A PROCESS FOR THE PRODUCTION OF AN EXTRACT OF DEDIFFERENTIATED STEM CELLS FROM CAMELLIA SINENSIS

Abstract

A process for the production of an extract of dedifferentiated stem cells from Camellia sinensis, the process comprising the following steps: (a) preparing a cell culture comprising dedifferentiated stem cells from Camellia sinensis; (b) performing an extraction in the cell culture, using ethanol and / or methanol as an extraction solvent, to produce extracts of dedifferentiated stem cells from Camellia sinensis.

Description

The invention relates to a process for producing a culture extract of dedifferentiated stem cells from Camellia sinensis, to a composition comprising such an extract, and to the use of said extract. People generally prefer to have healthy skin. However, environmental factors damage the skin and hair, undermining their health and resilience to stress, such as that caused by UV radiation and dryness. In general, consumers find it convenient to apply topical compositions to the skin and even use particular products to improve the appearance or protection of their skin. On the other hand, users have a general preference for those components that are natural and a general reluctance to use more synthetic compositions —particularly if they have to consume the composition. US patent document number US2014 / 0186315 describes a cosmetic composition containing a green tea stem cell extract. The composition is claimed to be an anti-aging composition. The green tea stem cell is produced by cultivating a totipotent callus in a cell culture. The active ingredient is said to be extracted from the cell culture. However, no details regarding the process are provided. extraction. Therefore, it would be advisable to move forward with improvements in this area. We have found that it is possible to use specific cell culture extracts from Camellia sinensis to improve skin health. More specifically, we have discovered that extracts from dedifferentiated stem cell cultures of Camellia sinensis offer resistance to environmental damage suffered by skin cells. Therefore, in a first aspect, the invention relates to a process for the production of an extract of dedifferentiated stem cells from Camellia sinensis, a process comprising the following steps: (a) Prepare a cell culture comprising dedifferentiated stem cells from Camellia sinensis; (b) Perform an extraction in the cell culture, using ethanol and / or methanol as an extraction solvent, to produce the extract from the dedifferentiated stem cell culture of Camellia sinensis. This extract of dedifferentiated stem cells has been found to provide amazing protection to skin cells against damage caused by dryness and ultraviolet light; it also offers an anti-inflammatory effect. Ethanol and methanol are believed to provide a higher concentration of certain active ingredients within the stem cell extract, such as flavanones, polyphenols, and terpenoids. Other solvents have been found to produce a stem cell extract that is significantly inferior. Thus, in a second aspect, the invention relates to a composition comprising an extract of dedifferentiated stem cells from Camellia sinensis, which can be obtained by the process as described in this document. Furthermore, it has been discovered that these dedifferentiated stem cell extracts have a marked and powerful protective effect on skin cells when They are exposed to dryness or UV radiation and also reduce inflammation. In a third aspect, the invention relates to the use of a stem cell extract. undifferentiated from Camellia sinensis, to protect the skin against damage caused by dryness and / or UV radiation and / or inflammation. Tea refers to one or more plants belonging to the Camellia sinensis var. sinensis and / or Camellia sinensis var. assamica family. Tea is the second most consumed beverage worldwide. It is a rich source of monomeric and polymeric forms of flavonoids and can represent up to 10-30% by weight of the flavonoids. In this invention, the extracts are prepared from dedifferentiated tea stem cells. Conveniently, the dedifferentiated tea stem cells can be prepared from callus, which is a wound response. A plant callus is a mass of disorganized parenchyma cells derived from plant tissues (explants). In plant biology, callus cells are those that cover a wound on a plant. Callus formation is induced from plant tissues after surface sterilization and placement on plates in an in vitro tissue culture medium. Plant growth regulators, such as auxins, cytokinins, and gibberellins, are added to the medium to initiate callus formation or somatic embryogenesis. In general, plant callus cells are obtained by seeding the cells in a culture medium. Plant callus material can be obtained and cut from an explant and transferred to a culture medium. Once in the culture medium, the cells can be cultured as desired until a sufficient quantity is obtained. The callus can be seeded on solid growth media, such as agar, and then transferred to liquid growth media to increase the volume of production. harvest the active components by extraction. The cell culture process can be carried out in any way known to the technique. Preferably, the cell culture medium comprises the hormones 2,4-dichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid (NAA), and 6-benzylaminopurine (BAP). Preferably, the cell culture medium has a pH between 5.6 and 6.0. Preferably, the composition of the invention comprises the stem cell extract at a concentration greater than 0.01% by weight. The composition of the invention comprises a cosmetically acceptable base, when it is a topical composition. The cosmetically acceptable base, according to the present invention, is a cream, lotion, gel, or emulsion. The cosmetically acceptable base preferably comprises a fatty acid or a silicone compound. When the cosmetically acceptable base comprises a fatty acid, it is preferably present in a percentage of 1 to 25% by weight of the composition. When the cosmetically acceptable bases are such that they allow obtaining a product in the form of a cream, lotion, or emulsion, they generally comprise a fatty acid. Of these forms, the most preferred is a cream or lotion, with greater Preferably, a cream. The base of the vanishing cream comprises 3 to 25%, more preferably 5 to 20% fatty acid, which is a preferred form of the composition of the invention. In this respect, the base preferably comprises 0.1 to 10%, more preferably 0.1 to 3% soap. Fatty acids C12 to C20 are especially preferred in the bases of vanishing creams, with fatty acids C14 to C15 being even more preferred. In the creams, the fatty acid is preferably a mixture of stearic and palmitic acids. The soaps present in the bases of the vanishing cream include an alkali metal salt of fatty acids, such as sodium or potassium salts. The soap is preferably the potassium salt of the fatty acid mixture.The fatty acid present in vanishing cream bases is often prepared using hystric acid, which is substantially (usually 90 to 95%) a mixture of stearic and palmitic acids (typically 55% stearic and 45% palmitic). Thus, the inclusion of hystric acid and its soap in preparing vanishing cream bases is within the scope of the present invention. It is particularly preferred that the composition comprise at least 6%, preferably at least 10%, and more preferably at least 12% fatty acid. The cosmetically acceptable base typically ranges from 10% to 99.9%, preferably from 50% to 99% by weight of the composition. Another preferred base is a lotion. Lotions typically comprise from 1% to 20% fatty acid. The cosmetically acceptable base preferably includes water.Water is included, preferably, in a percentage of 35 to 90%, more preferably 50 to 85%, and more preferably still 50 to 80% by weight of the composition. A cosmetically acceptable base, which is particularly suitable, is one comprising a water-in-oil emulsion with silicone oils as the continuous phase. Preferably, these water-in-oil emulsions comprise a mixture of cross-linked silicone elastomers. The inclusion of the silicone elastomer mixture in a water-in-oil emulsion can be used as a cosmetically acceptable base for preparing the compositions of the present invention. While silicone fluids can be used, cross-linked silicone elastomers are particularly preferred. The creation of cross-links between linear polymers, such as dimethicone, converts the linear polymer into a silicone elastomer. In contrast to silicone fluid polymers, the physical properties of cross-linked elastomers are enhanced. The properties of silicone elastomers generally depend on the number of cross-links, rather than molecular weight. Their ability to expand makes them ideal thickeners for oil phases. Elastomers impart a very soft and delicate feel when applied to skin or hair. They can also be used as delivery agents for fragrances, vitamins, and other additives in cosmetic formulations. The commercially available silicone elastomer blends or gels that are suitable for inclusion in the composition of the invention and that, as has been found, provide better stability, are the following: Dow Corning® EL-8051 IN organic silicone elastomer blend [INCI name [International Nomenclature of Cosmetic Ingredients]: isodecyl neopentanoate (and) Dimethicone / Bis-isobutyl PPG-20 crosspolymer]; EL-8050 [INCI name: isododecane (and) Dimethicone / Bis-isobutyl PPG 20 crosspolymer] DC 9040, DC9041, DC9045 (dimethicone crosspolymer); DC 9506, 9509 (dimethicone-vinyl-dimethicone crosspolymer); Shin-Etsu KSG-15, KSG-16, KSG-17 (dimethicone-vinyldimethicone crosspolymer). It is further preferred that the composition comprise between 5 and 50% silicone elastomer by weight of the composition. Preferably, it is possible to use protective agents against solar radiation, for example, inorganic sunscreens in the present invention. These include, for example, zinc oxide, iron oxide, silica such as fumed silica, and titanium dioxide. The total amount of sunscreen preferably incorporated into the composition according to the invention ranges from 0.1 to 5% by weight of the composition. The topical composition of the invention may additionally comprise a skin-lightening agent. The skin-lightening agent is preferably selected from a vitamin B3 compound or its derivative, for example, niacin, nicotinic acid, niacinamide, or other widely known skin-lightening agents, for example, aloe extract, ammonium lactate, azelaic acid, kojic acid, citrate esters, ellagic acid, glycolic acid, green tea extract, hydroquinone, lemon extract, linoleic acid, magnesium ascorbyl phosphate, vitamins such as vitamin B6, vitamin B12, vitamin C, vitamin A, a dicarboxylic acid, resorcinol derivatives, hydroxycarboxylic acids such as lactic acid and its ales, for example, sodium lactate and mixtures thereof. The vitamin B3 compound or its derivative, for example, niacin, nicotinic acid, niacinamide, are the most preferred skin-lightening agents according to the invention, niacinamide being the most preferred of all. When used, niacinamide is preferably present in an amount ranging from 0.1 to 10%, more preferably from 0.2 to 5% by weight of the composition. The topical composition according to the invention may also comprise diluents. Diluents act as a dispersant or carrier for other materials present in the composition, to facilitate their distribution when the composition is applied to the skin. Diluents other than water may include liquid or solid emollients, solvents, humectants, thickeners, and powders. The topical compositions of the present invention may comprise a wide range of other optional components. The CTFA, Personal Care Ingredient Handbook, Second Edition, 1992, which is incorporated herein by reference in its entirety, describes a wide variety of non-limiting pharmaceutical and cosmetic ingredients commonly used in the skin care industry and suitable for use in the compositions of the present invention. Examples include antioxidants, binders, biological additives, buffering agents, colorants, thickeners, polymers, astringents, fragrance, humectants, opacifying agents, conditioners, exfoliating agents, pH regulators, preservatives, and extracts. natural, essential oils, sensory capture agents (sensates) for the skin, skin softening agents and skin healing agents. When the composition is an oral composition, it may take the form of a food product or a beverage. Generally, such an oral composition may take any suitable form of a consumer product, such as a beverage, a bar, a meal, or a supplement. EXAMPLES Cell culture of tea calluses • Young leaves of Sri Lanka clone 2 (Camellia sinensis assimica) were sterilized as follows: o They were immersed in water with a few drops of Tween 20 for 30 minutes. They were washed in running water for 15 minutes, or the surface was sterilized in hydrogen peroxide for 2 minutes. or They washed in running water. The surface was sterilized in 70% ethanol for 2 minutes. or They washed in running water. or They were transferred to a flow hood and the surface was sterilized with a 10% household bleach product for 30 minutes. They were washed with distilled water (in the bell jar). The leaves were cut into squares (explants) and transferred to the media in Petri dishes. • Composition of solid media: or MS medium (salts and vitamins). or 30 g / 1 of sucrose or 0.8% agar. o pH is adjusted to 5.8 with 0.2 M KOH. o Hormones 0.25 mg Ll of 2,4-dichlorophenoxyacetic acid (2, 4- D), 0.25 mg L~1 of naphthaleneacetic acid (ΝΆΑ) and 1 mg L~1 of 6-benzylamino- -purine (BAP). The medium was sterilized by autoclaving at 121°C for 30 minutes. After autoclaving, the medium was cooled to 60°C before being poured into 9 cm Petri dishes (approximately 10 ml per dish). The agar hardened upon cooling to 4°C. • Five to ten tea leaf explants were grown in each Petri dish of solid medium at 25°C ± 2 in a 16-hour light and 8-hour dark cycle, in a plant growth cabinet. • The callus clusters that were developing on the injured areas of the explant were carefully removed, after they had developed sufficiently, and subcultured in a box fresh Petri dish from the solid medium. • The callus cell aggregate was subcultured onto fresh solid media every 4 weeks. The subculture was simply transferred from the callus culture to the fresh media. This is necessary because the solid agar gel medium dries out over time (approximately 3 to 4 weeks) and the developing callus must be kept hydrated. • After 12 weeks, the calluses had reached a sufficient size (approximately 1 to 2 cm in diameter) to continue with the experimentation. • At this stage, the callus was completely freeze-dried for 48 hours and stored at -20 °C until required. Preparation of tea callus cell extracts • A 20% suspension of the lyophilized callus material was prepared in 96% ethanol. • 0.1 g of the lyophilized material was mixed with 0.9 g of 96% ethanol. Note: This is not a 10 / 90 wt% callus:ethanol solution, as most of the callus material is insoluble and remains in suspension. By weighing the dry mass of the insoluble material after solvent extraction, the final concentration of the soluble extract was calculated to be approximately 10 mg / ml. Therefore, in the tests cell survival, 1% = 0.1 mg / ml of soluble extract. • The suspension was vigorously centrifuged for 1 minute and then placed in a sonication bath and sonicated for 30 minutes, at a temperature of 0-4 °C. • The suspension was centrifuged at 13,000 rpm for 10 minutes, at 4 °C, and the supernatant was retained (stem cell control solution extract). • The stem cell control solution was stored at -20 °C until use. Skin cell dryness protection assay • Human adult dermal fibroblasts (Cat. No. C-013-5C, Life Technologies) were cultured to approximately 90% confluence in 24-well plates containing 0.5 ml of medium 106 (Cat. No. M106-500, Life Technologies) supplemented with Lew Serum Growth Supplement (Cat. No. S-003-10, Life Technologies) at 37°C, 5% CO2. • Stem cell extract was added to the medium, until final concentrations of 0.1%, 0.5% or 1% were reached. EITHER. EITHER . • An experimental control containing 1% ethanol (carrier only) was also prepared. The cells were then cultured for another 24 hours. • The cells were dried by completely aspirating the medium from the wells and leaving them for 10 minutes in a laminar flow hood for cell cultures, at room temperature. • Undried controls were included for each condition. • Then 0.5 ml of fresh medium was added to each well and the cells were incubated at 37 °C, with 5% CO2. • After 1 hour, 50 μA of AlamarBlue cell viability reagent (Molecular Probes, Cat No. DAL1025) was added to each cavity. • After another 4 hours of incubation at 37 °C with 5% CO2, 200 µA of the medium were removed from each well and placed in a 96-well plate. The fluorescent intensity of the samples was then read at 550 nm excitation / 612 nm emission. AlamarBlue acts as an indicator of cell viability by converting a non-fluorescent dye (resazurin) to a highly fluorescent dye (resorufin) through reduction reactions in metabolically active cells. Ultraviolet Irradiation Protection Assay of Skin Cells • Human adult dermal fibroblasts (Cat. No. C-013-5C, Life Technologies) were cultured to approximately 90% confluence in Petri dishes 6 cm Petri dishes containing 1 ml of DMEM (Life Technologies, Cat. No. 21063-029) supplemented with 1 mM pyruvate, 2 mM glutamine and 10% fetal bovine serum at 37°C, 5% CO2. • A stem cell extract was added to the medium, until the following final concentrations were reached: 0.1%, 0.5% or 1%. • An experimental control containing 1% ethanol (carrier only) was also prepared. • The cells were then cultured for another 24 hours. • The Petri dishes were uncovered, and the cells were irradiated in a Uvacube 400 ultraviolet chamber (Honle UV technology) for 30 minutes. • The old media was removed and fresh media of the same type was added. • The cells were incubated at 37 °C, 5% CO2 for 24 hours. • 100 μA of AlamarBlue cell viability reagent (Molecular Probes, Cat. No. DAL1025) was then added to each well, and incubation continued for another 24 hours. • 200 μA were removed from the medium of each well and distributed onto a 96-well plate. The fluorescent intensity of the samples was then read at a Excitation at 550 nm and emission at 612 nm. AlamarBlue acts as an indicator of cell viability by converting a non-fluorescent dye (resazurin) into a highly fluorescent dye (resorufin) through reduction reactions in metabolically active cells. Anti-inflammatory assay • Human adult dermal fibroblasts (Cat. No. C-013-5C, Life Technologies) were cultured at a concentration of 60,000 cells per well in 12-well plates containing 1 ml of DMEMGlutaMAX (Gibco, Cat. No. 10566016), supplemented with 1% fetal bovine serum per well, at 37°C, 5% CO2. • Tea callus extract was added to each well, which contained 1 ml of the medium at a final concentration of 1%. At the same time, Phorbol 12-myristate 13-acetate (PMA, Cat. No. P8139 Sigma) was also added to each well, at a final concentration of 100 nm. • An experimental control was also prepared containing 1% ethanol (carrier only) and 100 nm PMA. • The cells were incubated at 37 °C, 5% CO2 for another 24 hours. The cell culture medium was then removed and centrifuged at 16,000 RPM for 1 minute. The supernatant • The supernatants from the used lysates were centrifuged at 16,000 minutes. They were then subjected to -20 °C, collected and stored until needed. • The cells were washed 3 times with PBS and then used by adding 1 ml per well of RIPA buffer for lysis and extraction (Cat. No. 8990, Thermo Scientific) for 30 minutes on ice. Cell phones were collected and RPMs were collected during 1; assay to determine total protein content, using a BCA protein assay kit (Cat. No. 23225, Thermo Scientific). • IL-6 content was measured in the supernatants of the cell culture media using the Quantikine Human IL-β ELISA Immunoassay Kit (Cat. No. D6050, R&D Systems). The total protein content for each well was used to normalize the measured IL-6 concentrations. This allowed for direct comparison of IL-6 levels in each sample. Skin cell dryness assay (tea extract) Table 1: Fluorescence measurements with AlamarBlue of human dermal fibroblast media, read at an excitation of 550 nm and an emission of 612 nm. Three replicates (samples 1-3) were performed for each condition. Fluorescence intensity of the medium and of AlamarBlue alone (average fluorescence). reference) was subtracted from the values Sample 1 Sample 2 Sample 3 Average Average Reference Standard Deviation 1% Ethanol 58628 56283 57194 57368 43116 1182 1% Ethanol (Dehydrated) 15997 14981 15763 15580 1328 532 1% Tea Callus Extract 49562 48734 49178 49158 34906 414 1% Tea Callus Extract (Dehydrated) 49698 46815 47935 48149 33897 1453 0.5% Tea Callus Extract 51018 53845 51933 52265 38013 1443 Tea callus extract 0.5% (Dehydrated) 52006 51307 50602 51305 37053 702 Tea callus extract 0.1% 53646 54813 52708 53722 39470 1055 Tea extract 0.1% (Dehydrated) 51737 52091 51445 51758 37506 323 Tea callus extract 0.01% 54996 55830 57119 55982 41730 1070 Tea callus extract 0.01% (Dehydrated) 29888 32796 32395 31693 17441 1576 Tea leaf extract, 1% 55931 53284 55993 55069 40817 1546 Tea leaf extract, 1% (Dehydrated) 26835 24405 24183 25141 10889 1471 These results demonstrate that, after dehydration, the number of viable cells in the samples treated with the tea stem cell extract is greater than that observed in the cells treated with the carrier (ethanol) alone. Furthermore, it is shown that levels above 0.01% by weight provide an effect very significant protector. A comparative example is also shown, in which a tea leaf extract is generated without creating a callus. While this achieves some protective effect, it is not as significant as the protective effect provided by the callus extract. Skin cell dryness assay (tea extract) using different extraction solvents • A 20% suspension of ground lyophilized callus material was prepared in the following solvents: ethanol, methanol, chloroform, ether, acetone, water. • The suspension was vigorously centrifuged for 1 minute and then placed in a sonication bath and sonicated for 30 minutes at 4 °C. • The suspension was centrifuged at 13,000 rpm for 10 minutes at 4 °C and the supernatant was retained. • The supernatant was dehydrated under vacuum to remove the solvent by evaporation. • The residual solid extract was dissolved in 1 ml of DMSO (vigorously centrifuged for 1 minute). • The extracts were then used in the skin cell dryness assay, at a concentration of 1% in the final media, as described. • An experimental control containing 1% DMSO (carrier only) was also prepared. Table 2: Fluorescence measurements with AlamarBlue of human dermal fibroblast media, read at an excitation of 550 nm and an emission of 612 nm. Three replicates (samples 1-3) were performed for each condition. The fluorescent intensity of the medium and of the AlamarBlue only (reference fluorescence) was subtracted from the average values. Sample 1 Sample 2 Sample 3 Average Average Reference Standard Deviation Ethanol 29326 31414 28234 29658 25556 1616 Methanol 25170 28037 26653 26620 22518 1434 Chloroform 17636 16629 16577 16947 12845 597 Ether 12086 11134 10611 11277 7175 748 Acetone 14513 13648 13109 13757 9655 708 Water 8318 8502 9652 8824 4722 723 1% DMSO 6473 7915 6600 6996 2894 798 It can be observed that skin cells treated with cell culture extracts obtained with the solvents ethanol and methanol provide results significantly better than those obtained with other solvents. Skin cell protection test against ultraviolet radiation (tea extract) Table 3: Fluorescence measurements with AlamarBlue of human dermal fibroblast media, read at an excitation of 550 nm and an emission of 612 nm. Three replicates (samples 1-3) were performed for each condition. The fluorescent intensity of the medium and of AlamarBlue alone (reference fluorescence) was subtracted from the average values. Sample 1 Sample 2 Sample 3 Average Average Reference Standard Deviation 1% Ethanol 79483 82725 78281 80163 68116 2231 UV, 1% Ethanol 45928 43754 40549 43410 31363 1760 Tea, 0.1% 83024 84153 80945 82707 70660 1607 Tea, 0.1%, UV 48142 49513 47035 48230 36183 1239 Tea, 0.5% 78582 84031 81842 81485 69438 1378 Tea, 0.5%, UV 55000 59546 56014 56853 44806 1845 Tea, 1% 85676 83877 81033 83529 71482 1551 Tea, 1%, UV 58303 64814 61758 61625 49578 1804 These results show that the number of viable cells post-UV irradiation in samples treated with tea callus cell extract is higher than for cells treated with the carrier (ethanol) alone. The effect appears to be dose-dependent. Anti-inflammatory trial Table 4: This table shows the concentration of interleukin-6 (pg IL-6 / µg of total cellular protein) produced by dermal fibroblasts from adult humans treated with the inflammatory pathway activator PMA and with either 1% tea tree callus extract or 1% ethanol (control). Three replicates (samples 1-3) were performed for each treatment. The results indicate that tea tree callus extracts lower the amount of IL-6 produced in cells treated with PMA, which is an indication of anti-inflammatory activity. Sample 1 Sample 2 Sample 3 Average Standard Deviation 1% Tea Callus Extract 9.32 8.65 8.34 8.77 0.50 + PMA 1% Ethanol + PMA 11.16 10.78 10.74 10.89 0.23 Chemical analysis of common gallate and non-gallate catechins in tea callus extract Samples: • Tea leaf: leaves harvested in June 2015. • Tea callus (old): the oldest batch of tea callus grown in early 2015. • Tea callus (new): the newest batch of tea callus grown towards the end of 2015 / beginning of 2016. A 10 μA aliquot was injected into a BEH C18 column (100 x 2.1 mm, 1.7 pm, Waters) on a Waters Acquity UPLC, using a Xevo Triple Quadrupole mass spectrometer. The mobile phase consisted of a methanol (0.1% v / v formic acid) / water (0.1% v / v formic acid) gradient (10:90 to 60:40 for 10 min; to 2:98 for 1 min; held for 2 min; to 10:90 for 1 min; held for 1 min) at a flow rate of 0.2 mL / min and a column temperature of 40 °C. Data were analyzed using Waters MassLynx software. 4.1. The amounts of each metabolite were established by monitoring the specific transitions in MRM mode (epicatechin / catechin: 289>245, epigallocatechin: 305>125, epicatechin gallate: 441>169, epigallocatechin gallate: 457>169, methyl gallate: 183.1>124, gallic acid: 169>125). This shows that the tea leaf sample has significantly more gallates than the callus samples (e.g., ~100 times more in the case of EGCG). Table 5: Measured levels of various tea components. These data are expressed in relative terms, as area under the curve (AUC). Tea leaf Callus(A) Callus(B) Gallic acid 19749 349 588 Gallic methyl 138289 684 1702 EGCG 697159 5798 7692 Epicatechin 62686 13078 68280 ECG 319658 4014 41479 EGC 106309 1187 3950 Catechin 278696 154930 172515

Claims

1. A process for the production of a dedifferentiated stem cell extract from Camellia sinensis, characterized in that the process comprises the steps of: (a) preparing a cell culture comprising dedifferentiated stem cells from Camellia sinensis; (b) performing an extraction from the cell culture using ethanol as the extraction solvent, to produce the dedifferentiated stem cell extract from Camellia sinensis. Three claims follow.