SERICINA INTEGRA A bio-adhesive molecule from

Luigi Rigano, Raffaello Leporatti, Nicola Lionetti, Lab Rigano* - Milano Cristina Mieli Keywords • Sericin • J. AND. C.** - Como Bioadhesion • Biopolymers • Cosmetic formulations

active ingredient, has been a INTRODUCTION conquest of modern technology. Biopolymers and health Biological materials such as wool, arious types of materials organized in the form of a silk, and cellulose are traditional V filament, consisting of a polymers of industrial use. repetitive molecular unit, are Biopolymers can be derived from produced in the animal world. A microbial systems, superior organisms like plants, or chemically silkworm’s cocoon, a spider’s web, a sheep’s wool, and a byssus of synthesized from biological many mollusks are familiar materials ( Table 1 ). Medical, examples. cosmetic, alimentary, and textile There are many purposes for this uses are common, while architecture: applications like biosensors or even data memory are very recent. Their  defense from potential aggressors in vulnerable salient property is their situations, biocompatibility. The term  traps in order to obtain food, biomaterials defines a non-living  thermal/mechanic shields, material used in medical devices that interacts with a biological  environment insertion systems. In each of these cases it involves a system. In other words, materials spun structure that is made of used in contact with living tissues, protein, very resistant to blood, cells, proteins, and every atmospheric agents, mechanically other living substance. The perfect, and re-constructible. So applications are numberless: suturing, fastening, adhering, complex that they are inimitable, in many cases, by synthetic materials. covering, occluding, inhibiting In the insect world, Lepidoptera are contact, and controlled release are tireless fabricators of silky fibers. In the most common functions. Other the case of silk, the woven filament uses contemplate the substitution of sick or non-functional tissues is a living example of co-extrusion, a genetic engineering wonder of (joints, cardiac valves, artificial nature. arteries, dental reconstruction, Sericin (INCI name: Sericin ) forms intra-ocular, and contact lenses), or a protective sheath that envelops assistance in the repair of tissues or the internal fiber of fibroin (INCI in normal sutures (in the reconstruction of tissues of crossed name: Fibroin ), then used in industrial woven threads. Cosmetic ligaments staminal cells of the applications of sericin, once patient are used today along with a considered a scrap material of the braided cord of silk fibroin. (1). woven thread industry, have been Silk (INCI name: Serica ) is biologically active. YQ Zhang found rather weak in the past. Today, rediscovering the essential that materials consisting of sericin properties of it along with keeping and its derivatives are usable as them unaltered with recent and degradable biomaterials, * Via Bruschetti, 1 - 20125 Milano special processing, in order to biomedical substances, and E-mail: [email protected] obtain a more intact and functional functional, fiber, and tissue ** Via Oltrecolle, 32 - 22100 Como membranes ( 2).

Table 1 Classification of natural principal Biopolymers (3) Polysaccharides (plants/algae) Strach (amylose/amylopectin), Cellulose, Agar, Alginate, Carrageenan, Pectin, Gum (guar), Konjac Proteins Silk, Collagen, Gelatin, Elastin, Soy, Zein,

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Gluten,Casein, Albumin Polysaccharides (animal) Chitin/chitosan, Jaluronic Acid Polysaccharides (bacterial) Xanthan, Destran, Gellan gum , Levan gum Polyfenols Lignin, Tannin, Humic Acid Polysaccharides (fungal) Pullulan, Elsinan, Yeast glucans

SILK: ORIGIN AND state, knit the sericin by means of a protects and lubricates it (fibroin and STRUCTURE gummy and transparent sheath. Once sericin detain up to 30% of their weight the cocoon (of variable colors from in water, 2/3 of which due to the As an animal protein fiber, consisting bright white to yellow, pink, orange, sericin). But it’s not just a simple of fibroin and sericin, silk is among the and green) is finished, after about a mechanical removal. Notable attractive few fibers produced in nature in the week, the larvae transform into forces (bonds to hydrogen, Van der form of a “filament,” by the caterpillar Bombyx mori (Mulberry silkworm), chrysalises. At the moment of Waals forces) act between the two emergence to become a butterfly (the proteins, which are responsible for the more commonly known as silkworm, morning is the preferred time, when the bio-adhesive capacity of the sericin. from the Silkworm Bombix family, first light passes through the cocoon), Silk’s chemical composition in the dry Lepidoptera order, and native to China to mate (it takes 12-24 hours for the state is depicted in Table 2 ( 4). The and then selected with numerous male to find the right female!) and elementary composition of the two crosses. It is a complete metamorphic deposit the eggs (about 500), and then protein fractions is very different than insect, with four mutation stages of concluding its life cycle, the insect the proteins of wool. Sulfur is form and structure: produces an alkaline substance to practically non-existant. (Table 3 ).  Egg break open the cocoon and open a  Larva or caterpillar passage ( Figure 1 ). Morphology and Chemistry of  Chrysalis or nymph Fibroin  Butterfly Composition of the filament It is extremely difficult to obtain pure Like other silk Lepidoptera, the In the serigene glands, two distinct fibroin, free from sericin, if not by silkworm, before transforming into a chrysalis, builds a cocoon. The larval types of proteins (fibroin and sericin, withdrawing the material directly from cycle lasts around 30 days at 25°C, like highly concentrated watery the serigene glands of the silkworm. solutions) are synthesized in two Diffraction with X-rays show the subdivided into five “ages” (periods separate areas and forced outward glycoprotein structure, with two distinct during which the larva eats Mulberry through special canals. The sericin protein components, of 370 and 25 kilo leaves day and night) and four forms a layer enveloping the nucleus, Daltons respectively, joined by “moltings” or “sleepings” (in which it the fibroin mass. A little before the covalent disulphide bridges. The remains immobile anchored with very expulsion, the filaments of fibroin primary structure is given by the amino fine filaments of silk on the support produced by the two glands, go to the acid sequence ( 5): (Gly-Ser-Gly-Ala- plan, and renews its tissue covering to sides and at the exit the crude silk is Gly-Ala) n with quantitative be able to get larger, from a few the sum of the two secretions: two relationships: Serin 12%, Alanin 30%, millimeters to 8-9 centimeters). Once filaments of fibroin enveloped in a layer Glycin 44%, amino acids characterized the fifth age is surpassed, the larva is of sericin. A real and true procedure: by different polarities. ready for the spinning of the cocoon the material must pass through an and searches for a quiet place to do it. orifice of a small diameter and the Figure 1 Breaking of the cocoon To protect itself, it builds a shell (from elevated cutting forces exercised after the butterfly’s emergence . 800 to 1500 meters of crude silk) denature the fibroin just secreted. In around its body, during two or three fact, it becomes insoluble and days, that will be the seat of passage assumes a characteristic spatial first to the chrysalis and then to the structure for crystallization, forming butterfly, emitting a silky slaver, in long-oriented fibrous chains. This order to form many rings in the form of eight overlappings ( 3). The slaver is thread has a triangular section, thus it has a luster, “hand” pleasing, and produced by two silk glands, arranged elegant characteristics. Instead, the at the sides of the intestine that sericin does not undergo structural gradually fill up with a viscous liquid transformations. It forms a made up of fibroin. The excretory concentrated soluble solution, with channels of the two silk glands reunite adhesive functions for the filaments of Glycin non-polar R=-H Hydrogen in an orifice that represents the natural Alanin non-polar R=-CH 3 Methyl fibroin, which is a load-bearing material spinneret of the Bombyx mori . In it, the Serin polar R=-CH 2OH Oxidril of the “coaxial cable” so formed, while two slavers of very thin fibroin, which the sericin agglutinates to it and come out of the silk glands in a pasty with glucidic groups, typical of all In the crystalline, the polypeptidic The form of the protein complex, glycoproteins ( 6). They are chains are arranged in an anti-parallel besides the disulphide bridges, is distinguished in ordered crystalline- beta-foil lamellar structure ( Figure 2 ). determined by strong covalent bonds type regions and shapeless regions.

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Figure 2 Chemical structure of beta Fibroin (anti-parallel chain)

Their development takes place in a parallel sense at the axis of the fiber. In this way, microfibrills are formed and then combined in bundles from which the silk thread has its origin. In the combining phase, the resolute contribution derives from the sericin. The anti-parallel beta-foils are joined by numerous hydrogen bridges (C=O....HN). The typical properties of saccharidic chain are also present most internal sericin, to interface with silk (luster, resistance, (3%) linked to the polypeptidic part fibroin, which has a higher crystallinity. weightlessness, and heat resistance) with O-glucosidic and N-glucosidic Two distinct distributions of molecular come from the structural arrangement links ( 9). weight exist: one correspondent to a of the fibroin. relative molecular mass of several

Morphology and Chemistry of PRODUCTIVE PROCESSES Sericin AND STRUCTURE Sericin is the second silk protein. Sericin is a globular protein. In its Besides the functions of covering, spatial conformation (examinable with adhesion and protection for the fibroin, X-rays) there are repetitive structural it lubricates and promotes the elements in regular intervals. The biological process of wrapping of the amino acidic structure of one of the thread in the construction of the RNA messengers deriving from the cocoon. The sericin serves as a Ser-1 gene has a polypeptidic block of bivalent cation donor and acceptor of 38 amino acids that repeat water molecules that free themselves themselves regularly ( 10 ). from the fibroin crystalline region. Also in this case, it is a complex Figure 3 Structure formula of anti- protein molecule. Five principal parallel serin fractions of sericin have been isolated Table 5 Amino acidic distribution of Sericin with glycoproteins of different molecular weight (65-400 kilo Daltons) Type % and other smaller figures. The native Polar oxdrilated amino acids 45.8 sericin, just produced by the serigene Polar non- oxdrilated amino acids 42.3 Non-polar amino acids 12.2 glands, is very different, in the traditional productive processes, than that coming from the boiling and gum phases of silk.

Table 2 Chemical composition (%) of silk Fibroin Sericin Waxy Mineral Substances Substances 72 - 76 22 - 23 1 - 3 0.3 - 1.6 Different than fibroin, there are large quantities of hydrophilic groups in the Table 3 Elementary composition (%) of silk proteins repetitive sequence. According to the Protein C H N O adopted method, many types of Fibroin 47.6 6.4 18.3 27.7 diverse molecular weight were Sericin 46.5 6.0 16.5 31.0 isolated: sericin A, B, I,II,III,IV, s1, s2, s3, s4, s5 ( 10a ). None of them distinguishes itself in a significant way in conformation or Amino acidic composition of sericin constitutive elements. They are all The amino acids that make up sericin, derivatives of an original sericin that which are more distinct than those in underwent various degradation and fibroin, are reported in Table 4 . In hydrolytic processes. It is probably the terms of polarity, different types of amino acids are found to be present Table 4 Amino acidic composition of Sericin (7) (number of residue per molecule) (Table 5 ) ( 8). To be noted is the high content of Polarity Amino acid % oxidrilated polar serine amino acids Non-polar aliphatic Alanin 6.0 (33.5%) ( Figure 3 ), which are non- Glycin 13.5 essential for human development and Isoleucin 0.8 an important source of carbon atoms Leucin 1.0 for the intercellular synthesis of purin, Prolin 0.7 as well as the scarce quantity of non- Valin 3.0 Non-polar aromatic Tirosin 2.6 polar amino acids. Sericin’s isoelectric Polar amidic Asparagin 13.7 point is pH 4.0. Sugars of the Glutamin 4.4 Polar basic Arginin 3.1 Istidin 1.3 Lisin 3.7 Polar oxdrilated Serin 33.5 Treonin 9.7 Figure 4 Distribution of molecular masses in Sericin after it has been spun Absorption (%)

millions and the other with values between 10,000 and 400,000 g/mole (Figure 4 ). In varying the extraction conditions (pH, temperature, time, additives, coadjutants, salts, etc.), there is a deterioration of the first fraction and the distributions of the molecular weights nears the second one. In the last one, the molecular weight also slips toward lower values. The working of the silk entails the The conscious use of proteins and In sericin for unrubberizing, covalent formation of wastes: their derivatives as cosmetic active bonds between the polypeptidic units  Fibrous, made up of slavers, and protective ingredients began 35 are absent. Sericin principally shows fibers of various lengths, fibroin years ago; while vitamins and up in an amorphous shape “in a ball” with or without sericin, or products hydrolyzed with amino acids (coiled ) and in smaller measure in a impurities of an animal or vegetal found satisfying use much earlier in lamellar shape with a flat beta-foil like nature; cleansing and hair products. The structure (like fibroin). The passage  Non-fibrous, made of solubilized success factors were two. First, the from the amorphous globular shape to sericin coming from the ability to tone down the active an organized lamellar shape takes unrubberization soakings that tensions, diminishing the formation of place following repeated processes of present problems of digestion like polar and covalent bonds with skin water absorption and release and flows of elevated organic charge , and, by this, improving the localized mechanic stress . (high COD and BOD) with high ability to rinse and reducing the costs of purification. incidents of irritation (the delicate Traditional industrial processes Many silk agencies are equipped with active tensions were just beginning to What is needed for the Bombyx mori ’s machinery for the recovery of sericin. appear). life cycle doesn’t work as well for the The unrubberization, in controlled Second, the vegetal origin evocative silk industry that needs to obtain a conditions (alkaline, acid, zymotic) of well-being and nourishment continuous thread. Once the forms sericin with a different molecular (proteins, building blocks of human chrysalises are suffocated by a weight than can be predetermined growth, quality element of foods). In heating action with a dryer, there is a (patented graded hydrolysis). In any the future, even the treatment creams sorting of the cocoons (they need to case, it is always sericin denatured by and leave-on products will adopt their be healthy, unspotted, layered tightly, the productive process and from use for their hydrating, filmogen, and resistant enough to pressure, and not impurities. smoothing powers and their velvety “deaf,” meaning you need to hear the touch. chrysalis inside). A following New productive process Even today, proteins of high molecular procedure (screening) divides the Years of study and experience have weight are interesting active cocoons according to their brought about a less aggressive and ingredients for both its filmogena and dimensions. faster purging method (dissolution of hydrating actions. Its acid-stopping You proceed to their maceration in sericin). In a new process, recently ability as well as its emulsifying ability water at 80°C to partly soak and developed by a unique agency in (which is still hardly utilized) are also eliminate the sericin covering and to Europe, sericin is recovered by waters interesting. render the thread less sticky. Through and freeze-dried. The thread of crude the mechanical brush action, you silk is treated in autoclaves in which it search for the main thread most is possible to control the pressure and Those with a low molecular weight act convenient for the unrolling of the temperature, with water and an active like emollients and skin revitalizers as cocoon. In this work of tension at pH 6.5 that works well as protectives with alkali action disentanglement, the slavers are as a seizer, at a temperature of (for example tioglycolates) ( 13 ). joined in numbers of 4 or 8 to form the 130°C for 30 minutes. The numerous sites of various twisted thread in a wrapped tangle by Besides the evident advantages charges (they are mixes of hydro- using a special spool. The number of soluble or hydro-dispersible in a silky slavers joined depends on the colloidial shape with hydrogen bridges title (thickness) of the thread that you and bonds which are stronger or less want to obtain ( 11 ). linked to a shorter working time, strong according to the acidity and Other procedures of spinning and the neutral pH does not ruin the temperature) allow the use in A/O or twisting follow until a crude thread is fibroin and does not alter the chemical O/A emulsifying products. Elastin, set for the fundamental operation of composition of the sericin. This allows collagen, milk proteins, almonds, unrubberizing, which has the purpose the recovery of unaltered sericin for grain, soy, and oats have found a of removing the excess cosmetic uses of high efficiency. The wide range of uses recently influenced treatment waters pushed through by the negative shroud from animal special micro-filters until a filtered epidemics. In Table 6 the functions concentrate is obtained with around that led to the vast use of protein in sericin. In fact, this hinders the tinting 10% sericin, then sent to the freeze- cosmetics are reported. of the silk and masks its luster. The dryer performed with pharmaceutical classic unrubberizing is performed at standards. The result is a white-cream COSMETIC PROPERTIES 95°- 98°C in a concentrated solution powder of pure sericin. OF SERICIN of water and vegetal soap of pH 9.5 -

9.8 per un tempo medio di 3 ore ( 12 ). PROTEIN IN COSMETICS

Recent studies ( 14 ) have shown that showed an evident attenuation of the hydrolyzed sericin inhibits the effects Affinity for keratin skin irregularities ( 15 ). of enzymes responsible for skin aging Sericin, exactly for its bio-adhesive and reduces spots and wrinkles. protective protein structure, has great CONCLUSIONS Today, it is preferable to use of whole affinity toward hydrophobic proteins proteins in which the action is not due (fibroin) and thus toward keratin of The new productive processes of to a non-structured amino acidic hair and the outer skin layer. sericin described allow you to “soup”, but to a spatial structure of the Fluorescent measures showed skin maximally respect its structure and the molecule and its bond coordination substantiality lasting two or three days distribution of molecular weights. Its and bio-adhesion to the skin’s and resistant to normal cleansing use in protective cosmetics and surfaces. In fact, the skin interactions habits after only one application. The cleansers with low levels of irritation, with polymers are not due to the affinity toward keratin is confirmed useful for sensitive skin and rapid chemical nature of constituent also in the treatment of hair. If it is resolution of skin abrasions, becomes monomers. The three-dimensional pretreated with solubilized sericin, it optional, just as in all the cases of structure determines areas which are shows a subsequent absorption of physiological reconstruction of the more or less polar, compensations or proteins inferior to untreated hair. The epidermic barrier. Applicative attractive magnifications, hydrogen active sites toward protein were examples of this renewed biological bonds, repeated bond or non-bond desirably saturated with sericin. material are reported in the areas that, interacting with the skin’s Hydrating and filmogen properties Attachment . surface (it also being polymeric and Sericin in a watery solution, applied formed of repetitive units) establish on the skin initially forms a sticky attractive and bio-adhesion effects, film that, after drying, gives the skin a which are impossible in the case of sense of being smooth, velvety, and BIBLIOGRAFIA monomers oligomers, which are healed. The filmogen abilities increase normal hydrolyzed proteins. the growth of molecular weight (the 1 Altman GH et al (2003) These incidents, now indicated even same thing happened with the fibroin Silk-based biomaterials in simple polisaccharides, are even fiber). For the conformation and Biomaterials 24 (3) 401-416 2005 8[1] • 19 Table 6 Functions used with proteins in cosmetics 2 Zhang YQ (2002) Applications of natural silk protein sericin Hair treatments Skin Treatments in biomaterials Protection (outside agents, action tensions, alkali) Protection (outside agents, action tensions) Biotechnol Adv 20 (2) 91-100

Substantiality Hydration 3 Barbera A, Albertazzi PG (1990) Strengthening (against breakage) Ant-wrinkle, regenerating Guida informativa di merceologia e chimica Elasticizing Improvement of sensorial perception tessile. Il mondo delle fibre in tavole Filmogen Filmogen sinottiche Zanichelli, Bologna

4 Kaplan D L et al ( 1994) Natural Occurring Biodegradables Polymers. more important for proteins, with a presence of numerous hydrophilic In: Polymer: Systems-Synthesis very complex and polarity variable areas and areas of oligosaccharidic and Utility Swift G Narayan R Eds, Hanser Publishing, New York, NY-USA repeated unit. chains, the sericin is similar to Then in glycoproteins you sum up: glycosaminoglycans, thus able to 5 Lee YW (1999) 1. 1 the large possibility to improve the structure of skin Silk Reeling and Testing Manual FAO Agricultural Services Bulletin coordinate many water hydration. Not the corneometry, static N.136, Seoul, Korea molecules, value, but another type of test, the 2. 2 the effect of inter-molecular dynamic measure of the Trans 6 Fournier A (1979) bonds and geometric tendencies Epidermal Water Loss (TEWL), Quantitative Data on the Bombyx mori L. Silkworm of polypeptidic chains, relative to the exchanges of humidity Biochimie 61 283-320 3. 3 the polarization induced by the between skin and the external amidic bonds of keratin and by environment, has given satisfying 7 Sinohara H, Asono Y (1967) Carbohydrate content of fibroin and their spatial distribution. results. Sericin has a prominent Sericin of the silkworm, Bombyx mori Security, sensorial pleasure, and hydrating effect, with a long-lasting J Biochem 62 129-130 efficacy have been greatly proven for effect, to improve the epidermic sericin. Experiments tests ( 15 ) have barrier. The hydrating action is linked, 8 Pizzichini A et al (2004) Innovazione tecnologica nell’industria shown: above all, to the filmogen effect, di lavorazione delle uova - the affinity toward hydrophobic without obstruction ( 15 ). www.tiscali.it/chemengworld/fles/uova.pdf proteins such as keratin ENEA, Settore Biotecnologie e Agricoltura, Dip. Innovazione (protective effect), Anti-wrinkle properties - the improvement of the Sericin has also shown a marked 9 Komatsu K (1972) applicative phase and of the final smoothing effect on skin irregularities Chemistry and Structure of Silk sensations (after the absorption and an effective anti-wrinkle action. JARQ 13 (1) 64-72

of the product), The depth and direction of the 10 Gamo T, Inokuchi T, Laufer H (1977) - the filmogen property (anti-wrinkle wrinkles were assessed with a mold of Polypeptides of fibroin and sericin secreted effect), skin reliefs ( Silflo technique ) both from the different sections of the silk gland in Bombyx mori Insect Biochem 7 285-295 - the ability to hold water (hydrating before and after the 21 day treatment effect). with sericin cream. The results

10a Michaille JJ, Couble P, Proudhomme JC, Studio Cinetico della Solubilizzazione et al (1998) Garel A (1986 ) conAlcali della Sericina Tesi di Laurea, Proteins: Classic Additives and Actives A single gene produces multiple sericin Facoltà di Chimica di Milano for Skin and hair Care messenger RNAs in the silk gland (Sede di Como) Cosm Toil 113 (11) 69-73 of Bombyx mori Biochimie 68 1165-1173 13 Challoner NI, Chahal SP, Jones RT 15 Voegeli R, Meier J, Blust R 11 Quaglierini C (1989) (1997) et al (1993) Manuale di merceologia tessile Cosmetic protein for skin care Sericin Silk Protein: Unique Structure Zanichelli, Bologna Cosm Toil 112 51-63 and Properties Cosm Toil 108 (12) 101-108 12 Brischetto FP (1994-95) 14 Griesbach U, Klingels M 2 Attachment 0 2005 8[1] Protective CREAM for sensitive skin FIXATIVE Emulsion GEL The filmogen properties of sericin and polydecene are important in Emulsifying formula; the lipidic phase produces beneficial and softening preventing contact between the skin and potential aggressive impurities oils (polydecene, UV filter), while the hydrophilic phase transports, of the environment of formula. A cosmetic designated for sensitive skin, besides betainem a protein complex in which the sericin rises with its with non-traditional preservatives, delicate polymeric emulsifiers, and capacity to adhere to the hair and carry out protective and restructuring fragrance free of allergens, strengthens the hydrating, smoothing, and actions. anti-wrinkle abilities with sericin, Phase Ingredients (INCI name ) % (p/p) Phase Ingredients (INCI name ) % (p/p) A Aqua 35.70 A Water 71.25 Glycerin 2.00 Disodium EDTA 0.10 Panthenol 0.50 Allantoin 0.20 Disodium EDTA 0.10 Glycerin 2.00 Betaine 0.50 Methylpropanediol 2.00 A1 Aqua 39.70 A1 Xanthan Gum 0.50 Carbomer 0.80 A2 Acrylates/C10-30 Alkyl Acrylate Crosspolymer 0.30 B Polysorbate 60 5.00 B Hydrogenated Polydecene 5.00 Hydrogenated Polydecene 5.00 PPG-15 Stearyl Ether 1.00 Cetyl Alcohol 1.00 Butyrospermum parkii 4.00 Ethylhexyl Ethylhexanoate 2.00 Cetyl Alcohol 1.80 Ricinus communis 0.10 Phenyl Trimethicone 1.50 Tocopheryl Acetate 0.50 Glyceryl Caprylate 2.00 Methylparaben 0.20 Phenoxyethanol 0.60 Propylparaben 0.20 Tocopheryl Acetate 0.50 C Sericin 1.00 C Glycyrrhetinic Acid 0.05 Hydrolyzed Corn Protein, Hydrolyzed D Aqua, Sodium Hydroxide (Sol 10%p/p) 1.50 Wheat Protein, Hydrolyzed Soy Protein 1.00 E Sericin 2.20 D Aqua 3.00 F Aqua, Butylene Glycol, Glycerin, Betaglucan 1.50 Diazolidinyl Urea 0.30 G Aqua, Aloe barbadensis 1.50 E Triethanolamine 1.20 H Sodium Levulinate 0.50 F Parfum 0.20

Procedure • Mix evenly the components of phase A. • Add A1 and A2 in order, mixing for 5-10 minutes. Before adding Procedure the second, heat to 70°C with turboemulsor in act ion until • Prepare phase A. • Disperse completely the polymer (Carbopol Ultrez 10) in the complete dispersion of polymers. • Heat B to 75°C and add to (A+A1+A2) with the same agitation water of phase A1. regimen. • Add phase A with A1 under agitation and heat to 80°C. • Add C in the same conditions. • Add under turbo phase B, priorly heated to 80°C. • Cool to 40°C and add C, D in order. • Cool to 40°C and add D, E, F, G, H, one after ano ther, mixing for • Add phases E and F in order (turboemulsor in action). a few minutes after each addition. • Cool to room temperature under agitation. Discard. • Cool to room temperature.

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Emulsion SHAMPOO for oily hair Restructuring hair MASK Shampoo in emulsion: cleanses, conditions, and leaves hair shiny. Thanks Sericin, together with polydecene, biotin, and panthenol ensures a product to its filmogen property and to its substantiality, sericin acts as a reparative of protective, hydrating, reparative and restructuring action on hair and and normalizing smoother on possible disturbances of the scalp, in scalp. The presence of quaternary ammonium reinforces the conditioning association with Panthenol and Vitamin E acetate. action.

Phase Ingredients (INCI name ) % (p/p) Phase Ingredients (INCI name ) % (p/p) A Aqua 42.40 A Aqua 68.10 Acrylates/C 10-30 Alkyl Acrylate Crosspolymer 1.10 Panthenol 0.20 Glycerin 5.00 Disodium EDTA 0.10 Panthenol 0.10 Glycerin 2.00 Betaine 1.00 Betaine 2.00 Disodium EDTA 0.10 A1 Polyacrylamide, C 13-14 Isoparaffin, Laureth-7 3.00 Diazolidinyl Urea, Methylparaben, B Hydrogenated Polydecene 5.00 Propylparaben, Propylene Glycol 1.00 Phenyl Trimethicone 5.00 B Hydrogenated Polydecene 3.00 Tocopheryl Acetate 0.50 Tocopheryl Acetate 0.50 Phenoxyethanol 0.60 PEG-20 Methyl Glucose Dioleate 5.00 Stearamidopropyl Dimethylamine 4.00 C Polysorbate 20 1.50 C Aqua 3.00 Aqua, Cocamidopropyl Betaine 2.00 Aminomethyl Propanol, Aqua 0.10 Aqua, Sodium Laureth Sulfate (27% was) 33.00 Sericin 1.00 D Triethanolamine 1.00 Diazolidinyl Urea 0.30 E Sericin 1.00 Biotin 0.10 F Aqua, Polyquaternium-39 2.00 E Polyquaternium-39, Aqua 4.80 G Parfum 0.30 F Parfum 0.20

Procedure Procedure • Disperse the polymer (Carbopol ETD 2020) in phase A and heat to 50°C. • Heat phase A to 70°C. • Heat phase B to 50°C and add it to A under agitat ion. • Pour slowly into A1 under agitation. • Cool to 40°C and introduce C, D, E, F, and G in o rder. • Heat phase B to 70°C and add to (A+A1) under agit ation. • Cool to room temperature under agitation. • Cool to 40°C and add C, D, E and F in order. • Cool to room temperature under agitation.

Delicate FOAM cleanser Delicate and effective self-foaming cleanse suitable for daily use on hair and Delicate conditioning SHAMPOO scalp. Micro-emulsion between polydecene, panthenol, vegetal extracts, sericin, The mild combination of polydecene, sericin, fetoprotein, and active tensions and a mix of delicate cleansers able to perform a delicate cleansing and gives way to a rich, dense, stable foam that is easy to distribute. Ready to decisive conditioning action on hair. Sericin bio-adheres to the keratin of the rinse, it leaves no residue. hair and protects it from active tension. It leaves hair soft and shiny. A supply device can change the product from a transparent liquid form to a Phase Ingredients (INCI name ) % (p/p) soft and thick foam. A Hydrogenated Polydecene 2.00 PEG-20 Methyl Glucose Distearate 4.00 Phase Ingredients (INCI name ) % (p/p) PEG-200 Hydrogenated Glyceryl Palmate, A Aqua, Sodium Laureth Sulfate (27% was) 23.00 PEG-7 Glyceryl Cocoate 4.00 Decyl Glucoside 3.80 Tocopheryl Acetate 0.50 Aqua, Cocamidopropyl Betaine 7.70 Ethoxydiglycol 1.00 Hydrogenated Polydecene 1.50 B Aqua, Sodium Laureth Sulfate (27%was) 43.00 Laureth-4 2.00 Aqua, Cocamidopropyl Betaine (32%was) 14.00 B Aqua, Hamamelis virginiana 3.80 C Aqua, Tilia cordata 11.00 Sericin 1.50 Aqua, Sodium Hydroxide 0.13 C Diazolidinyl Urea, Methylparaben, Cocamide DEA 5.00 Propylparaben, Propylene Glycol 0.76 Laureth-23 10.00 Aqua, Propylene Glycol, Cucumis sativus , D Sericin 1.00 Phosphoric Acid 0.76 Panthenol 1.00 Aqua 53.33 Diazolidinyl Urea, Methylparaben, Panthenol 0.50 Propylparaben, Propylene Glycol 1.00 D Allantoin 0.10 Aqua, Ethoxydiglycol, Propylene Glycol, Aqua, CI 42051 0.10 Butylene Glycol, Humulus lupulus 1.07 Aqua, Cetrimonium Chloride 1.00 E Aqua, Cetrimonium Chloride 1.00 Parfum 0.15 F Parfum 0.30

Procedure Procedure • Heat phase A to 45°C. • Heat phase A to 60°C. • Add phases B, C, D, and E in order and under agitation. • Introduce phase A to B under agitation. • Cool to room temperature. • Add phase C, heated to 60°C, to the mixture (A+B) continually under agitation. • Cool to 40°C and introduce D, E, and F in order. • Cool to room temperature.

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Restructuring emulsion CONDITIONER Based on a fixative action of traditional resins, this hydro-alcoholic gel An emulsifying conditioner that combines quartemary ammonium and lipidic appeals especially to the bio-adhesiveness of whole sericin to perform a emollient substances to the proteinic and restructuring strength of sericin. preventative action of protection and restructuring on hair.

Phase Ingredients (INCI name ) % (p/p) Phase Ingredients (INCI name ) % (p/p) A Aqua 50.90 A1 Carbomer 1.20 Panthenol 0.20 A Sorbitol 1.00 Disodium EDTA 0.10 Aqua 63.43 Glycerin 2.00 B Triethanolamine 0.90 Betaine 2.00 C Alcohol 20.00 A1 Aqua 15.40 Phenoxyethanol 0.50 Acrylates/C10-30 Alkyl Acrylate Crosspolyme 0.30 PVP 2.00 B Octyldodecyl Ricinoleate 15.00 PVP/VA Copolymer 4.00 Phenyl Trimethicone 10.00 Aqua 3.00 Tocopheryl Acetate 0.50 D Hydrogenated Polydecene 1.00 Phenoxyethanol 0.60 Soja, Juglans regia 0.50 Dimethiconol Beeswax 1.00 Ethylhexyl Methoxycinnamate 0.10 C Aminomethyl Propanol 0.20 E Aqua 3.00 D Sericin 0.50 Sericin 1.00 E Diazolidinyl Urea 0.30 PPG-26-Buteth-26, PEG-40 Hydrogenated F Biotin 0.10 Castor Oil 0.50 G Aqua, Polyquaternium-39 1.00 Parfum 0.30 H Parfum 0.30 Ethylhexyl Methoxycinnamate 0.15 G Mica, Titanium Dioxide 0.02 Procedure H Aqua, CI 42051 (Soluz 1%) 0.40 • Heat phase A to 60°C. • Prepare phase A1 separately. Disperse with turboemulsor. Procedure • Heat phase B to 60°C. • Sprinkle phase A1 into phase A under adequate agitation. • Add A1 to A under agitation. • To the even mixture, neutralize with B and form the gel. • Pour B into (A+A1) slowly and under turbo and pale action. • Introduce phase C complete and uniform, prepared separately. • Pour C into (A+A1+B) slowly and under agitation. • Under turbo and pale action, introduce phase D and then E, F, G, • Cool to 40°C and introduce D, E, F, G, and H in o rder. Assure a and H one at a time and in continuous pale action with perfect dispersion before adding the next one. turboemulsor until complete dispersion of each one. • Bring to room temperature and discard.

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STYLING GEL