GABRIEL BITARNew molecular mechanisms that prevent sagging and wrinkles were discovered in the Spanish Needle (Bidens pilosa). This finding has resulted in an active cosmetic ingredient that is similar to retinol, which is synthesized from vitamin A. On October 19, the project was named as the best scientific paper presented at the 20th Latin American and Iberian Cosmetic Chemists Congress held at Isla Margarita in Venezuela. The paper combined the knowledge of pharmacologist and phytobotanist Luiz Claudio Di Stasi, a professor at Paulista State University (Unesp) in Botucatu, State of São Paulo, and the experience of the Chemyunion company, from Sorocaba, also in the State of São Paulo. The plant, viewed as being an invasive weed in the fields, was tested and showed that it produces benefits similar to those offered by retinoid, commonly prescribed by dermatologists for its cell regeneration and collagen synthesis properties. Unlike retinoid, however, the plant does not cause side effects, such as skin rash, peeling, and burning.
The plant is being tested by a Brazilian cosmetics company and joins other recently discovered active ingredients and plant additives produced by Chemyunion in partnership with universities. These active ingredients are now being used by such prominent Brazilian and international cosmetics companies as Natura, O Boticário, L’Oréal, Estée Lauder, Victoria’s Secret, Johnson & Johnson and Medley, among others.
Aquasense, launched in 2008, was one of the first innovative products to stand out in the fiercely competitive beauty care market. The product was developed with the support of FAPESP, through a project with funding from the Program for Innovative Research at Small Companies (Pipe). Prior to this development, Chemyunion, which has been in the market for 19 years, had resorted to raw materials used by pharmaceutical and cosmetics companies. Aquasense is an extract made from the bark of the branches of the angico-branco (Piptadenia colubrina), a big tree found in the Mata Atlântica Rain Forest. This extract is used in creams, lotions and other products, to moisturize skin. Argentina, Colombia, Russia and the United States are among the countries that purchase this raw material, based on aquaporins – proteins that move water among the cells. Aquaporins were discovered by an American scientist, Peter Agre, who was awarded the Nobel Prize for Chemistry in 2003 for his discovery. “When it enters the skin, Aquasense stimulates the cell to express aquaporin and the water begins to move, thus generating moisturizing”, says Gustavo Dieamant, the company’s research and development manager.
The focus of the research study, which began in 2004, was to find an active moisturizing ingredient in a plant found in Brazilian biodiversity, capable of stimulating the synthesis mechanism of aquaporins. To this end, Maria Del Carmen Velasquez Pereda, one of the partners in the company, contacted professor Di Stasi, of the Pharmacology Department of Unesp’s Biosciences Institute. Professor Di Stasi has written several books on medicinal plants from the Amazon Region and from the Mata Atlantica Rain Forest. The researcher, who joined the company as a consultant of the company’s scientific team, researched native Brazilian plants with the potential to moisturize and repair skin.
Angico-branco was the plant of choice for this end. However, it was necessary to prove in practical terms that the plant would actually be able to express the mechanism of interest. In the case of the angico-branco, the bark of the branches is crushed into tiny fragments, providing an extract that contains a class of polysaccharides called arabinogalactans. These stimulate the cells to express aquaporins. Thus, the cells move more water to the skin and moisturize it. The moisturizing mechanism of the angico-branco extract resulted in the publication of an article in scientific journals and in awards granted at congresses, such as the one organized by the Brazilian Cosmetology Society in 2008.
Thanks to the first Pipe Project funded by FAPESP, submitted in 2006 by Chemyunion, the company was able to purchase the equipment necessary to extract and test the new active ingredients. Among these pieces of equipment is a supercritical fluid extractor that uses carbon gas (CO2) in the transition phase from the liquid to the gaseous state to obtain high purity plant extracts, and a device called Laser Doppler, which identifies skin microcirculation and sensitive skin characteristics in real time.
Another product has been developed by the company with the support of the Pipe project. More specifically, the product – currently being tested – is an active ingredient extracted from the camapu plant (Physalis angulata) – a native shrub from Brazil’s North, Northeast, and Amazon Regions. The active ingredient acts in similar manner to that of anti-inflammatory corticosteroids without the side effects, such as drying and aging of the skin caused by long-term corticosteroid use. The active ingredients of the camapu and of the Spanish Needle are extracted through the so-called supercritical CO2 extraction process. “When the carbon gas is injected into the equipment, it goes through the plant and drags the active ingredients with it”, says Dieamant. Elimination of the gas prevents any residues from remaining, as is the case with traditional extraction processes. A paste is thus obtained from the plant; the paste, when mixed with the appropriate medium, allows the triage process to begin.
“In the case of the camapu, the proposal was to look for active ingredients with anti-inflammatory properties, similar to those of corticoids, widely used nowadays to treat skin and scalp conditions, such as itching, eczema, and dandruff”, says biologist Juliana Tibério Checon, who studied the plant as part of her master’s degree in pharmacology program at Unesp in Botucatu; Di Stasi was her advisor at the time. At present, Juliana is part of the company’s research and development team. “We studied native Brazilian plants to find active ingredients that would produce the same benefits as those of the corticoids currently being used for skin treatment, but without the side effects”, says Juliana. The first step in the research study was to conduct a triage of the plants in vitro to evaluate if – when used for cosmetic purposes – they had the same anti-inflammatory effects as those produced by the traditional use of these plants by populations from regions where these plants come from.
Several of the plant extract’s action mechanisms were investigated; these ranged from anti-heat properties and collagen replacement to possible side effects. In vitro studies of the active ingredient’s effects when exposed to heat were conducted with skin fragments discarded in cosmetic surgeries, such as eyelid surgery, and obtained with the permission of the ethics committee. Some of the skin fragments were treated with 0.1% of the product, while other fragments were not. The fragments were then placed in an oven at 40oC for 90 minutes, simulating the heat due to exposure to the sun, a situation which leads to the development of an inflammatory process, and, consequently, to hyper pigmentation and wrinkles. All fragments were marked by using immunofluorescence with antibodies for collagen. The antibody, in contact with the skin, emits light when it binds with the collagen and thus it is possible to evaluate qualitatively whether a larger or smaller amount of this protein is in the skin.
“When comparing two kinds of fragments, it is possible to see that the camapu extract was able to prevent cell loss and the rupture of collagen fibers”, says Juliana. The side effects produced by corticoids were also analyzed. In this analysis, skin cell cultures were treated with commercially available corticoids and with the active plant ingredient. “In five days, the commercial corticoids began to degrade the collagen of the skin, whereas the active ingredient that we developed continued to stimulate collagen production.”
The need to thoroughly explore the actual effects of skin care products is due to the requirements of the cosmetics industry. Cosmetic companies must be certain of the actual efficacy of active ingredients before they enter into any kind of commercial agreement. Chemyunion has invested 7%, of its revenues in research and development, on average, to meet the needs of a market hungry for novelties. In 2010, revenues came to R$ 41 million. The R & D team is comprised of 15 people dedicated to research, development, and innovation; three team members have doctorates and four team members have master’s degrees. The group includes researchers with expertise in various fields, such as synthesis and extraction of active ingredients from plants, nanotechnology, cell biology, molecular biology, and immunotoxicology. In addition to the partnership with Unesp, the company is involved in research work in collaboration with the School of Pharmacy of the University of São Paulo (USP) and with the Chemistry Institute of the State University of Campinas (Unicamp).
In the course of the research studies on the Spanish Needle, coordinated by Dieamant, the phytochemical analysis of the plant extract revealed the existence of phytol and fatty acids, which signal possible anti-inflammatory and anti-oxidant properties, and stimulation of the synthesis of the extracellular matrix, all linked to an action mechanism that is similar to that of retinoid. Based on this evidence, the researchers evaluated the anti-oxidizing activity (through the action of specific enzymes), the anti-inflammatory activity (through the quantification of inflammatory mediators such as prostaglandin) and the retinoid-like activity (through the measurement of the growth factor of the extracellular matrix’s components, such as collagen and elastin). The findings showed that the plant extract works in similar manner to that of classic retinoid, which acts on skin rejuvenation, by promoting speedy and efficient cell renovation, reducing wrinkles and spots, and increasing elasticity.
“In vitro evidence was conducted with antibodies that mark the retinoid receptors in the skin”, says Juliana. Retinoid can act on two distinct receptors – the acids (RARs) and the non-acids (RXRs). The acid receptors produce a stronger biological response, which can unleash adverse effects. As the active ingredient of the Spanish Needle has an insignificant effect on this receptor, it stands to reason that this might explain why its biological benefit is similar to that of retinoic acid, albeit on a smaller scale and without the side effects.
WSBG, a small company from the city of São Carlos, in the State of São Paulo, also had the support of Pipe to develop active ingredients for cosmetics. One of these products is an enzyme-based hair straightener. This product will soon leave the test bench on its way to retail shelves. The formula uses a specific type of enzyme called keratinase, whose structure is similar to that of hair keratin. This enzyme had attracted the interest of Biominas, an institution that bridges the gap between the companies that develop technologies and the companies with the means to produce the technologies on a large scale. “There is no keratinase enzyme-based hair straightening product on the market yet”, says pharmacist Monica Cristina Salvagnini, a partner in WSGB. The company has filed patent request for the formula. Another project that the company is working on – also with the support of FAPESP – explored the possibility of modifying titanium dioxide for sunblocks. “Our prototypes did not produce the whitened effect of sunblock containing high solar protection factors”, says Monica. However, the delay in transforming this lab-produced innovation into a commercial product has made the development obsolete and economically unfeasible, as other companies had already launched similar products in the market, at lower prices than those of the product produced by WSGB. Still concerning sunblocks, São Paulo’s Technological Research Institute (IPT) has recently developed a nanostructure that protects skin from UVA rays, which cause premature aging of the skin, and from UVB rays, which cause sunburns and skin cancer due to extended exposure to the sun. A patent request for this product has already been filed, but tests will begin only when companies manifest their interest in this innovation.
In the State of Rio Grande do Sul, expertise in nanotechnology has also migrated to the cosmetics industry. In June 2008, two professors from the Federal University of Rio Grande do Sul (UFRGS), Adriana Pohlmann, from the Chemistry Institute, and Silvia Guterres, from the School of Pharmacy – partnered with two of their former students – Renata Raffin, who has a doctorate in nanotechnology, and Candice Felippi, who has a master’s degree in the same subject – to establish a company called Inventiva. Their aim was to provide the market with a company prepared to nanoencapsulate active ingredients for the cosmetics industry. Some of the equipment used by the company, established in the city of Porto Alegre, had been developed in collaboration with engineers. “We developed our own process for the production of nanoparticles”, says Renata. The support came from the Financiadora de Estudos e Projetos (Finep) promotion agency in the form of a grant. The grant was instrumental for the development of the products and for the clinical, stability, and product safety tests.
“The particles that we produce are nanoencapsulated active cosmetic ingredients, such as vitamins, oils, and plant extracts”, says Renata. The nanoparticles, made from lipophilic (fatty) materials, are sold to cosmetics companies, which use these as active ingredients in their own formulas for skin, hair and nail care products. All the products developed by Inventiva are sold with instructions on how to use them. One of these is a nanoparticle comprised of five active anti-cellulite ingredients. The anti-wrinkles product, in turn, has two vegetable oils (grape seed and linseed) and two vitamins (A and E). At present, sales are concentrated on the domestic market, but the company is looking for distributors with business abroad to expand their own business.
“The company’s leading product is an anti-cellulite product called NanoSlim Complex”, says Renata, who heads the development of new formulas. The first line launched by the company comprised 13 products. Nowadays, the company has a portfolio of 23 products, and 3 more products in the pipeline, which will soon be ready for launching. “New products are launched every six months”, she says. Clients always want special launches. “We don’t sell huge quantities – we sell a variety of products”. At present, company personnel comprise the two partners, two employees, and seven grantees linked to the National Council for Technical and Scientific Development (CNPq), involved in the creation and development of formulas.
The average size of the nanoparticles produced by Inventiva ranges from 130 to 150 nanometers. The nanoparticles are submitted to strict quality control. According to the nanosciences standards established by the European Commission, 50% of a nanomaterial’s particles have to be smaller than 100 nanometers. Visually, the product looks like a sort of milk, but it needs no refrigeration. The product’s safety trials were conducted according to European laws, which do not allow cosmetics to be tested on animals. “We conducted tests that are equivalent to in vitro testing, which mimics organs or parts of the body and specifies if the product is potentially toxic, irritating, or aggressive”, says Renata. Tests proved that the products are safe, do not irritate the eyes, do not cause cell death and do not increase cell oxidation when exposed to light. Based on these results, the company started to conduct clinical trials on human beings, via companies accredited by the National Health Surveillance Agency (Anvisa). At this stage, the product is tested to check whether it causes accumulated irritation after being used for a few days, and whether it has allergenic potential. In addition, the product’s reaction to sun exposure is tested, along with its potential to produce pimples and blackheads. The results were negative for all items, for all the products tested. In addition, an efficacy test was conducted to check whether the products delivered what they had promised. “Everything was duly proven”, says Renata.
The company’s sales increased from April onwards, thanks to an agreement with two major distributors that represent cosmetics, chemical, pharmaceutical, and raw materials industries. In addition to selling its products to the industrial market, Inventiva sells its raw materials to compounding pharmacies.
1. Study of immunomodulating and anti-inflammatory activity of Physalis angulata extracts from hydroglycolic and supercritical carbon dioxide extractions and possible application thereof (nº 2006/56552-8); Modality Program for Innovative Research at Small Companies (Pipe); Coordinator Márcio Antonio Polezel – Chemyunion; Investment R$ 465,692.30 (FAPESP)
2. Evaluation of the moisturizing capacity of active ingredients extracted from native Brazilian plants via expression of aquaporin 3 (nº 2006/51824-0); Modality Program for Innovative Research at Small Companies (Pipe); Coordinator Maria Del Carmen Velazquez Pereda – Chemyunion; Investment R$ 494,639.44 (FAPESP)
3. Study of the phytochemical composition and pharmacological activity of polar and nonpolar fractions of Bidens pilosa obtained by means of supercritical carbon dioxide extraction for application in dermatology and cosmetology (nº 2007/59310-8); Modality Program for Innovative Research at Small Companies (Pipe); Coordinator Gustavo de Campos Dieamant – Chemyunion; Investment R$ 88,058.36 (FAPESP)
4. Biotechnological product for the modification of hair fibers (nº 2006/60459-3); Modality Program for Innovative Research at Small Companies (Pipe); Coordinator Valeria Fernandes Monteiro – WSGB; Investment R$ 152,196.60 (FAPESP)
PEREDA, M.C.V. et al. Expression of differential genes involved in the maintenance of water balance in human skin by Piptadenia colubrina extract. Journal of Cosmetic Dermatology. v. 9, p. 35-43. 2010.