Tricholastyl by Beauty Creations ™ Anti-hair loss: preserving hair youthfulness Tricholastyl™ After the development of traditional anti-hair loss care, demand nowadays is moving towards innovative treatment based on clearly identified mechanisms of action. Strengthened by their experience in the field of skin ageing, Beauty Creations have developed a new hair concept: anti-ageing of the scalp, designed to slow hair loss and preserve hair youthfulness. Hair loss represents the end of the hair life cycle. The cycle takes place in three successive phases lasting very different times: - anagenic growth phase (3 years), - catagenic phase representing a regression phase (3 weeks), followed by - telogenic phase, resting and then loss of hair (3 months). Some regions of the head are more affected than others, particularly the occipital area. In addition, a seasonal variation has been seen as it appears that physiological hair loss is greater in the post summer period. Although hormonal factors are undisputedly involved in this process, other factors influence and aggravate hair loss, particularly ageing of the scalp. An important fundamental study performed by Beauty Creations has demonstrated that glycation, which is already recognised in the skin, also occur in the scalp and is a triggering factor for hair loss. Development of rigidity of the tissues surrounding the bulb leads to poor anchoring of the hair and its premature expulsion. It appears to make it more difficult to implant new hair. In addition, inflammation has been blamed by some authors for the effects of, particularly male, alopecia. In this context a new active substance has been developed, which is designed to produce a natural solution to this problem of ageing of the scalp and its associated hair loss: Tricholastyl™. It is prepared from an extract of an India tree (Pterocarpus marsupium), and proposes to combat this effect. Its anti-glycation activity is supplemented by anti-inflammatory and anti-free radical effects. A clinical trial has proven that when used regularly for 6 months, Tricholastyl™ helps to delay hair loss and therefore preserve youthfulness of hair. Definition / Composition Tricholastyl™ is prepared principally from an extract of the bark of Pterocarpus marsupium, a tree originating from India. This is associated synergistically with glutamic acid which acts as a substrate for many cell reactions, and sodium succinate, a stimulant of cell metabolism. Hair benefits Tricholastyl™ has good anti-glycation activity (glycation being the major cause of ageing of the scalp). Its activity is supplemented by anti-inflammatory and anti-free radical properties, which are widely involved in cell ageing processes. A 6 month clinical trial over supplemented by in vitro tests, has demonstrated good anti-hair loss effect on both the frontal and occipital areas. Tricholastyl™ helps to reduce the severity of seasonal loss observed in summer. By protecting and strengthening the bulb and restoring a normal growth cycle, Tricholastyl™ protects hair youthfulness. Cosmetics use Anti-hair loss preventing care: • in intensive treatments of some weeks, 1 to 2 times a year, • in complementary care, as shampoo and/or hair tonic. Dosage / Solubility / Mode of incorporation 1. Dose of use: - in intensive treatments: 10% - in complementary care: 2 - 5% 2. Solubility: soluble in water, insoluble in oils. 3. Mode of incorporation: Tricholastyl™ is incorporated into the cosmetic product at room temperature. Analytical characteristics 1. Aspect: brown-red liquid with a characteristic odor. 2. Specifications: upon request. 2. Main components: flavonoids, disodium succinate, glutamic acid. Tolerance Good. Efficacy Test summaries overleaf. Storage In its original packaging, at 15 - 25°C. INCI name Tricholastyl™ LS9912 Glucerin (and) Water (and) Sorbitol (and) Pterocarpus Marsupium Bark Extract (and) Disodium Succinate (and) Glutamic Acid. Anti-glycation effect on collagen (in vitro) Aim To evaluate the capacity of Tricholastyl™ to reduce the non enzymatic glycation of collagen in vitro. During ageing, reducing sugars like glucose or saccharose, fix on aminated functions of some macro-proteins like collagen to form Schiff bases. This non enzymatic glycation (at the opposite of natural enzymatic glycation of glycoproteins) is responsible for destructuration of collagen fibers, leading to an atrophy of dermis by inactivation of fibroblasts. Glycation also exists at the scalp level, where it induces hair-loss. In this test, aminoguanidine is used as a «control» inhibitor of glycation. Protocol Human dermal fibroblasts in culture, DMEM, 37°C, 5% CO2 At confluence, incubation in presence of vitamin C for 4 days to stimulate the collagen synthesis Cells are lysed and only the collagen fibers synthetized by fibroblasts are kept Incubation for 15 days of collagen fibers + [3H]-glucose + products to be tested Extraction of proteins Measurement of incorporated radioactivity (liquid scintillation counter) Fig. 1 - Schema of protocol. % of glycation / versus control put to 100% Results 150 125 100 p=0.10 100 75 79 50 p<0.01 25 p<0.01 46 28 0 Glycated control Aminoguanidine 1mg/ml Tricholastyl™ 1% Mean ± SEM of 3 measurements Anova at 1 factor PLSD of Fisher (NS) not significant Tricholastyl™ 3% Fig. 2 - Inhibition of collagen glycation by Tricholastyl™. Conclusion Tricholastyl™ has clearly reduced the rate of glycated collagen, and the effect is dose-dependent. This confirms the anti-glycation effect of Tricholastyl™, hence its anti-ageing activity at the scalp level. Anti-free radical effect (in tubo) Aim The anti-free radical capacity of Tricholastyl™ was investigated by a battery of chemical and biochemical tests. These tests encompass the initial radical forms and induced reactive forms of oxygen. Protocol Cell membrane lipids Anoxia Oxidative stress UVB inflammation Arachidonic acid Nucleic bases Hypoxanthine Lipoxygenase XOD Leucotrien Uric acid Luminol O• NBT SOD H2O2 Fenton reaction: iron, copper, etc... Legend: DPPH° = diphenyl pycryl hydrazyl XOD = xanthine oxidase O2• = superoxide anion H2O2 = hydrogen peroxide SOD = superoxide dismutase ROO° = peroxyl radical HO° = hydroxyl radical R° = free radical in blue = in tubo test HO° Deoxyribose then reaction with thiobarbituric acid and OD at 532 nm ROO° O2 UVB UVA + chromophore DPPH°-test (OD at 513 nm) R° Proteins Unsaturated lipids Fig. 3 - Anti-free radical screening. Results Results in % of inhibition / control 80 78 60 61 64 62 40 20 23 17 Mean ± SEM on 2 or 3 assays 0 DPPH° 0.1% 0.3% 1% 3% 42 38 18 Tricholastyl ™ Tricholastyl ™ Tricholastyl ™ Tricholastyl ™ HO° (Fenton reaction) O2• from lipoxygenase activity Fig. 4 - Results of chemical and biochemical tests in tubo. Conclusion These tests show the anti-free radical activity of Tricholastyl™, complementing its anti-ageing capacity. Anti-inflammatory effect (in vitro) Cutaneous inflammation is a process that involves different mechanisms, at different levels. Two different specific tests showing the anti-inflammatory effect have been performed with Tricholastyl™. Test on keratinocytes In this model, UV-B irradiation is used to trigger inflammation. Inflammation has a toxic effect on cells (decrease of living cell number) and induces release of lactate deshydrogenase (LDH), prostaglandin E2 (PGE2) and interleukine IL1α. Protocol Seeding of human keratinocytes in a standard medium Incubation for 3 days at 37°C, CO2= 5% Treatment Hanks + product to be tested Hanks Irradiation UVB: 0 and 30 mJ/cm² Incubation for 1 day at 37°C, CO2= 5% Measurement of cell number, and released LDH, PGE2, and IL1a Fig. 5 - Schema of protocol. Results 110 100 90 80 Results 70 ** * ** 60 50 40 30 20 ** 10 0 ** ** ** Cell number Control UVB at 30 mJ/cm 2 UVB + aspirin at 0.03% Tricholastyl ™ 1% Tricholastyl ™ 2% Tricholastyl ™ 5% ** ** ** Released LDH ** ** ** Released PGE2 ** ** Released IL 1a Mean ± SEM on 5 assays in triplicate Student’s t test (*) p<0.05 (**) p<0.01 Fig. 6 - Measurement of the cytophotoprotective and anti-inflammation effect of Tricholastyl™. Conclusion Tricholastyl™ has a good protective effect of cells against UV-B toxic effects and significantly decreases photo-induced inflammation. This effect is particularly clear on IL1α. Test on polymorphonuclear neutrophilic granulocytes (PMN) During the inflammation process, PMN produce pro-inflammatory cytokines, enzymes and reactive oxygen species (ROS). This phenomenon, called “respiratory burst”, amplifies the inflammatory reaction and is responsible for associated tissue damage. Protocol Making up of suspensions of human PMN: 1 million of cells by ml, with products to be tested Control (without) Minocyclin Product Incubation for 24 hours (37°C, CO2 = 5%) Numeration of cells in suspension Activation of PMN with 0.1 ml of zymosan Incubation for 0.5 hour (37°C, CO2 = 5%) Measurement of ROS with luminol: recording of the luminescence sending out during 60 seconds Legend: ROS = reactive oxygen species (H2O2, superoxide anion, hydroxyl radicals and singulet oxygen) PMN = polymorphonuclear neutrophilic granulocytes Fig. 7 - Schema of protocol. Results 110 Rate of release ROS in % / control 100 90 Control Minocyclin at 0.001% Tricholastyl ™ 0.05% Tricholastyl ™ 0.1% Tricholastyl ™ 0.2% 100 80 70 * 65 60 50 40 30 20 ** 31 10 0 ** 49 ** 31 Mean ± SEM on 3 assays in triplicate Student’s t test (*) p<0.05 (**) p<0.01 Released ROS Fig. 8 - Measurement of the cytophotoprotective and anti-inflammation effect of Tricholastyl™. Conclusion Tricholastyl™ shows a significant capacity to reduce ROS production during respiratory burst, corresponding to a clear anti-inflammatory effect. This efficacy will counteract the inflammatory process involved in hair loss. Anti-hair loss preventive activity (clinical test) To show the protective effect of Tricholastyl™ on the hair youthfulness by application of a hair tonic with 10 % active during 6 months. Protocol 2 groups of 20 men with androgenetic alopecia D0 : trichogram Ratio A/T < 3 (frontal and/or occipital zones) Treatment 3 times per week 1 group with placebo lotion 1 group with lotion containing 10% Tricholastyl™ 6 months D56 - D112 - D168 : trichogram Fig. 9 - Schema of protocol. Results Aspect of hair bulbs (x 25) Example of trichogram Telogen hair (x 40) Anagen hair Fig. 10 - Study on 30 to 50 hair, to determine the rate of anagenesis (A) and telogenesis (T). Calculation of A/T ratio, and the % of telogenic hair. Evolution of the % of telogenic hair. Absolute variation of telogenesis % Frontal zone Occipital zone 25 80 20 70 15 60 10 50 5 40 0 30 -5 20 -33% -10 10 -15 0 -20 -10 0 1 2 3 Time (month) 4 -51% 5 6 -48% 0 1 2 3 Time (month) 4 5 6 Placebo tonic (n = 15) Tonic with 10% Tricholastyl ™ (n = 15) Fig. 11 - Anti-hair loss of Tricholastyl™, especially during the seasonal peak of hair loss. Conclusion After a 6 month treatment: • With placebo: Strong and significant worsening of hair loss in occipital and frontal zones -> peak of seasonal hair loss. • With the 10% Tricholastyl™ hair tonic: Stabilization of hair loss. The evolution of the percentage of telogenic hair confirms the significant activity of Tricholastyl™ in preventing hair loss at the appropriate time, and protecting hair youthfulness. EUROPE BASF Beauty Creations 49, avenue Georges Pompidou 92593 Levallois-Perret Cedex France Tel: +33 (0) 1.49.64.53.97 Fax: +33 (0) 1.49.64.53.85 [email protected] AMERICAS BASF Corporation 361 Sheep Pasture Road East Setauket, NY 11733 USA Tel: +1 (631) 380 2652 [email protected] JAPAN & ASIA-PACIFIC BASF Japan Ltd. 21F Roppongi Hills Mori Tower, 6-10-1 Roppongi, Minato-ku, Tokyo, 106-6121 JAPAN Tel: +81 (0) 3-3796-9214 Fax: +81 (0) 3-3796-9299 [email protected] Edition March 7, 2013 Although all statements and information in this publication are believed to be accurate and reliable, they are presented gratis and for guidance only, and risks and liability for results obtained by use of the products or application of the suggestions described are assumed by the user. THERE ARE NO WARRANTIES OF ANY KIND. ALL EXPRESS AND IMPLIED WARRANTIES ARE DISCLAIMED. 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