Mint Eco extract is produced from the leaves of Mentha piperita, coming from organic crops.

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1 Mint Eco BOTANY Mentha piperita L. This plant belongs to the Lamiaceae family. It is commonly known as mint, peppermint, lamb mint or brandy mint. The plant is a perennial, 50 to 90 cm high. The usually branched stems are normally glabrous, but sometimes they are gray-tomentose and often tinged violet. The leaves are short-petioled, oblong-ovate and serrate. The plant has over- and underground runners. The flowers are false spikes with numerous inconspicuous bracts. The calyx is tubular, with a ring of hair. The corolla is violet, glabrous inside and has an almost even margin divided into four parts. Mentha piperita is thought to be a natural hybrid between spearmint (Mentha spicata) and water mint (Mentha aquatica). Mint is common in Europe and is usually cultivated the U.S. Mint Eco extract is produced from the leaves of Mentha piperita, coming from organic crops. CHEMISTRY Phenolic acids The main phenolic acid of mint is rosmarinic acid (3% of dry weight), but it also has other hydroxycinnamic derivatives like caffeic acid and cinnamic acid. V 01-03/

2 Flavonoids Various flavonoids are present including luteolin and its 7-glycoside, rutin, hesperidin, eriocitrin and highly oxygenated flavones. Essential oil The main active component is essential oil (1-3%), of which the principal constituent is usually menthol, in the form of (-)-menthol (usually 35-55%) with smaller amounts of stereoisomers such as (+)- neomenthol and (+)-isomenthol, together with menthone (10-35%), menthyl acetate, menthofuran, cineole, limonene and other monoterpenes. Small amounts of sesquiterpenes occur in the oil, notably viridoflorol. Other active principles Other constituents include tannins, phenolic acids and small amounts of triterpenes (ursolic acid, oleanolic acid). TRADITIONAL USES the oral mucosa and throat. Mentha piperita is one of the world s oldest medicinal herbs. Ancient Greek, Roman, and Egyptian cultures used the herb in cooking and medicine. Mint is taken internally as a tea, tincture, oil, or extract, and applied externally as a rub or liniment. Herbalists consider mint an astringent, antiseptic, antipruritic, antispasmodic, antiemetic, carminative, diaphoretic, mild bitter, analgesic, anticatarrhal, antimicrobial, rubefacient, stimulant, and emmenagogue. Mint oil vapour is used as an inhalant for respiratory congestion. Mint tea is used to treat coughs, bronchitis, and inflammation of It has traditionally been used to treat a variety of digestive complaints such as colic in infants, flatulence, diarrhea, indigestion, nausea and vomiting, morning sickness and anorexia, and as a spasmolytic to reduce gas and cramping. Mint is currently used to treat irritable bowel syndrome, Crohn s disease, ulcerative colitis, gallbladder and biliary tract disorders, and liver complaints. Mint oil is used to relieve menstrual cramps. Mint oil is used externally for neuralgia, myalgia, headaches, migraines and chicken pox. V 01-03/

3 COSMETIC PROPERTIES Antioxidant activity Several studies show the antioxidant activity of mint, which is due to flavonoid and hydroxycinnamic derivatives. The hydroalcoholic extracts of some medicinal plants with a high content in hydroxycinnamic derivatives (rosmarinic acid was found in more that 3-6% of dry weight) showed a significant antioxidant activity as 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical scavengers. The antioxidant action was attributed to the high concentration of rosmarinic acid in these plants (Al-Sereiti M.R., 1999). Kosar et al. in 2004, performed a study where 9 mint extracts were examined. The caffeic acid, eriocitrin, luteolin-7-o-glucoside and rosmarinic acid were identified as potential free radical scavengers, being tested by HPLC-DPPH method (Kosar, 2004). In another study made by Dorman et al. in 2009, 7 different extractions of Mentha piperita leaves were done, one of them being the aqueous extract. In these extracts, the most abundant components are eriocitrin and rosmarinic acid, and 6 different assays were done to see the antioxidant activity of different detected actives. The aqueous extract is one of the most potent of the 7 tested, because it has been demonstrated that it can scavenge and chelate different free radicals to work as an antioxidant. To sum up, we can consider that Mint Eco extract is useful to formulate cosmetic products designed to preserve skin and hair integrity against oxidative processes. Anti-inflammatory activity A dry 80%-ethanolic extract from mint leaf showed anti-inflammatory activity against acute and chronic inflammation in rodents. After oral administration it reduced xylene-induced ear oedema in mice (acute model) by 49% at 200 mg/kg and 50% at 400mg/kg (both p<0.05). After intraperitoneal administration in the cotton pellet granuloma test in rats (chronic model), only the higher dose of 400mg/kg had a significant inhibitory effect (p<0.01); (ESCOP, 2003). Rosmarinic acid showed an anti-inflammatory activity in rat models, where carragenin was used to induce plantar edema. Experimentally, it was shown that rosmarinic acid acts on prostaglandin formation (PGE 2 ), similarly to non-steroidal anti-inflammatory drugs. It also caused complement-c3 factor inhibition, an inflammatory process mediator not involving cyclooxygenase activity or Prostacyclinsintase. Also, it reduced leukotriene B4 reduction in human polymorphonuclear leukocytes (Alonso J., 2004). V 01-03/

4 In order to evaluate the potential role of L-menthol as anti-inflammatory, preclinical in vitro-investigations were performed using LPS-stimulated monocytes from healthy volunteers. Arachidonic acid metabolism was assessed by measuring LTB subset4 and PGE subset2 as indicators for both the lipoxygenase and the cyclooxygenase pathways respectively. In addition, the anti-inflammatory effects of the two terpenes on IL-1beta production were analysed. - L-menthol significantly suppressed the production of each of the three inflammation mediators by monocytes in vitro. LTB subset4 decreased by /- 10%, PGE subset2 by /- 8%, and IL-1beta by /- 7% respectively at L-menthol concentrations within the presumed therapeutic range of about 10 superset-7 g/ml (Juergens, UR et al., 1998). Therefore, Mint ECO extract is recommendable to formulate cosmetic products with anti-irritant activity. Vessel-protection and venotonic activities Flavonoids are vein-active and vessel-protective agents because they reduce the permeability and increase the resistance of blood capillaries. Flavonoids are used in the treatment of blood vessels disorders such as varices, chronic venous insufficiency (CVI), low capillary resistance, etc. Their protective effect is due to their high affinity for proline-rich proteins, such as collagen and elastin. Since these proteins are structural components of veins, their degradation weakens the blood vessels, inducing edema and swelling of the lower limbs. Oral administration of flavonoids has been observed to effectively improve the capillary resistance in animal models. The RELIEF study ( Reflexus asessment and quality of life improvement with micronized flavonoids in chronic venous insufficiency ) is a controlled multi-centre study carried out between March 1997 and December 1998, in 23 countries around the world, in which more than 10,000 patients suffering CVI participated. These patients were treated with a purified flavonoid fraction for six months. The results of this study showed that the flavonoid treatment significantly improved the cases of pain, tired legs, swelling, cramps and edema (when it was present) (Spignoli G., 2000). Thus, Mint ECO extract is of use to formulate cosmetic products aimed at improving general blood circulation. Antimicrobial activity Antiviral In vitro data: mint has significant antiviral activity. Menthol is virucidal against Influenza, Herpes and other viruses in vitro. Aqueous extracts of mint leaves were antiviral against Influenza A, Newcastle V 01-03/

5 disease virus, Herpes simplex virus, and Vaccinia virus in egg and cell-culture systems (Gardiner, P., 2000). Antibacterial In vitro data: mint oil and menthol have moderate antibacterial effects against both Gram-positive and Gram- negative bacteria. Mint extracts are bacteriostatic against Streptococcus thermophilus and Lactobacillus bulgaricus. Menthol is bactericidal against Staphylococcus pyogenes, S. aureus, Streptococcus pyogenes, Serratia marcescens, Escherichia coli, and Mycobacterium avium (Gardiner, P., 2000). Furthermore, rosmarinic acid showed its antibacterial properties against Grampositive and Gramnegative bacteria. It is a bacteriostatic, especially against S. aureus and S. epidermis (Mencherini, 2007). Antifungal In vitro data: menthol and mint oil are fungicidal against Candida albicans, Aspergillus albus and dermatophytic fungi (Gardiner, P., 2000). Rosmarinic acid is evaluated against Candida albicans and shows its fungicidal activity (Mencherini, 2007). Thus, Mint ECO extract is recommendable to formulate cosmetic products with purifying and antiseptic activity. BIBLIOGRAPHIC EFFICACY STUDY Mint is rich in flavonoids and hydroxycinnamic acids, such as rosmarinic acid. The following study shows that rosmarinic acid and flavonoids of mint extract have antioxidant activity. 1. Experimental method In a study conducted by Dorman et al. in 2009, the antioxidant capacity of different Mint extracts, including an aqueous extract was evaluated. Several actives with this antioxidant ability were detected by HPLC, such as rosmarinic acid. Several assays were performed to evaluate different aspects of antioxidant activity: The ability of polyphenolic compounds to participate in electron transfer and hydrogen atom transfer reactions is an important mechanism for antioxidant activity. To assess the extracts ability to participate in these reactions, their iron (III) reducing efficacy was determined. Another test was performed to evaluate the capacity of chelating the available iron, to assess their V 01-03/

6 protective role against oxidative damage catalized by available metals. The EC50 data (concentration required to chleate 50% of the available iron)of different extracts was determined. Cell membranes are highly susceptible to oxidative degradation via free radical-mediated chain reactions. The β-carotene-linoleic acid bleaching assay was used to evaluate the extracts capacity as primary antioxidants, acting like free radical scavenging to prevent these oxidation reactions. Free radicals are involved in the process of lipid peroxidation, involved in premature aging. The DPPH radical test was used to show the free radical scavenging activity. In this assay, the concentration required to scavenge 50% of the avalaible DPPH (IC 50 ) was determined. To further confirm the free radical scavenging activity of the extracts, the ABTS radical test, which evaluates the free radical scavenging capacity, was used. If the extracts are able to scavenge ABTS, it means that radicals are estabilized before reacting, and that is why they don t cause oxidative damage 2. Results In the assay to assess the iron reduction, several extracts showed activity, like the aqueous extract. Therefore, it is proved that extracted soluble components may be able to transform reactive free radical species into more stable non-radical products. In the iron chelation test, only three extracts were active at reasonable concentrations. Again, the aqueous extract showed efficacy. Observing the results, it was also proved that extracts inhibited the bleaching of β-carotene (Fig. 1.), so they can be considered as primary antioxidants. In this assay the aqueous extract was the most effective. Fig. 1. β-carotene-linoleic acid bleaching inhibitory activity of the extracts: (1) AscA, (2) BHA, (3) Trolox, (4) PE, (5) CH 2Cl 2, (6) ACN, (7) EtOAc, (8) MeOH, (9) n-buoh y (10) H 2O V 01-03/

7 The positive results of DPPH radical test suggest that mint extracts are able to scavenge free radicals at physiological ph and thus may be able to prevent the initiation/propagation of free radical-mediated chain reactions. The aqueous extract was one of the most efficient in this assay. This result was confirmed by ABTS radical test (Fig. 2), where is observed the capacity to scavenge it, and it means that reactive species are going to be stabilized before they can participate in deleterious reactions 3. Conclusions Fig. 2. ABTS free radical scavenging activity of the extracts: (1) AscA, (2) BHA, (3) Trolox, (4) PE, (5) CH 2Cl 2, (6) ACN, (7) EtOAc, (8) MeOH, (9) n-buoh y (10) H 2O The data confirm that the leaves of Menta piperita contain high levels of polyphenolic compounds, which includes rosmarinic acid, caffeic acid and various flavonoids. Results revealed that the extracts show iron (III) reductive and free radical scavenging activities because of their constituents ability to donate electrons/hydrogens. Therefore, mint extracts can be described as primary antioxidants, what suggests they can play a beneficial role of preventing the initiation/propagation of free radical-mediated chain reactions and consequently, prevent the oxidative damage on skin and avoid premature skin aging. V 01-03/

8 COSMETIC APPLICATIONS Action Active Cosmetic application Antioxidant Anti-inflammatory Hydroxycinnamic derivatives Flavonoids Essential oil Rosmarinic acid Anti-aging Anti-irritant Vessel-protection and venotonic Flavonoids Blood flow activator Antimicrobial Essential oil Purifying Antiseptic RECOMMENDED DOSE The recommended dose is between 0.5% and 5.0%. BIBLIOGRAPHY Alonso, Jorge. Tratado de Fitofármacos y Nutracéuticos. Barcelona: Corpus, 2004, p: ; Al-sereiti, M.R. et al. Pharmacology of rosemary (Rosmarinus officinalis Linn.) and its therapeutic potentials. Indian Journal of Experimental Biology.1999, 37(2): Dorman, D. et al. Phenolic profile and antioxidant evaluation of Mentha x piperita L. (Peppermint) extracts. Natural Product Communications. 2009, 4(4): E.S.C.O.P. (Ed). Monographs. Second Edition. Exeter (UK), 2003; p: (633.8(031) ENC). Gardiner, P. Peppermint (Mentha piperita). Longwood Herbal Task Force, Revised May 2, 2000 [ %22; consultado en Agosto de 2006]. Juergens UR, Stober M, Vetter H. The anti-inflammatory activity of L-menthol compared to mint oil in human monocytes in vitro: a novel perspective for its therapeutic use in inflammatory diseases. Eur J Med Res Dec 16;3(12): Koşar M. et al. Screening of Free Radical Scavenging Compounds in Water Extracts of Mentha Samples Using a Postcolumn Derivatization Method. Journal of Agricultural and Food Chemistry. 2004, 52(16): V 01-03/

9 Spignoli G. Protective effects of dietary flavonoids on cardiovascular system and circulation. European Bulletin of Drug Research, 2000; 8 (1): 1-8. V 01-03/