) having a purity of 96.2% or more was purchased from Morinaga Milk Industry Co. , Ltd, Tokyo, Japan. Black tea polyphenol (polyphenone-B) is available from Mitsui Norin Co. , Ltd., Ltd. , Courtesy of Tokyo, Japan. The composition of Polyphenon-B was the same as described above. Epicatechin (0.4%), epigallocatechin-3-gallate (1.4%), epicatechin-3-gallate (0.1%), gallocatechin-3-gallate (0.2%), Free theaflavin (0.32%), theaflavin monogallate-A (0.14%), theaflavin monogallate-B (0.15%), theaflavin digallate (0.21%), tannin (35.6%) And caffeine (4.9%). All other reagents used were analytical grade.

Animals and Diet Experiments were performed using 8- to 10-week-old male Syrian hamsters weighing 100 to 110 g obtained from the Central Animal House, Annamalai University, India. Five animals were housed in one polypropylene cage, provided food and water ad libitum and maintained under temperature and humidity controlled conditions in alternating light / dark cycles. The animals were maintained according to the guidelines of the National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, India, and approved by the Annamali University Ethics Committee. Experimental meals were prepared daily by mixing chemopreventive agents, either alone or in combination, into a pre-measured standard solid meal (Mysore Snack Feed, Mysore, India). Diet was replenished daily and diet consumption was recorded.

Treatment plan Hamsters were randomly divided into experimental and control groups and divided into 8 groups of 20 animals in each group. In Group 1, 0.5% DMBA liquid paraffin solution was applied to the right buccal sac of a hamster 3 times a week with a No. 4 brush. Each application leaves about 0.4 mg DMBA. Group 1 hamsters received no further treatment. In groups 2 to 4, the right cheek pouch with DMBA applied as in group 1 is further supplemented with a basic diet containing 0.2% bLF and 0.05% polyphenone-B, and 0.2% bLF and 0.05% polyphenone. Each diet containing a combination with -B was received. Groups 5 to 7 animals were administered bLF, polyphenone-B alone and combinations thereof, respectively. Group 8 animals served as controls. The experiment was terminated at 14 weeks and all animals were sacrificed by cervical dislocation after an overnight fast. Prior to slaughtering the animals, the right pouch was macroscopically assessed for precancerous lesions or tumor development and photographed. Tumor volume was calculated by multiplying the average tumor volume (4 / 3πr3) (r = 1/2 tumor diameter (mm)) by the average number of tumors. The buccal sac tissue was subdivided and processed variously for distribution to each experiment. Tissues were fixed in 10% formalin, embedded in paraffin, and placed on polylysine-coated glass slides. One section from each specimen was stained with hematoxylin and eosin. Residual sections were used for immunohistochemical staining.

Immunohistochemistry Tissue sections were deparaffinized by heating at 60 ° C. for 10 minutes and then washed three times in xylene. After gradual hydration with special grade alcohol, the slides were incubated in citrate buffer (pH 6.0) in a microwave oven for 5 minutes × 2 cycles for antigen activation. The sections were allowed to cool for 20 minutes and then rinsed with Tris buffered saline (TBS). Sections were treated with 3% H ₂ O ₂ in distilled water for 15 minutes to inhibit endogenous peroxidase activity. Nonspecific antibody binding was reduced by incubating sections with normal goat serum for 25 minutes. The sections were then incubated overnight at 4 ° C. with PCNA, p53 and cytokeratin AE1 / AE3 mouse monoclonal antibodies (Dako, Carprinteria, Calif., USA). The slides were washed with TBS and then incubated with anti-rabbit and anti-mouse biotin-labeled secondary antibodies (DAKO, Carprinteria, CA, USA) followed by streptavidin biotin-peroxidase for 30 minutes each at room temperature. Immunoprecipitation was visualized by treatment with 3,3′-diaminobenzidine (Sigma) and counterstaining with hematoxylin. For negative controls, the primary antibody was replaced with TBS. Positive controls for each antibody were also processed simultaneously. The labeling index for PCNA was expressed as the number of cells with positive staining per 100 counted cells. Data for cytokeratin were graded as I = stain limited to some evidence of basal or suprabasal region staining, II = positive staining through basal and / or suprabasal region. p53 expression was determined according to the number of positively stained cells per 100 counted cells, I = 5% to 25%, II = 26% to 50%, III = 51% to 75% and IV = 75. Graded as over%.

SDS-PAGE and Western Blot Analysis About 50 mg of each tissue sample is taken in a sample buffer containing 62.5 mM Tris (pH 6.8), 2% SDS, 5% 2-mercaptoethanol, 10% glycerol and bromophenol blue. It was subjected to solubilization. The protein concentration of the lysate was measured by the Bradford method. SDS-PAGE was performed using an equivalent protein extract (55 μg) from each sample according to the Laemmli method. Degraded proteins were electrophoretically transferred to polyvinylidene difluoride membranes (Immobilion, Millipore, Bedfore, MA, USA). Membranes were incubated in TBS (150 mM NaCl / 50 mM Tris, pH 7.4) containing 5% non-fat dry milk to block non-specific binding sites for 1 hour. Blots were incubated overnight at room temperature with a 1: 1000 dilution of anti-Bcl-2, anti-Bax, anti-p53, anti-NF-κB, anti-Fas, and anti-caspase 3 antibodies (NeoMarkers, USA). The blot was washed thoroughly with TBS containing 0.1% Tween-20 (TBS-T) and then at room temperature with a 1: 1000 dilution of horseradish peroxidase labeled secondary antibody (Santa Cruz Biotechnology, CA, USA). Incubated for 30 to 45 minutes. After extensive washing in TBS-T, the protein was visualized by treatment with 3,3′-diaminobenzidine (Sigma). Densitometry was performed on an IISP flatbed scanner and quantified with Total Lab 1.11 software.

Colorimetric evaluation of caspase 3 activity DEVD-specific caspase 3 activity was assayed using the CASP-3-C colorimetric kit (Sigma, St. Louis Mo, USA) according to the manufacturer's instructions. The cytoplasmic extract was prepared by homogenizing the tissue in solubilization buffer containing 50 mM HEPES (pH 7.4), 5 mM CHAPS and 5 mM DTT. The supernatant was collected as an enzyme source. The caspase 3 colorimetric assay is the release of the p-nitroaniline (pNA) moiety as a result of hydrolysis by the caspase 3 of the peptide substrate acetyl-Asp-Glu-Val-Asp-nitroanilide (Ac-DEVD-pNA). Is based on what happens. The concentration of pNA released from the substrate is calculated from the absorbance value at 405 nm or from a calibration curve created with a pNA defined solution.

Statistical analysis Data are expressed as mean ± SD. Tumor incidence was analyzed using a χ2 test combined with Yates correction. Statistical analysis was performed on data regarding tumor burden, PCNA labeling index and densitometric analysis using analysis of variance (ANOVA), and group means were compared by Tukey-Kramer test. Statistical analysis on the data for CK and p53 was performed using the χ2 test. Results were considered statistically significant when the p-value was less than 0.05. The nature of the interaction between the effects of the combination of bLF and polyphenone-B was evaluated as described by Yokoyama et al. Briefly, the expected value of the effect of the combination between bLF treatment and polyphenone-B treatment is [(observed bLF treatment value) / (control value)] × [(observed polyphenone-B treatment value). / (Control value)], and the combination index was calculated as the ratio of expected value / observed value. A ratio greater than 1 indicates a synergistic effect, and a ratio less than 1 indicates less than an additional effect.

Results Table 6 in FIG. 14 summarizes food consumption, tumor incidence, tumor burden and SCC incidence in the experimental and control hamster cheek pouches. At the end of 14 weeks, the tumor incidence of group 1 was 100%. These tumors were exoproliferative and were clearly defined with an average tumor volume of 172.97 mm ³ . Administration of bLF and polyphenone-B alone and in combination (Group 2 to Group 4) significantly reduced tumor incidence, tumor burden and incidence of SCC, but the combination was more effective than a single agent It was the target. Furthermore, a combination index ratio of 1.46 for the combination of bLF and polyphenone-B to tumor incidence indicates that the combination has a synergistic effect in suppressing HBP carcinogenesis. Tumors were not observed in Groups 5-8. The amount of food consumed in groups 1 to 8 was not significantly different.

  FIG. 15 (A) and Table 7 in FIG. 16 show the immunohistochemical analysis of cytokeratin in various groups. In animals that received DMBA (Group 1), cytokeratin expression was significantly higher than in control animals. Furthermore, we observed that infiltrating cancer cells showed strong expression of cytokeratin in group 1 animals. The combination of bLF and P-B significantly reduced cytokeratin expression than either drug alone. Administration of chemopreventive agent alone (Group 5 to Group 7) did not significantly affect cytokeratin expression compared to the control group (Group 8).

  FIG. 15B and Table 7 in FIG. 16 show immunohistochemical analysis of PCNA labeling index in hamster buccal sac of control and experimental animals. Topical application of DMBA (Group 1) significantly increased the mean PCNA labeling index compared to the control group (Group 8). Co-administration of bLF and P-B reduced the PCNA labeling index more significantly than either agent alone compared to Group 1. Administration of bLF and PB alone and in combination (Group 5 to Group 7) did not significantly affect the PCNA labeling index compared to the untreated control group (Group 8).

  The results of immunohistochemical analysis of mutant p53 protein expression and representative micrographs of p53 immunostaining are shown in Table 7 of FIG. 15 (C). In animals that received DMBA, p53 expression was significantly increased compared to the control group (Group 8). Administration of bLF and P-B significantly reduced p53 expression over either drug alone. In animals that received chemopreventive agent alone (Group 5 to Group 7), p53 expression was not significantly different from the control group.

  FIG. 17 shows representative Western blot analysis of Bcl-2, Bax, NF-κB, p53, Fas and caspase 3 and caspase 3 activity in the buccal sac of control and experimental animals. Expression of Bcl-2, Bax, NF-κB, p53, Fas and caspase 3 was detected as molecular weight bands of 25 kDa, 21 kDa, 65 kDa, 53 kDa, 48 kDa and 32 kDa, respectively. Average protein expression from the lysate of the control group was expressed as 100% in the graph. Each bar graph represents the mean protein expression ± SD of 10 measurements per treatment. Topical application of DMBA significantly increased Bcl-2 / Bax ratio and p53 and NF-κB expression, and reduced Fas and caspase 3 expression and caspase 3 activity compared to the control group (group 1). It was. Co-administration of bLF and P-B significantly reduced Bcl-2 / Bax ratio, p53 and NF-κB expression, Fas and caspase 3 expression and caspase 3 activity more significantly than either drug alone Increased. No significant changes in the expression of these proteins were observed in groups 5 to 7 compared to control animals.

  Therefore, administration of a combination of bLF and black tea polyphenol P-B was more effective than either drug alone in suppressing DMBA-induced HBP cancer development, as evidenced by a decrease in tumor incidence. Should be understood. This is a substantial reduction in cell proliferation and significant enhancement of apoptosis, as demonstrated by downregulation of PCNA, NF-κB, p53 and Bcl-2 and upregulation of Bax, Fas and caspase-3 expression. Was accompanied. The anti-proliferative and apoptotic effects of bLF and P-B could potentially alleviate the carcinogenic effects of DMBA, thereby reducing the invasion ability of HBP cancers as reflected by cytokeratin expression.

  The chemopreventive effect of the combination of bLF and P-B reflects the anti-proliferative and apoptotic effects of individual drugs. bLF has been reported to down-regulate PCNA expression in 4-nitroquinoline 1-oxide-induced tongue carcinogenesis. It has been discovered that the inhibitory effect of bLF on azoxymethane-induced colon carcinogenesis is mediated by induction of the death receptor Fas and the pro-apoptotic members Bid and Bax of the Bcl-2 family. Tea polyphenols have been shown to suppress cell growth detected by in situ Brdu incorporation, PCNA downregulation and G1 phase arrest.

  The results presented here clearly demonstrate that the combination of bLF and various tea polyphenols, especially black tea polyphenols, acts as an inhibitor by inhibiting cell proliferation and inducing apoptosis. To do. An interesting finding is the sensitivity of discrimination between HBP cancer and normal cells for growth control and apoptosis. Several in vitro studies have also demonstrated that tumor cells are more sensitive to the anti-proliferative and apoptotic effects of tea polyphenols than normal cells. These findings support the fact that dietary drugs that cause tumor cells to undergo apoptosis and lead normal cells to the survival pathway are ideal chemopreventive agents. Based on these observations, our data suggest that the combination of bLF and P-B may have unlimited potential in human oral cancer prevention strategies.

  We further describe the effects of lactoferrin and various black tea polyphenols (polyphenone B (P-) on the cellular redox status and regulation of carcinogen metabolizing enzymes in 7,12-dimethylbenz [a] anthracene-induced hamster buccal sac (HBP) carcinogenesis. The effect of the combination with B)) was evaluated.

Chemicals DMBA was purchased from Sigma Chemical Company, St. Purchased from Louis, MO, USA. BLF (lot number 020119) with a purity of 96.2% was purchased from Morinaga Milk Industry Co. , Ltd, Tokyo, Japan. Black tea polyphenol (polyphenone-B) is available from Mitsui Norin Co. , Ltd., Ltd. , Courtesy of Tokyo, Japan. Polyphenone B is epicatechin (0.4%), epigallocatechin-3-gallate (1.4%), epicatechin-3-gallate (0.1%), gallocatechin-3-gallate (0.2% ), Free theaflavin (0.32%), theaflavin monogallate-A (0.14%), theaflavin monogallate-B (0.15%), theaflavin digallate (0.21%), tannin (35.6) %) And caffeine (4.9%). All other reagents used were analytical grade.

Animals and Diet Experiments were performed using 8- to 10-week-old male Syrian hamsters weighing 100 to 110 g obtained from the Central Animal House, Annamalai University, India. Five animals were housed in one polypropylene cage, provided food and water ad libitum and maintained under controlled temperature and humidity conditions in alternating light / dark cycles. Animals were maintained according to the guidelines of the National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, India, and approved by the Annamali University Ethics Committee. Experimental meals were prepared daily by mixing chemopreventive agents, alone or in combination, into a weighed standard solid meal (Mysore Snack Feed Ltd, Mysore, India). Diet was replenished daily and diet consumption was recorded.

Treatment plan Hamsters were randomly divided into experimental and control groups and divided into 8 groups of 20 animals in each group. In Group 1, 0.5% DMBA liquid paraffin solution was applied to the right buccal sac of a hamster 3 times a week with a No. 4 brush. Each application leaves about 0.4 mg DMBA. Group 1 hamsters received no further treatment. In groups 2 to 4, the right cheek pouch with DMBA applied as in group 1 is further supplemented with a basic diet containing 0.2% bLF and 0.05% polyphenone-B, and 0.2% bLF and 0.05% polyphenone. Each diet containing a combination with -B was received. Groups 5 to 7 animals were administered bLF, polyphenone-B alone and combinations thereof, respectively. Group 8 animals served as controls. The experiment was terminated at 14 weeks and all animals were sacrificed by cervical dislocation after an overnight fast. Prior to sacrifice of the animals, the right pouch was visually inspected to assess pre-neoplastic lesions or tumor development and photographed. Tumor volume was calculated by multiplying the average tumor volume (4 / 3πr ³ ) (r = 1/2 tumor diameter (mm)) by the average number of tumors. The buccal sac and liver tissue were subdivided and variously processed for distribution to each experiment.

Micronucleus test To assess chromosomal damage in experimental group animals, a bone marrow micronucleus test was performed according to Schmid. A total of 2500 polychromatic erythrocytes were scored from a single slide per animal to determine the frequency of polychromatic erythrocytes with micronuclei. All slides were scored by the same observer.

Biochemical assay Biochemical evaluation was performed using freshly homogenized tissue. Cytochrome P450 was assayed by the method of Omura and Sato using a carbon monoxide difference spectrum between 400 nm and 500 nm using an absorption coefficient of 91 cm ² M-1m-1. Glutathione S-transferase (GST) activity was measured from the increase in absorbance at 340 nm using 1-chloro-2,4-dinitrobenzene as a substrate as described by Habig et al. The activity of DT-diaphorase (DTD) was assayed using NADPH as an electron donor and 2,6-dichlorophenol-indophenol as an electron acceptor as described by Ernster. Lipid peroxidation was assessed as evidenced by the formation of thiobarbituric acid reactive substances (TBARS), conjugated dienes and lipid hydroperoxides. TBARS was assayed in tissue by the method described by Ohkawa et al., Lipid hydroperoxides by Jiang et al., And conjugated dienes by Rao and Recnagel, respectively. Reduced glutathione (GSH) was measured by Anderson's method based on yellow coloration when 5,5′-dithiobis (2-nitrobenzoic acid) was added to a compound containing a sulfhydryl group. Oxidized glutathione (GSSG) was evaluated based on Anderson's method at 340 nm after oxidation of NADPH with glutathione reductase. Glutathione peroxidase (GPx) activity was assayed by utilizing hydrogen peroxide according to the method of Rotruck et al. Protein content was evaluated by the method of Lowry et al.

Statistical analysis Data are expressed as mean ± SD. Body weight was analyzed by Student's t test. Tumor incidence was analyzed using a χ2 test combined with Yates correction. Analysis of variance (ANOVA) was used to analyze the statistical analysis on data regarding tumor volume, tumor multiplicity, bone marrow micronucleus incidence and biochemical assays, and group means were compared by the Tukey-Kramer test. Results were considered statistically significant if the p value was less than 0.05. The nature of the interaction between the effects of the combination of bLF and polyphenone-B was evaluated as described by Yokoyama et al. Briefly, the expected effect of the combination between the first treatment (bLF) and the second treatment (polyphenone-B) is expressed as [(first treatment value observed) / (control value)] × [( Observed second treatment value) / (control value)] and the combination index is calculated as the ratio of (expected value) / (observed value). A ratio greater than 1 indicates a synergistic effect, and a ratio less than 1 indicates less than an additional effect.

Visual and Microscopic Observation Table 8 in FIG. 18 shows the average body weight, food consumption, and bone marrow micronucleus frequency for the various groups. Topical application of DMBA for 14 weeks significantly reduced the average body weight of group 1 animals compared to the control group (group 8). No significant difference in body weight was observed in groups 2 through 8. There was no significant difference in the amount of food consumed in Groups 1-8. The frequency of polychromatic leukocytes with bone marrow micronuclei (MnPCE) was significantly higher in animals (Group 1) coated with DMBA compared to the control group (Group 8). Dietary administration of bLF and polyphenone-B alone (groups 2 and 3 respectively) significantly reduced the incidence of MnPCE compared to group 1, and the combination of bLF and polyphenone-B (group 4) The inhibition was greater (43.33%) compared to the other groups. Administration of bLF and polyphenone-B alone and in combination (group 5 to group 7) showed no significant change in the incidence of MnPCE compared to the control group (group 8).

Table 9 in FIG. 19 summarizes tumor incidence, tumor multiplicity, average tumor burden and histopathological changes in the experimental and control hamster cheek pouches. The tumor incidence in group 1 was 100% and the multiplicity was 1.55 tumors per hamster. These tumors are exoproliferative and are clearly defined with an average tumor volume of 172.97 mm ³ . Administration of bLF and polyphenone-B alone and in combination (Group 2 to Group 4) significantly reduced tumor incidence, tumor multiplicity, and tumor volume as well as pathological changes. Among groups 1 to 4, the combination of bLF and polyphenone-B (group 4) significantly reduced these changes. Furthermore, a combination index ratio of 1.46 for the combination of bLF and polyphenone-B to tumor incidence indicates that the combination can have a synergistic effect in suppressing HBP carcinogenesis. Tumors were not observed in Groups 5-8.

Biochemical Findings FIG. 20 shows the effect of treatment with bLF and polyphenone-B alone and in combination on the activity of phase I and phase II carcinogen metabolizing enzymes in the buccal sac and liver. Topical application of DMBA (Group 1) significantly enhanced enzyme activity in the sac, whereas in the liver, an increase in cytochrome P450 significantly increased phase II enzyme activity compared to the control group (Group 8). With a decrease (p <0.001). Dietary administration to animals coated with DMBA, alone or in combination with bLF and polyphenone-B, significantly suppressed the activity of cytochrome P450 in the sac and liver compared to group 1, and the activity of phase II enzymes (P <0.05, p <0.01 and p <0.001 for animals in groups 2 to 4 respectively). Administration of dietary drugs alone and in combination (groups 5 to 7) did not induce any significant changes in cytochrome P450, but the activity of the phase II enzyme was significantly enhanced compared to group 8. It was.

  FIG. 21 shows the effect of treatment with bLF and polyphenone-B alone and in combination on DMBA-induced changes in lipid peroxidation in hamster cheek sac and liver. Topical application of DMBA for 14 weeks reduced lipid peroxidation in the buccal sac, whereas the extent of lipid peroxidation increased in the liver compared to the control group (p <0.001). Dietary administration of bLF and polyphenone-B alone and in combination significantly mitigated DMBA-induced changes in lipid peroxidation in the buccal sac and liver compared to group 1 (p for animals in groups 2 to 4 respectively) <0.05, p <0.01 and p <0.001). Administration of dietary drugs alone and in combination significantly reduced the degree of lipid peroxidation in the buccal sac and liver of the animals of Groups 5 to 7, respectively, compared to Group 1. The GSH, GSSG, GSH / GSSG ratio and GPx activity in the hamster sac and liver of the control group and experimental group are shown in FIG. In animals applied with DMBA (Group 1), concentrations of GSH and GSH / GSSG ratio and GPx activity were enhanced in the buccal sac, whereas in the liver, all antioxidants were significant compared to the control group (P <0.001). Treatment with bLF and polyphenone-B alone and in combination significantly increased all antioxidants in the sac and liver compared to group 1 (p <0.05 for group 2 to group 4 animals, respectively). p <0.01 and p <0.001). Administration of bLF and polyphenone-B alone and in combination (group 5 to group 7) significantly enhanced the antioxidant status in the buccal sac and liver compared to the control group (group 8). Overall, the combination of bLF and polyphenone-B has been found to be more effective than either agent alone in modulating carcinogen metabolizing enzymes and lipid peroxidation and antioxidants. .

  As is apparent, topical application of DMBA to HBP for 14 weeks resulted in fully developed SCC, with a very high average tumor burden. This was further accompanied by an imbalance in the increased frequency of phase I and phase II xenobiotic metabolizing enzymes, intracellular redox status, and bone marrow micronuclei. Of note, co-administration of bLF and black tea polyphenol (polyphenone-B) resulted in HBP with regulated equilibrium and redox state modulation in the phase I and phase II enzymes in the sac and liver. The incidence of cancer was significantly reduced. Such balance and regulation is believed to mitigate the mutagenic and carcinogenic effects of DMBA, thereby minimizing the frequency of bone marrow micronuclei.

  The inventor has further found that bLF and polyphenone-B function as dual-acting agents by inhibiting phase I enzymes and enhancing the activity of phase II enzymes. Dual-acting drugs are recognized as more promising as chemopreventive agents for cancer because they simultaneously inhibit metabolic activation of carcinogens while promoting detoxification and excretion. The chemopreventive ability of the combination of bLF and polyphenone-B can also be attributed to its antioxidant properties. In the buccal sac, bLF and polyphenone-B reversed the sensitivity to lipid peroxidation, while at the same time increasing the GSH / GSSG ratio and GPx activity, while in the liver, the degree of lipid peroxidation is an antioxidant defense Decreased with the rise of the system. Thus, differential oxidative events induced in tumors and host liver by bLF and polyphenone-B can possibly reduce cell proliferation in HBP and block tumor development. These results of this study show that administration of a combination of bLF and polyphenone-B suppresses HBP carcinogenesis, carcinogen metabolizing enzymes and redox status compared to the case where either drug is used alone. Suggests that it is more effective in regulating These findings further suggest that the combination of bLF and polyphenone-B can act synergistically in suppressing HBP carcinogenesis.

(III) Effect of Green Tea Polyphenol-Lactoferrin Combination We have further demonstrated that bovine lactoferrin in combination with various green tea polyphenols (polyphenone E) and black tea polyphenol (polyphenone E) is human squamous cell carcinoma of the tongue (CAL-27) and Anti-proliferative effects and apoptosis-inducing effects on normal human gingival fibroblast (HGF) cells were evaluated.

BLF (Lot No. 020119) with a purity of 96.2% or higher was purchased from Morinaga Milk Industry Co. , Ltd, Tokyo, Japan. The iron content of bLF was 18 mg / 100 g. Green tea polyphenols (polyphenone-E: PE) and black tea polyphenols (polyphenone-B: P-B) are available from Mitsui Norin Co. , Ltd., Ltd. , Courtesy of Tokyo, Japan. The compositions of Polyphenon-E and Polyphenon-B are the same as described above. Polyphenone-E (PE) contains epigallocatechin-3-gallate (64.6%), epigallocatechin (4.3%), epicatechin (9.4%), epicatechin-3-gallate ( 6.4%), gallocatechin-3-gallate (3.5%), catechin-3-gallate (0.2%), galactocatechin (0.2%), catechin (1.1%) and caffeine ( 0.7%). Polyphenone-B (P-B) has the following composition: epicatechin (0.4%), epigallocatechin-3-gallate (1.4%), epicatechin-3-gallate (0.1%), Gallocatechin-3-gallate (0.2%), free theaflavin (0.32%), theaflavin monogallate-A (0.14%), theaflavin monogallate-B (0.15%), theaflavin digallate (0 .21%), tannin (35.6%) and caffeine (4.9%). All other reagents used were analytical grade. Stock solutions of bLF and PE were prepared in phosphate buffered saline (PBS). P-B was dissolved in PBS containing 0.5% dimethyl sulfoxide (DMSO). The stock solution was then diluted with media before use to obtain the desired concentration. The final concentration of DMSO in the medium was less than 0.01%, demonstrating that this concentration has no detectable effect on cell growth. Dulbecco's Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were purchased from GIBCO. Dithiothreitol (DTT), 3,3-diaminobenzidine tetrahydrochloride (DAB), 2 ′, 7′-dichlorofluorescein diacetate (DCFH-DA), 3- (4,5-dimethylthiazole-2 bromide) -Yl) -2,5-diphenyltetrazolium (MTT), propidium iodide, protease K, fluoride-phenylmethanesulfonyl (PMSF), rhodamine 123, and RNase A were obtained from Sigma Chemical Company, St. Purchased from Louis, MO, USA. Dr. D. A. Normal human gingival fibroblast (HGF) cell line and cell culture medium and human tongue squamous cell carcinoma (CAL-27) cell line courtesy of Tipton, University of Tennessee, College of Dentry, Memphis, TN, USA Were used in this study. Cells were grown in DMEM supplemented with 10% FBS, 50 U / ml penicillin G, and 50 μg / ml streptomycin sulfate. The culture was maintained at 37 ° C. in a humidified atmosphere of 5% CO 2 in air. Exponentially growing cells were used for all experiments.

Cytotoxicity Assay Cytotoxicity was assessed by an MTT assay based on the reduction of live cells to violet formazone product by mitochondrial dehydrogenase. Briefly, cells were diluted in growth medium and seeded in 24-well plates (5 × 10 4 cells / well). After growing overnight, the growth medium is exposed medium (including FBS) containing the indicated doses of bLF, PE, PB alone and bLF and PE or a combination of PB. Not DMEM). After 24 hours, cells in each well were washed with 200 μL PBS and incubated at 37 ° C. with 100 μL 500 μg / mL MTT in PBS for 3 hours. MTT-formazone product dissolved in 200 μl DMSO was evaluated by measuring absorbance at 570 nm in an ELISA plate reader. Cell survival was expressed as the percentage of viable cells in the treated sample relative to the control sample. All dietary drugs were tested 3 times and the experiment was repeated at least 3 times.

Nuclear Morphology CAL-27 cells were plated on 6-well chamber slides at a density of 5 × 10 4 cells per well. After 80% confluence, CAL-27 cells were treated with dietary agents alone and in combination for 24 hours. The cells were then washed with PBS, fixed in methanol: acetic acid (3: 1 (v / v)) for 10 minutes, and stained with 50 μg / ml propidium iodide for 20 minutes. The morphology of apoptotic cell nuclei with concentrated / fragmented nuclei was examined under a fluorescence microscope and at least 1 × 10 3 cells were counted to assess apoptotic cell death.

Cell cycle analysis Percent cell distribution and measurement of apoptotic cells were performed by flow cytometry. After the treatment, the cells floating in the medium were combined with the adherent cells collected by trypsin treatment. Cells were then washed with cold PBS and fixed in 80% ethanol in PBS at -20 ° C. After 12 hours, the fixed cells were pelleted in the presence of 37 ° C. for 30 minutes RNA degrading enzyme A (20μg / ml) and stained with propidium iodide (50 [mu] g / ml), about 10 ⁴ events Becton Dickinson Analyzed with a FACScan flow cytometer. Cell cycle histograms were analyzed using Cell Quest software. Apoptotic cells were distinguished from non-apoptotic intact cells by a decrease in the DNA content of these cells, as measured by the lower propidium iodide staining intensity appearing in the region under their sub-G0 / G1 phase. .

Measurement of ROS generation In order to evaluate the occurrence of intracellular ROS, the oxidation sensitive fluorescent probe DCFH-DA was used. Briefly, after treatment, CAL-27 cells were collected and suspended in 0.5 mL PBS containing 10 μM DCFH-DA at 37 ° C. in the dark for 15 minutes. DCFH-DA is taken up by cells and deacetylated by cellular esterase to form non-fluorescent product DCFH, which is converted to green fluorescent product DCF by intracellular ROS produced by treated CAL-27 cells. did. The intensity of DCF fluorescence was measured by flow cytometry with excitation and emission settings at 488 nm and 530 nm, respectively. A total of 104 events were counted and the histogram was analyzed using Cell Quest software and compared to a histogram of untreated cells in the control group.

Measurement of Mitochondrial Membrane Potential Difference The change in mitochondrial membrane potential difference (ΔΨm) was measured by incorporation of the mitochondrial specific lipophilic cationic dye rhodamine 123. After treatment, CAL-27 cells were pelleted by centrifugation at room temperature for 10 minutes and washed with PBS. The pelleted cells were incubated with 1 mL of exposure medium containing 10 μg / mL rhodamine 123 at room temperature in the dark for 30 minutes, washed and resuspended in PBS. The sample (104 events) was then immediately subjected to flow cytometry analysis at an excitation wavelength of 488 nm and an emission wavelength of 545 nm. Histograms were analyzed using Cell Quest software and compared to histograms of untreated cells in the control group.

Immunofluorescence CAL-27 cells cultured to approximately 80% confluence in 6-well chamber slides were exposed to dietary drugs alone and in combination for 24 hours. Following treatment, cells were fixed for 5 minutes in acetone precooled to 4 ° C. Fixed cells are permeabilized with 0.1% Triton X-100 in PBS and warmed overnight at 4 ° C. with a 1: 1000 dilution of anti-Bcl-2 and anti-Bax antibodies (Dako, Carpinteria, Calif., USA). I put it. The protein was then detected by incubation with fluorescein isothiocyanate (FITC) labeled secondary anti-mouse IgG antibody (Dako, Carpinteria, CA, USA) and visualized using a fluorescence microscope.

Western blot After treatment with dietary drug, CAL-27 cells were washed twice with ice-cold PBS and lysis buffer (50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 150 mM sodium chloride, 0.5% nonidet). P-40, 0.5 mM PMSF and 0.5 mM DTT) at 4 ° C. for 30 minutes and the supernatant was collected by centrifugation at 12,500 × g for 20 minutes. Approximately 50 μg of total protein from the supernatant as measured by the Bradford protein evaluation kit was separated on 10% SDS-PAGE and then transferred to a nitrocellulose membrane using a semi-dry transfer system (BIORAD). Non-specific binding sites by incubating the membrane in Tris buffered saline (TBS) (150 mM / L sodium chloride, 50 mM / L Tris, pH 7.4) containing 5% non-fat dry milk for 1 hour. Shut off. This block was then incubated overnight at a 1: 1000 dilution of anti-Bcl-2 and anti-Bax (NeoMarkers, USA). Thoroughly wash this block with TBS containing 0.1% Tween-20 and incubate with the corresponding horseradish peroxidase labeled secondary antibody (1: 2000) at room temperature for 60-90 minutes. To detect the protein. After extensive washing in TBS containing 0.1% Tween-20, the transferred protein was visualized using DAB. Densitometry was performed on an IISP flatbed scanner and quantified with Total Lab 1.11 software.

Colorimetric evaluation of caspase-3 activity Caspase-3 activity was assayed using the CASP-3-C colorimetric kit (Sigma Chemical Company, St. Louis, MO, USA). After treatment, CAL-27 cells were solubilized in solubilization buffer containing 250 mM / l HEPES (pH 7.4), 25 mM / L CHAPS and 25 mM / L DTT. The supernatant was used as the enzyme source. The caspase-3 colorimetric assay is based on the hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp-nitroanilide (Ac-DEVD-pNA) by caspase-3 and the p-nitroaniline (pNA) moiety. Is based on the release of. The concentration of pNA released from the substrate was calculated from the absorbance value at 405 nm or from a calibration curve prepared with a pNA defined solution.

Statistical analysis Cytotoxicity data were obtained as the mean percentage of control group ± SEM. Presented as D, IC50 values were calculated using linear regression analysis. Statistical analysis on data on cytotoxicity on CAL-27 and HGF cells in tea polyphenol alone and in combination with bLF was performed using analysis of variance (ANOVA). The nature of the interaction between the effects of the combination of tea polyphenols and bLF was evaluated as described by Yokoyama et al. The synergistic effect is calculated from the ratio of expected value (E) / observed value (O) of the combination treatment of bLF and tea polyphenol, and a ratio exceeding 1 indicates a synergistic effect, and E value of bLF and tea polyphenol = [(bLF (O value of cells in control group)] × [(O value of tea polyphenol) / (O value of cells in control group)] × (O value of cells in control group). Data on nuclear morphology, cell cycle analysis, Bcl-2 / Bax ratio and caspase 3 activity were analyzed by Student's t-test. If the p-value was less than 0.05, the result was considered statistically significant.

Results Cytotoxicity Assay We first examined the inhibitory effects of various concentrations of PE, PB and bLF on the proliferation of CAL-27 and HGF cells (FIG. 23). Both P-E and P-B showed a dose-dependent suppression of the growth of CAL-27 cells with IC50 values of 20 μg / mL and 40 μg / mL, respectively. However, HGF cells had IC50 values of 70 μg / mL and 120 μg / mL, respectively, and were more resistant to the growth inhibitory effects of PE and P-B. Treatment with bLF did not induce any cytotoxic effect on either CAL-27 cells or HGF cells. We next examined the growth inhibitory effect of tea polyphenols in combination with bLF on CAL-27 cells and HGF cells. We selected PE and PB IC50 values for CAL-27 cells and tested this in combination with increasing bLF concentrations. FIG. 24A shows U-shaped growth inhibition of CAL-27 cells by a combination of PE with increasing bLF concentration. A significant synergistic effect was observed with the combination of PE (20 μg / ml) + bLF (40 μg / ml) in a ratio of 1: 2. HGF cells appeared to be less affected by growth inhibition than CAL-27 cells, which show preferential growth inhibition of CAL-27 cells by the combination of PE and bLF. FIG. 24B shows the effect of treatment with a combination of P-B and bLF on the proliferation of CAL-27 cells and HGF cells. Simultaneous treatment with P-B and bLF significantly reduced the sensitivity of CAL-27 cells to growth inhibition compared to P-B alone. These results suggest that bLF suppresses the anticancer effect of P-B. Overall, the results of the MTT assay suggest that dietary drugs preferentially inhibit the proliferation of CAL-27 cells compared to normal HGF cells, and the degree of cytotoxic effects of dietary drugs is P- It suggests that the combination of E and bLF (ratio of 1: 2)>P-E> P-B. Since the dietary drug preferentially inhibited the growth of CAL-27 cells and the addition of bLF suppressed the anticancer effects of P-B, PE (20 μg / ml) was investigated to explore the mode of cell death. ), P-B (40 [mu] g / ml) and bLF (40 [mu] g / ml) alone and in combination with PE (20 [mu] g / ml) and bLF (40 [mu] g / ml), CAL-27 cells Further studies were conducted only on.

Induction of apoptosis in CAL-27 cells by dietary drugs To determine whether inhibition of cell proliferation by dietary drugs is due to apoptosis, the fluorescent form-binding agent propidium iodide is used to form nuclear morphology. Observed. Incubation of CAL-27 cells with a 24-hour dietary drug significantly increased the number of apoptotic cells compared to the control group, as evidenced by nuclear fragmentation and aggregation (FIG. 25). . Treatment of CAL-27 cells with P-E (20 μg / ml) and P-B (40 μg / ml) alone significantly increased the number of apoptotic cells compared to the control group (p <0. 0 respectively). 01 and p <0.05). bLF alone did not induce apoptosis, but simultaneous treatment with PE (20 μg / ml) and bLF (40 μg / ml) significantly increased the number of apoptotic nuclei to 60.24% (control group). And p <0.005 and p <0.05 compared to P-E alone treated cells, respectively. In particular, 40 μg / ml bLF increased the ability of 20 μg / mL PE to induce apoptosis by ˜1.9 fold. The degree of apoptosis-inducing ability of dietary drugs was PE and bLF (1: 2)>PE> PE.

Effect of dietary drugs on cell cycle regulation of CAL-27 cells To study whether dietary drugs have a role in cell cycle control, we used a fluorescence-excited cell sorter to Changes in cell cycle characteristics were analyzed. As shown in FIG. 26, cells with reduced DNA content (sub-G0 / G1 or A0 peak) showing apoptosis lost by cells in G1 phase by incubation with CAL-27 and a dietary drug for 24 hours. The proportion of was increased significantly. The loss of DNA that is characteristic of apoptosis occurs as a result of diffusion of DNA degradation outside of the cell after endonuclease cleavage, and after staining with propidium iodide, these cells are almost unstained and sub-G0 / Appears at the G1 or A0 peak, ie, to the left of the G0 / G1 peak. Incubation of CAL-27 cells with PE and PB alone resulted in DNA content of 8.36% (control group) to 35.99% (PE) and 24.01% (PB ) Significantly decreased the proportion of cells that were reduced. bLF alone did not significantly change the ratio of sub-G0 / G1 cells, but co-treatment with 20 μg / ml PE and 40 μg / ml bLF resulted in a sub-G0 / G1 cell ratio of 72. Significantly increased to 86% (p <0.005 and p <0.01 compared to control group and cells treated with PE alone, respectively). Specifically, 40 μg / ml bLF increased the ability of 20 μg / ml PE to induce apoptosis by ˜2 fold. The degree of apoptosis-inducing ability of dietary drugs was the same as for nuclear morphology analysis.

Effect of dietary drugs on intracellular ROS development To study whether ROS is involved in mediating apoptosis induced by dietary drugs, we used the fluorescent probe DCFH-DA to Intracellular development was measured. Treatment with dietary drugs significantly increased ROS generation in CAL-27 treated cells compared to the untreated control group, the order of ROS generation being P-E + bLF>P-E> P-B (FIG. 27A). The average of DCF fluorescence was Ma = 381 (control group) to Mb = 938 (PE treatment group), Mc = 1229 (PE and bLF, ratio of 1: 2) and Md = 649 (PB treatment). Group). Exposure of CAL-27 cells to bLF alone did not induce ROS generation (data not shown).

Effect of dietary drugs on mitochondrial membrane potential difference (ΔΨm) With increasing evidence that changes in ΔΨm are linked to apoptosis, we have examined the effects of dietary drugs alone and in combination on ΔΨm in CAL-27 cells. It was examined by flow cytometry using the probe rhodamine 123. Rhodamine 123, a cationic stain, is selectively taken up by mitochondria to the extent that it is directly proportional to ΔΨm. As shown in FIG. 27B, CAL-27 cells exposed to tea polyphenol for 24 hours showed a loss of ΔΨm as evidenced by reducing the fluorescence intensity of rhodamine 123 compared to the control group. . The average fluorescence of rhodamine 123 decreased from Ma = 4636 (control group) to Mb = 2456.12 (PE treatment group) and Md = 2659.88 (PB treatment group). bLF alone did not show any significant change in ΔΨm (data not shown), but treatment with the combination of PE and bLF was compared to control group cells and PE alone treated cells. It was shown that the fluorescence intensity decreased more sharply to 2098.75.

Effect of dietary drugs on the Bcl-2 / Bax ratio We have played an important role in controlling apoptosis by modulating ΔΨm using immunofluorescence (data not shown) and Western blotting. The effects of dietary drugs on the expression of 2 and Bax were examined. Bcl-2 / Bax protein compared to the untreated control group by incubation of CAL-27 cells with PE, P-B and a combination of PE and bLF (ratio 1: 2). The order of Bcl-2 / Bax that significantly reduced expression and reduced the efficacy of dietary drugs was P-E + bLF> PE-P-B (FIG. 28A).

  Effect of dietary drugs on caspase 3 activity: Assay of caspase 3 in CAL-27 cells treated with dietary drugs revealed that apoptosis induction was mediated through activation of caspase 3 activity (FIG. 28B). Incubation of CAL-27 cells with PE, P-B and a combination of PE and bLF (1: 2 ratio) significantly increased caspase 3 activity compared to the control group. Although bLF had no effect on caspase 3 activity, simultaneous treatment with bLF and PE enhanced enzyme activity by ˜1.46 fold.

  These results suggest that tea polyphenols preferentially inhibit the growth of human tongue squamous carcinoma (CAL-27) cells in a dose-dependent manner. The data further shows that PE is more effective than P-B in suppressing the growth of CAL-27 cells. Furthermore, the combination of PE and bLF (1: 2 ratio) exhibits a significant synergistic effect in suppressing tumor growth, as reflected by the U-shaped growth curve, Simultaneous treatment with bLF inhibited the anti-carcinogenic effects of P-B. Overall, the order of preferential suppression of proliferation and antiproliferative effects of CAL-27 cells was PE + bLF (1: 2)> PE> P-B. The greater efficacy of PE in suppressing the growth of CAL-27 cells clearly reflects the higher concentration of EGCG. The results also show that green tea and black tea polyphenols suppress the growth of CAL-27 cells by inducing apoptosis as evidenced by the nuclear morphology and characteristic changes in the A0 peak. There is increasing evidence that apoptosis induced by chemopreventive or chemotherapeutic agents is associated with changes in specific phases of the cell cycle. As shown in FIG. 4, 24-hour incubation of CAL-27 cells with tea polyphenols resulted in cell loss in the G0 / G1 phase with a concomitant increase in the appearance of apoptotic cells, which was caused by G0 / G1 by tea polyphenols. CAL-27 cells arrested in the phase suggested that they selectively experienced apoptosis. Although bLF alone did not induce apoptosis of CAL-27 cells, simultaneous treatment with PE and bLF increased the ability of PE to induce apoptosis by a factor of ˜2. The order of the apoptosis-inducing ability of the dietary drugs was the same as that of the growth inhibitory effect, that is, P-E + bLF (1: 2)> PE-P-B.

  Mitochondria, which play a central role in apoptosis, are the main site of ROS development. Over-production of ROS opens the mitochondrial permeability transition pore and decreases ΔΨm, resulting in cytochrome c release from the intermembrane space to the cytoplasm leading to caspase cascade activation and apoptotic cell death. Can occur. The results of this study show that incubation of CAL-27 cells with P-E, P-B and P-E + bLF (1: 2 ratio) increases ROS generation, which in turn is evident by a decrease in ΔΨm. Thus, it is shown that apoptosis was induced by disrupting the function of mitochondria and caspase-3 was activated. EGCG has been reported to induce apoptosis in Jurkat cells by the development of ROS. Several studies have provided evidence for ROS involvement, loss of ΔΨm and caspase activity during apoptosis induced by EGCG and tea polyphenols. Together these results suggest that tea polyphenols induce apoptosis through ROS-induced mitochondrial destruction.

  In summary, the present results indicate that tea polyphenols exert a growth inhibitory effect on CAL-27 cells mediated through apoptosis. The data shows an important role for ROS and Bcl-2 / Bax in mitochondria-mediated apoptosis by eliminating ΔΨm and activating caspase-3. This study also highlights the greater efficacy of both PE alone and in combination with bLF. Since green tea polyphenols have already been included in clinical trials in patients at high risk for liver cancer and prostate cancer, the same applies to patients with oral premalignant lesions to evaluate the chemopreventive efficacy of PE. Planning a clinical trial will be valuable.

  Thus, specific embodiments and applications of tea polyphenols have been disclosed. However, it will be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. Accordingly, the subject matter of the present invention should not be limited except in the spirit of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms “comprising” and “including” should be construed as referring to elements, components or steps in a non-exclusive manner, which refers to the referenced element, component or step. Indicates that it may be present, utilized or combined with other elements, components or steps not expressly referenced. Furthermore, provided herein that a definition or use of a term in a reference incorporated herein by reference is inconsistent with or opposite to the definition of said term provided herein. The definition of the term applies and the definition of the term in the reference does not apply.

FIG. 1 contains two tables listing various tumor and histopathological parameters of animals treated with black tea polyphenol compositions. FIG. 2 shows various photomicrographs of animal tissues (H and E stained) treated with black tea polyphenol composition. FIG. 3 is a graph showing quantitative analysis of PCNA expression in animals treated with black tea polyphenol compositions. FIG. 4 shows various micrographs (PCNA stained) of the animal tissue of FIG. FIG. 5 is a table listing various biochemical parameters of animals treated with black tea polyphenol compositions. FIG. 6 shows various graphs showing enzyme activity in selected organs of animals treated with black tea polyphenol compositions. FIG. 7 shows various graphs showing further enzyme activity in selected organs of animals treated with black tea polyphenol compositions. FIG. 8 shows various graphs showing still further enzyme activity in selected organs of animals treated with black tea polyphenol compositions. FIG. 9 is a table listing various tumor parameters for animals treated with black tea polyphenol compositions. FIG. 10 shows a table listing the expression of various tumor associated proteins in animals treated with black tea polyphenol compositions. FIG. 11 shows various photomicrographs of animal tissues (stained for PCNA, NF-κB, cytokeratin) treated with black tea polyphenol composition. FIG. 12 shows various micrographs of animal tissues (stained for Bcl-2, Cyc-c, caspase 3) treated with black tea polyphenol composition. FIG. 13 is a graph showing caspase 3 activity in the animal of FIG. FIG. 14 is a table listing various tumor parameters for animals treated with a combination of black tea polyphenol composition and lactoferrin. FIG. 15 shows micrographs of selected tissues (stained for cytokeratin, PCNA, and p53) of animals treated with a combination of black tea polyphenol composition and lactoferrin. FIG. 16 is a table showing the results of quantitative analysis for the animal of FIG. FIG. 17 shows Western blots and their densitometric analysis for selected proteins of animals treated with a combination of black tea polyphenol composition and lactoferrin. FIG. 18 is a table listing the results for the measurement of micronucleated polychromatic erythrocytes in animals treated with a combination of black tea polyphenol composition and lactoferrin. FIG. 19 is a table listing additional tumor parameters for animals treated with a combination of black tea polyphenol composition and lactoferrin. FIG. 20 shows various graphs showing enzyme activity (Cyt-C, GST, diaphorase) in selected organs of animals treated with a combination of black tea polyphenol composition and lactoferrin. FIG. 21 shows various graphs showing various biochemical parameters (TBARS, LOOH, CD) in selected organs of animals treated with a combination of black tea polyphenol composition and lactoferrin. FIG. 22 shows various graphs showing additional biochemical parameters (GSH, GSSG, GSH / GSSG ratio, GPx) in selected organs of animals treated with a combination of black tea polyphenol composition and lactoferrin. FIG. 23 shows a graph showing the viability of human (cancer and normal) cells as a function of exposure to lactoferrin, green tea polyphenols and black tea polyphenols. FIG. 24 shows a graph showing the viability of human cells (cancer cells and normal cells) as a function of exposure to lactoferrin and a combination of green tea polyphenols or black tea polyphenols. FIG. 25 shows micrographs of cells stained for apoptotic micronuclei as a function of treatment with a combination of lactoferrin and green tea polyphenols or black tea polyphenols and their quantitative analysis. FIG. 26 shows various graphs showing the cell cycle distribution of human oral cancer cells treated with a combination of lactoferrin and green tea polyphenols. FIG. 27 shows various graphs showing the effect of the combination of lactoferrin and green tea polyphenols on ROS and mitochondrial membrane potential in human oral cancer cells. FIG. 28 shows a Western blot of Bcl-2 / Bax and caspase 3 and their densitometric analysis in human oral cancer cells as a function of treatment with a combination of green tea polyphenol composition and lactoferrin.

Claims (24)

A method of improving oral cancer treatment or chemoprevention using oral administration of tea polyphenol composition or lactoferrin,
Confirming that oral administration of the tea polyphenol composition or the lactoferrin alleviates oral cancer with the first effect, and oral administration combined with the tea polyphenol composition and the lactoferrin has the first effect Providing information to alleviate oral cancer with a second effect that exceeds the method of improving oral cancer treatment or chemoprevention.   The method of claim 1, wherein the tea polyphenol composition comprises a composition comprising a plurality of catechins, optionally polymerized catechins. The tea polyphenol composition comprises a mixture of a plurality of catechins derived from green tea,
The method of claim 1. The lactoferrin is bovine lactoferrin, the tea polyphenol composition is composed of a plurality of catechins or a composition containing polymerized catechins, and the combination of lactoferrin and catechins is liquid, capsule, tablet, powder The method of claim 1, optionally formulated.   The method according to claim 1, wherein the weight ratio of tea polyphenol composition to lactoferrin in the combined oral administration is 1: 1 to 1: 3.   6. The method of claim 5, wherein the weight ratio is synergistic.   The method according to claim 1, wherein the oral administration is simultaneous administration in a single dosage unit form.   A dietary supplement or pharmaceutical comprising a combination of a tea polyphenol composition and lactoferrin in an amount effective to alleviate oral cancer with effects that exceed those achieved by oral administration of tea polyphenol composition or lactoferrin alone.   9. The dietary supplement or pharmaceutical product according to claim 8, wherein the tea polyphenol composition contains at least a mixture of a plurality of catechins derived from black tea or green tea and is optionally formulated as a liquid, capsule, tablet or powder.   9. A dietary supplement or pharmaceutical product according to claim 8, wherein the tea polyphenol composition comprises at least one synthetic catechin.   9. A dietary supplement or pharmaceutical product according to claim 8, wherein the tea polyphenol composition is present at 100 mg to 600 mg per unit dose and the lactoferrin is present at 200 mg to 1200 mg per unit dose.   The dietary supplement or pharmaceutical product according to claim 8, wherein the dietary supplement or pharmaceutical product is a non-liquid dietary supplement formulated to contain the tea polyphenol and the lactoferrin.   The dietary supplement or pharmaceutical product according to claim 8, wherein the dietary supplement or pharmaceutical product is a dietary supplement, and at least one of the tea polyphenol and the lactoferrin is contained in a liquid carrier. 9. A dietary supplement or pharmaceutical product according to claim 8, wherein the greater effect is a synergistic effect.   Use of a combination of tea polyphenols and lactoferrin in the manufacture of a dietary supplement or medicament for the treatment or chemoprevention of oral cancer.   16. Use according to claim 15, wherein the tea polyphenol composition and the lactoferrin are present in a weight ratio of 1: 1 to 1: 3.   17. Use according to claim 16, wherein the weight ratio is a synergistic weight ratio.   16. Use according to claim 15, wherein the tea polyphenol composition comprises a mixture of a plurality of catechins derived from at least black tea or green tea.   16. Use according to claim 15, wherein the combination is optionally formulated as a liquid formulation, capsule, tablet or powder in a single dosage unit.   16. Use according to claim 15, wherein the dietary supplement or medicament is a medicament, optionally formulated as a liquid formulation, capsule, tablet or powder.   A method for preventing oral cancer, comprising administering a combination of tea polyphenol and lactoferrin.   13. The method of claim 12, wherein the step of administering comprises oral administration and the combination is formulated in a single dosage unit.   13. The method of claim 12, wherein the tea polyphenol comprises a mixture of a plurality of catechins derived from at least black tea and green tea.   13. The method of claim 12, wherein the combination is formulated into a product selected from the group consisting of liquid nutritional supplements, capsules, tablets, and powder formulations.

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