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(11) EP 3 124 030 A1

(12) EUROPEAN PATENT APPLICATION

(43) Date of publication: (51) Int Cl.: 01.02.2017 Bulletin 2017/05 A61K 35/583 (2015.01) A61Q 19/00 (2006.01) A61K 8/98 (2006.01) A61P 17/02 (2006.01) (2015.01) (21) Application number: 15386024.2 A61K 35/618

(22) Date of filing: 30.07.2015

(84) Designated Contracting States: • PAVLIDIS & Partners Co. AL AT BE BG CH CY CZ DE DK EE ES FI FR GB 54248 Thessaloniki (GR) GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR (72) Inventors: Designated Extension States: • Staikou, Alexandra BA ME GR-54124 Thessaloniki (GR) Designated Validation States: • Garefalaki, Marina-Elena MA GR-54633 Thessaloniki (GR)

(71) Applicants: (74) Representative: Petsis, Christos • Aristotle University Of Thessaloniki-Research 4-6, Kyparissias Committee 542 49 Thessaloniki (GR) 54636 Thessaloniki (GR)

(54) METHOD FOR PRODUCING A EXTRACT FROM AND ITS EXTRACT

(57) Method for snail extract acquisition, more par- conditions including moisture, temperature and duration, ticularly of Cornu aspersum, comprising at least two yielding a quantity of clear pedal mucus, which is re- steps, being remarkable in that a first step for obtaining moved from said surface means and placed into recep- the extract consists of a non stressful snail extract acqui- tacles. It is followed by a further step consisting of drying sition, wherein a batch of is selected which are with a subsequent resuspension step of the produced brought to move by crawling on a set of surface means, snail extract, resp. of a centrifugation of said snail extract. particularly glass plates, under a set of predetermined EP 3 124 030 A1

Printed by Jouve, 75001 PARIS (FR) 1 EP 3 124 030 A1 2

Description onstrated that aspersa extract is a natural, safe and effective alternative treatment in open wound manage- Field of the invention ment by accelerating skin regeneration and decreasing pain (Tsoutsos et al 2009). [0001] The present invention relates to a method for 5 [0005] Life-cycle and biology of the Cornu aspersum producing a snail extract from a selected member of the also play an important role. Cornu aspersum, also known gastropod family, particularly Cornu aspersum. as Helix aspersa, Cantareus aspersus and Cryptomph- alus aspersus, is a terrestrial pulmonate gastropod with Background of the invention as common name ’garden snail’. It is native to the Med- 10 iterranean area but has been spread by humans to nu- [0002] Snails have always been considered as a nat- merous areas all over the world and is therefore consid- ural source having numerous therapeutic and biological ered as an anthropochorus species. The garden snail is properties. Land snails have been used in medicine since herbivore: it feeds on plants only, notably on numerous antiquity and were introduced by Hippocrates, Pliny, and types of fruit trees, vegetable crops, garden flowers and Galen for their medical use. Snails were prepared ac- 15 cereals. In addition to humans, it is a food source for cording to several formulations for internal or external many animals. Adult snails bear a hard, thin use. Boiled, grilled or crushed with their shell and heated shell with 4 or 5 whorls. The body of the snail is retracted with wine, they appeared curative against several dis- entirely into the shell when the animal is inactive or threat- eases such as vertigo, fainting fits, anthrax and for the ened, while during times of activity the head and foot spasms of spitting blood accompanying tuberculosis,20 emerge. The head bears four tentacles, which can be and for the urine ardor of nephritis. External use of snail retracted into the head. The mouth is located beneath extracts was considered to treat pain related to burns, the tentacles, and contains a chitinous which the abscesses and other wounds. Thus, eating or drinking snail uses to scrape and manipulate food particles. The snails, or applying them to wounds provided a very ef- of the shell is sealed with a thin membrane of fective treatment based on natural products. 25 dried (mucus know as an , which helps the [0003] The 19th century revealed new insights in the snail retain moisture under stressful condition of humidity pharmaceutical and medical use of snails with more ad- and temperature. Thermal death by overheating poses vanced snail preparations, such as tablets, syrup, snail a danger for these animals in hot regions. Thus restricting paste and snail chocolate. They were applied orally and activity to favorable time periods with respect to temper- were indicated for resistant colds, tuberculosis and30 ature and humidity can be seen as a further behavioral against most inflammations. They were especially indi- adaptation of terrestrial snails ( Staikou 1999). In addition, cated against lung diseases and infections, against path- many species use summer during ological disorders of the stomach, and some skin diseas- times of disadvantageous conditions in order to save wa- es (Baron-Barthelemy 1855, Bonnemain 2003). ter and energy (Herreid 1977; Barnhart & McMachon [0004] The interest in therapeutic and biological prop- 35 1987; Withers, Pedler & Guppy 1997). Hibernation rep- erties of snail extract continued in the 20 th century when resents the inactive phase of snails during the winter due more than 30 enzymes within the digestive mucus were to low temperatures When hibernating, Cornu aspersum identified, and several enzymes in the pancreostomach, avoids ice formation by altering the osmotic components the muscle and lymphatic fluid, had demonstrated muc- of its blood (or haemolymph), and can survive tempera- olytic and spasmolytic activity for patients with chronic 40 tures as low as -5°C (Ansart e.a.2002). During aestiva- bronchitis (Quevauviller et al 1953, Bonnemain 2003). tion, the collar has the unique ability to change Current concern on snail research includes a more de- its permeability to water (Machin, 1966). In combination tailed approach and has recognized E 2 prostaglandin as with an osmoregulatory mechanism similar to that-seen a broncho-relaxant substance (Pons et al 1999 ), a lectin duringhibernation, this allows Cornuaspersum to survive from as a prognostic factor for several can- 45 several months of aestivation. cers (Dwek et al 2001, Brooks & Leathem1991 ), whereas [0006] Cornu aspersum is a land snail with diverse sci- hundreds of neurotoxins derived from sea snails have entific interest. The biology and behavior of this snail has been evaluated by the FDA for their powerful anesthetic many applications in biological research and it is used effect (Webster et al 2001). Interestingly, the molecular as model organism in numerous fields of biology, includ- basis of the regenerative properties of Helix aspersa se- 50 ing ethology, neurobiology, and reproductive biology. In- cretion has been recently identified as it exhibited anti- terestingly, due to the fact that it is an edible land snail, oxidantSOD and GST activities (Brievaet al2008). More- farming of this species has gained attention the last dec- over, an up to date investigation revealed the regenera- ades notably in the Mediterranean. Therefore, under- tive properties of the secretion of Helix aspersa as it also standing the reproductive cycle and all aspects of the promotes proliferation, migration and survival of kerati- 55 reproductive biology of Cornu aspersum is a prerequisite nocytes and dermal fibroblastsin vitro (Iglesias-de la in order to organize a successful snail farm. Cornu as- Cruz et al 2012). The topical application of a cream based persum is a simultaneous reciprocal and obligatory out- on snail extract of Helix aspersa on dermal burns dem- crossing with internal fertilization, which

2 3 EP 3 124 030 A1 4 means that each individual acts in both sexual roles dur- active changes and predative threat through feedback ing , by exchanging sperm, whereas self-fer- from sensoryarray integration by the central nervoussys- tilization never occurs. Cornu aspersum mates repeat- tem (CNS) (Deyrup-Olsen 1996 et al 1992). Release of edly with different partners, i.e. multiple , in the pedal mucus from the pedal gland is in the form of mem- course of a reproductive season. It has been reported to 5 brane encased mucus granules which are ruptured by mate on average three times up to maximum seven various cues including mechanical shearing forces. Mol- times, (Fearnley 1993, 1996; Madec & Daguzan 1991, luscs produce 60-400 mg of dry mucus/hour/g body 1993). Cornu aspersum from Greek populations mated weight which has a calorific value of about 24 J/mg dry on average three times up to maximum seven times, with weight (Schlichter 1981, Calow 1978). This mucus con- at maximum five different partners, during the reproduc- 10 veys information about the animal’s sexual state and di- tive period (Koemtzopoulos 2007, Garefalaki 2010). Fur- rection of locomotion to conspecifics and predators (Den- thermore, mating frequency was consistent in F 1 gener- ny 1989). The uniqueness of an individual mollusc’s mu- ation snails reared in the lab (Koemtzopoulos & Staikou, cus at species level has been supported by findings using 2007). Interestingly, multiple mating leading to multiple SDS-PAGE electrophoresis -seven snail species-, which paternity of the offspring has been related to behavioral 15 showed that there were few similarities between the in- -mating order- and anatomical -epiphallus length-repro- dividual species’ carbohydrate and protein profiles (Cot- ductive traits in this species (Garefalaki et al. 2010). In trell et al 1993). the field, snails aestivate during the dry period charac- [0008] Other glandular secretions of snails are strongly terizing the Mediterranean climate ranging from June to related to reproduction such as mucus that derives from September. With the first autumnal rainfalls starting from 20 a pair of digitiform glands and covers the . Dart early October, they emerge and immediately start mat- shot during copulation acts as a hypodermic device to ing. The reproductive period lasts for 2 months. Mating deliver the mucus to the interior of the recipient (Koene in this species includes elaborate courtship behaviour & Chase 1998). The mucus contains a biologically active with optional dart shooting(Chung, 1987; Adamo & substance that could influence the female organs in- Chase 1988). The female part of the 25 volved in the processing of foreign sperm. Thus the suc- includes a sperm digesting -bursa copulatrix- and a cessful dart shooter may increase the chance that his sperm storing [fertilization pouch-spermatheca complex sperm will fertilize eggs. (FPSC), Tompa, 1984] organ.Cornu aspersum copu- [0009] Another gland of the reproductive system is the lates for variable time periods ranging from 2 to 12 h albumen gland which provides numerous nutrients to the (Koemtzopoulos& Staikou 2007) during which each snail 30 fertilized ova before oviposition (Tompa 1984). The al- transfers a spermatophore containing from 106 to 14 X bumen gland of land snails is the organ that fluctuates 106 (Rogers & Chase 2001) sperm into its partner. The most in size during different stages of the reproductive spermatophore is formed in the epiphallus and flagellum cycle; it is largest immediately before egg laying, when which are the male parts of the reproductive system. The it can be 6% of the total body weight (Runham & Laryea majority of sperm transferred is digested in the bursa 35 1968), and may contain more than 85% of the total body copulatrix, whereas only a small portion of about 0,025% polysaccharides, resp. 100% of the galactogen. Imme- escapes and travels up the spermoviduct to reach the diately after egg laying, the size of this organ is reduced. tubules of the spermatheca(Rogers & Chase 2001) Apart from its role in galactogen synthesis and nutrient where it is stored until fertilization. The fertilization of the enrichment of the ova, it has been proposed to contain ova generally occurs several hours or days after mating 40 antimicrobial substances (Renwrantz & Berliner 1978). (Madec et al 1998). The albumen gland provides numer- In Cornu aspersum the fertilization pouch-spermatheca ous nutrient substances to the fertilized eggs such as complex (FPSC) is an organ of the reproductive system proteins, lipids, polysaccharides. After about two weeks, embedded in the albumen gland, playing a fundamental approximately 80 spherical pearly-white eggs are laid in- role in sperm storage (allosperm) and fertilization of the to crevices in the topsoil. The young snails take one to 45 ova. As sperm is stored for prolonged periods up to over two years to reach maturity. a year, the FPSC must contain factors that promote cell [0007] Snails produce mucus which is crucial in de- viability, whereas growth factors inducing cell division fense and locomotion of molluscs that are exposed to may also be present in this organ for the fertilized ova. the external environment(Denny 1980, Davies & A recent study revealed the potential effect of spermath- Hawkins 1998). Pedal mucus is composed of a variety 50 eca gland extract of a marine snail on wound healing in of molecules in an aqueous carrier medium, including an animal model (Kumar et al 2008). mainly charged mucopolysaccharides, glycoproteins, [0010] The digestive tract and the digestive gland is proteins, uronic acid, sialic acid, hexosamine, gly- composed of calcium, excretory and digestive cells, cosaminoglycans (GAGS), lectins, and proteoglycans which absorb and secrete food material, store glycogen (Skingsley et al 2000). It is produced from a series of 55 and fat, and play a role in shell regeneration(Sumner glands used to aid locomotion of the animal over a variety 1965). In addition, snail products thus provide skin re- of terrestrial and aquatic terrain. Stimulation of mucus generation. Skin regeneration due to ageing, environ- secretion is based on sensation of mechano- and chemo- mental factors, such as UV light exposure, or injury is a

3 5 EP 3 124 030 A1 6 complex procedure which involves alterations in the re- proven beneficial in the regeneration of burnt skin (Badiu modeling process of the extracellular matrix (ECM), pro- et al 2008) and in the management of open wounds liferation, migration and survival of new cells, a task car- (Tsoutsos et al 2009). Furthermore, a recent study dem- ried out by dermal fibroblasts and keratinocytes (Ashcroft onstrated that the secretion from Cornu aspersum stim- et al 1995). Proliferation and migration of fibroblasts is 5 ulates fibroblast proliferation, rearrangement of the actin activated and ECM components such as elastin, collagen cytoskeleton and stimulates ECM assembly (Brieva et al and fibronectin are secreted to renew the elasticity and 2008). Additionally, a more recent study revealed the ef- consistency of the skin. In healthy skin, physiological re- fect of this secretion on proliferation, migration and sur- generative events include proliferation and migration of vival of keratinocytes and dermal fibroblasts in vitro (Ig- keratinocytes,which isa layer ofcells attached toa carpet 10 lesias-de la Cruz et al 2012). Therefore, the attention of of specialized extracellular matrix, the basal lamina. The many pharmaceutical companies has been directed, establishment of new adhesions cell-cell and cell-sub- through extensive research programs, on identifying nat- strate (basal lamina), includes interactions between pro- ural products that stimulate skin regeneration using the tein molecules such as E-cadherin (ECD), β-catenin, in- secretion of snails as a possible treatment of skin dis- tegrin, laminin (basal lamina), hemidesmosomes and in- 15 eases. termediate filaments of the cytoskeleton (cytokeratins). [0012] Until now, the most commonly used method in E-cadherin (ECD) is a transmembrane protein that me- snail extract acquisition consists of two steps, a first cen- diates intercellular adhesion through other intracellular trifugation of the snails in order to obtain the extract and anchor proteins such as β-catenin in adhesion junctions a further centrifugation of the snail extract as disclosed (Knudsen & Soler 2000). Hemidesmosomes anchor to 20 inpatent US 5538740.According to this method, the tech- the laminin in the basal lamina through specific integrins nique used to obtain the extract from the live gastropod belonging to the β1-subfamily for the cell-substrate junc- does not change the biological structure of the derived tions (Yamada et al 1995), while cell-cell adhesions product. Physical stimulus of the snail was induced by through hemidesmosomes include interactions with the centrifugation. Before the physical stimulation, snails ECM and intermediate filaments of the cytoskeleton (cy- 25 werefasted for 5days toensure the absence ofany toxins tokeratins). These interactions are important not only for present in the secreted fluid. Preferably, a force of about the integrity of the skin but also for signal transduction 2000 rpm was used for about 1 minute at 20°C. This leading to cell migration and proliferation, e.g. phospho- procedure was repeated twice, which has the drawback rylation of the Tyr kinase focal adhesion kinase (FAK) to possibly result in snail mortality. The snails, which were (Yamada et al 1995, Zouq et al 2009). Finally, in terms 30 still alive, were removed and the snail extract was ob- of survival signals, elevated levels of β-catenin have also tained, yet at the expense of snail mortality. Snail extract been associated with improved cell survival, as it is in- obtained in this way was further processed, on a second corporated to the transcription of genes related to cell step, by centrifugation at 5000 rpm for about 10 min. The proliferation (Fuchs et al 2005). resulting supernatant was the final extract acquired. 35 [0013] Said US patent yet deals with both Helix and Prior Art non-Helix forms which are suitable to obtain glandular secretions, indicating a positive effect in case of infec- [0011] The interest in skin regeneration properties of tions of the organ. It is stated that preferred numerousnatural substances obtainedeither from plants include a number of 21 species within the genus Helix. or animals including snails, has increased the last few 40 However, Helix aspersa (Cornu aspersum) is not men- years.Snails secrete largeamounts of mucoussubstanc- tioned at all therein, whereas it refers to centrifuging a esas a defensive response inorder to protect themselves live "Helix gastropod". from harmful conditions such as radiation, infections, me- chanical stress, increased salinity, temperature, sound Aim of the invention vibrations, hyperbaric pressure, and hypoxia (Dittbren- 45 ner et al 2009, Kuang et al 2002). Moreover, snails per- [0014] The aim of this invention is to propose an inno- form a relatively rapid reconstitution of their broken shell, vative method for acquisition and further processing of whereas they never suffer from skin infections or cancer, snail extract, wherein an attempt is made to develop an despite the exposure of their skin to damaging factors alternative method for both steps of the already used such as UV light. The main mechanism of UV induced 50 process of snail extract acquisition evoked above, on the photo-ageing is a marked increase in the production of one hand, in order to avoid both snail centrifugation (first reactive oxygen species (ROS) which causes photo-ox- step), which has the drawback to cause snail death, and idative damage and decreases cell migration and prolif- snail extract centrifugation (second step) during which eration.Interestingly, the secretionfrom Cornuaspersum potentialbioactive substances may remain in the residue, has been proven to have antioxidant activity(Brieva et 55 which is an additional drawback. An additional object of al 1999) and to induce skin regeneration after wound- the invention is thus to test the bioactive effect of specific healing impairment from acute radiodermatitis (Ledo et glandular extracts of Cornu aspersum. al 1999). In this regard, the secretion of some snails has [0015] A further object of the invention is to evaluate

4 7 EP 3 124 030 A1 8 an improved technique, thereby targeting the optimum cells, as well as the expression of protein-markers of cell one, that can be applied in a large scale, at industrial survival, cell-cell and cell-substrate adhesion in vitro, level, such as in cooperation with snail farms, wherein compared to said known centrifuged extracts. the effect ofthe bioactivesubstances in theobtained snail [0021] According to a main significant embodiment of extracts, on the other hand, on regenerative properties 5 the invention, a snail extract is derived from the proximal of the HaCaT human keratinocyte cell line is evaluated. part of the digestive tract of the snails, wherein the snail [0016] In the frame of this invention, an object thereof type Cornuaspersum is selected from thegastropod fam- is therefore the preparation of an extract from Cornu as- ily, wherein a fraction is extracted from said snail parts, persum that brings a solution to the abovementioned wherein a snail extract is derived from said fraction. problems of snail mortality and extract centrifugation no- 10 [0022] The invention further concerns the evaluation tably. In this respect, the dermatoprotective properties of of the effect of specific glandular extracts, including al- extracts of Cornu aspersum are investigated. When look- bumen gland of the reproductive system and proximal ing for a suitable substance therefor, such as for skin part of the digestive tract of the snails, on regenerative diseases or disorders, said proposed substance from the properties of the HaCaT human keratinocyte cell line. relevant group was tested. Another object of the invention 15 The effect of the specific extracts on the HaCaT cell line thus consists of the investigation of their regenerative is evaluated by estimating the proliferation rate, the mi- properties and the use thereof based upon the statement gration and wound healing potential, as well as the ex- that snails are a natural source with therapeutic proper- pression of protein-markers of cell survival β( -catenin), ties. cell-cell adhesion (E-cadherin) and cell-substrate adhe- 20 sion (integrin-β1). Furthermore, in order to evaluate the Summary of the invention potential protective or therapeutic role of bioactive sub- stances in snail extracts, the response of the HaCaT cell

[0017] Thus according to the invention, a method is line treated with snail extracts under H2O2 induced oxi- proposed for snail extract acquisition comprising at least dative stress is estimated. two steps essentially. The first step for obtaining the ex- 25 [0023] A second aspect of the invention thus consists tract consists of a non stressful snail extract acquisition, of the evaluation of the effect of specific glandular ex- wherein a batch of snails is selected, which are brought tracts, both albumen gland of the reproductive system to crawl on a set of glass surface means yielding a clear and proximal part of the digestive tract of the snails, on pedal mucus. It is followed by a further step consisting regenerative properties of the HaCaT human keratinoc- of drying and a subsequent resuspension step of the pro- 30 yte cell line. For the first time, the bioactive potential of duced snail extract. the glandular extract from the proximal part of the diges- [0018] In this respect, a method that is non stressful, tive tract of Cornu aspersum is identified at all levels of in contrast with snail centrifugation, was thus tested in this process. This extract appears to induce significantly order to obtain clear pedal mucus from snails crawling the proliferation rate, the migration and wound healing on surfaces, preferably made of inert material notably 35 potential, as well as the expression of all protein-markers glass. Then drying and a subsequent resuspension of tested. the snail extract is tested comparatively to extract cen- [0024] Among all extracts tested, only this specific trifugation, on both snail extracts obtained either by cen- glandular extract obtained by snails of all sampling sea- trifuged or crawling snails. sons including October with reproductive activity, Febru- [0019] Snail mucus obtained either by centrifugation 40 ary with hibernation, & May with activation, demonstrated or by snail crawling on glass plates, yet exhibit a signifi- a significant therapeutic role on cells under H 2O2 induced cant positive effect in the regenerative properties of the oxidative stress in vitro. Based on the annual life cycle cells in vitro. However, according to the results of the of Cornu aspersum and in order to evaluate the optimum tests performed according to the invention, further treat- sampling season, snail extracts were thus acquired at ment of the snail extract, that is dried versus centrifuged 45 said three sampling seasons: October as a period of re- snail extract increased significantly the proliferation rate, productive activity, February as a hibernation period, and the migration and wound healing potential, as well as the May as activity period. Noticeably, although this extract expression of protein-markers of cell survival, cell-cell obtained from hibernating snails did not appear to influ- and cell-substrate adhesion. This is presently attributed ence cell proliferation or migration, it was effective after to the presence of growth factors that are more effective 50 strong chemical oxidative stress as a therapeutic factor. -in terms of composition or concentration- and/or antimi- This initial paradox observation is presently assumed to crobial substances in dried snail mucus, compared to the be attributed to the physiological role of this gland during supernatant of centrifuged snail mucus. hibernation as it could maintain an antioxidant role in [0020] According to this invention, said further step is snails with low metabolic rates. In addition, the therapeu- thus remarkable in that it consists of drying with a sub- 55 tic role of this extract obtained from active snails is as- sequent re-suspensionstep of theproduced snail extract. sumed to be due to the presence of regenerative factors Dried snail extracts increase significantly the proliferation in its glandular secretions, as regeneration of epithelial rate, the migration and wound healing potential of HaCaT cells is mandatory.

5 9 EP 3 124 030 A1 10

[0025] According to an additional embodiment of the [0034] The invention also relates to an animal extract invention, the bioactive potential and therapeutic role of obtained according to a method as set out above for use this glandular extract is yet apparent in extremely low in protecting said organ of a mammal, i.e. for preventing concentrations. said organ disorder therein [0026] It is thus made clear by the developments of the 5 [0035] The point is that the extraction method of said invention that regenerative properties of the human ke- extract from Cornu aspersum proposed here is not ratinocyte cell line HaCaT appear to be positively influ- known, since the related animal Cornu aspersum is used enced by factors in (A) dried snail extracts and as set out for numerous purposes, including others than those men- above, on the one hand, but also (B) in snail extracts of tioned here. The uniqueness of this invention refers to the proximal part of the digestive tract and gland, on the 10 the use of the DG extract, which is not disclosed any- other hand, as set out hereinafter. where else until now, as a therapeutic means after oxi- As skin ageing, skin exposure to various damaging en- dative stress. vironmental factors, or even skin burns could result in The scope of the solution which is aimed for thus lies in oxidative stress of human epithelial cells, this glandular the deliberate and targeted selection of some indications extract is further proposed according to the invention to 15 thereof. There are references yet to numerous products be further used not only in cosmetology but also as a including these, but there is no mention of the said ex- pharmaceutical product. tract, which is clearly responsible for the activity in the [0027] According to an alternative embodiment of the specific animal Cornu aspersum against said organ dis- invention, said further step may consist of a centrifugation orders, and with the additional advantage that it is present of the snail extract. 20 herein in considerable amounts, as a significant constit- [0028] According to a limited embodiment of the inven- uent thereof. tion, in a first instance, specific animal parts ofCornu [0036] According to an additional embodiment of the aspersum are used, especially those less or not at all invention, said animal extract is used for treating a said affected by the season. The result is that harvesting these organ disorder in a mammal, in particular one resulting offers the advantage of providing availability year-around 25 from chemical causes or caused by infections, in partic- thanks to appropriate storage conditions, and it also has ular by bacteria or viruses, as well as physical or immu- been experimentally established and implemented in the nologic causes. meaning that the extract can be stored first and used [0037] Additional features and particularities of the later without losing its properties. method according to the invention are defined in the ad- [0029] According to a specific embodiment of the in- 30 ditional sub-claims. Further details are incorporated in vention, the weight of the extract obtained ranges from the following description of some preferred exemplary 1% to 10%, of live snail weight. embodiments of the method according to the invention, [0030] According to a yet more advantageous embod- which is illustrated based on the attached drawings. iment of the invention, a certain quantity of said extract, in particular about 1% of snail live weight, is obtained 35 Brief description of the drawings from snails which have been allowed to crawl for two hours on glass plates. In this case, subsequent mortality [0038] observed four days after snails’ use was zero. [0031] A certain quantity of said extract, in particular Fig. 1 shows a schematic representation of experi- about 10% of snail live weight, is obtained from snails 40 mental design for the method according to the inven- which have been subjected to two subsequent centrifu- tion. gations at 2000 rpm for one minute. In this case subse- Fig. 2 shows a graphical representation of the pro- quent mortality observed four days after snails’ use liferation rate of HaCaT cell line under the effect of ranged from 33% to 63%. snail extracts obtained from live adult snails during [0032] Further increase of crawling time or centrifuga- 45 their reproductive period (GB). tion repeats would lead to an increase in the quantity of Fig. 3 shows likewise an analogue representation the snail extracts obtained, in case of commercial exploi- thereof during hibernation (A). tation. Fig. 4 shows a graphical representation of the per- [0033] In contrast, there is no description in said US- centage of ’Wound’ healing (mean 6 standard devi- document of the use of an extract from saidCornu as- 50 ation) potential of the human keratinocyte cell line persum containing said extract for protecting the said or- HaCaT after treatment with snail extracts obtained gan function, that would indicate any method for prevent- from live adult snails during the reproductive and ac- ing any of said organ disorder, even less for treating tivity period. same, and certainly not a natural animal with the use of Fig. 5 shows a graphical representation of the rela- said specific extract or any extract exactly containing this 55 tive expression of E-cadherin,β -catenin and in- component, being referred to here to the extract of the tegrin-β1 in the human keratinocytes cell line HaCaT digestive tract, whereas said US patent contains no ref- after the effect of extract from (A) centrifuged snails erence to such an extract. from the 1st sampling period further being treated

6 11 EP 3 124 030 A1 12

by centrifugation (CGB CF) or drying (CGB dry), and expression of protein-markers of cell survival ( β-catenin), (B) from crawling snails from the 1st sampling period cell-cell adhesion (E-cadherin) and cell-substrate adhe- further being treated by centrifugation (WGB CF) or sion (integrin-β1). Furthermore, in order to evaluate the drying (WGB dry). potential protective or therapeutic role of bioactive sub- Likewise Fig. 6 shows a similar representation there- 5 stances in snail extracts, the response of the HaCaT cell

of, but after the effect of extract from centrifuged (CA line treated with snail extracts under H2O2 induced oxi- dry) and crawling (WA dry) snails from the 2nd sam- dative stress was estimated. pling period further being treated by drying. [0040] An optimal method of snail extracts acquisition Again Fig. 7 shows an analogue representation from Cornu aspersum and investigation of their regener- thereof but after the effect of mucus from centrifuged 10 ative properties is tested. (CE dry) and crawling (WE dry) snails from the 3rd [0041] Regenerative properties of the human keratino- sampling period further being treated by drying. cyte cell line HaCaT appear to be positively influenced Fig. 8 similarly shows a graphical representation of by factors in (A) dried snail extract and(B) extracts of the proliferation rate of HaCaT cell line, but under the proximal parts of the digestive tract from Cornu as- the effect of the albumen (AL) and digestive tract and 15 persum snails. gland (DG) extract obtained from snails during their [0042] Said known method of snail extract acquisition, reproductive period. consists of two steps, including a first centrifugation of Fig. 9 shows an analogue representation thereof as the snails in order to obtain the extract and a further cen- in Fig. 8, but under the effect of the DG extract -di- trifugation of the snail extract. The supernatant of the luted by a factor 1/10- obtained from snails during 20 second centrifugation is used in cosmetology. The effect (A) hibernation (DGA), (B) activity (DGE) of the bioactive substances in the obtained snail extracts Fig. 10 shows a graphical representation of the per- on regenerative properties of the HaCaT human kerati- centage of ’wound healing’ -mean 6 standard devi- nocyte cell line is evaluated. ation- potential of the human keratinocyte cell line [0043] The results obtained with this invention reveal HaCaT after treatment with snail extracts obtained 25 that both processes of snail extract acquisition being snail from the albumen (AL) and digestive tract and gland centrifugation vs. snail crawling on glass plates, yield ex- (DG) of (A) the reproductive period, and (B) their tracts that exhibit a positive effect in the regenerative period of activity. properties of the cells in vitro. It’s the further treatment Fig. 11 shows a relative expression of E-cadherin, of both snail extracts that differentiate their regenerative β-catenin and integrin- β1in the human keratinocytes 30 effect. Dried snail extract according to the invention in- cell line HaCaT after the effect of DG extract from A. crease significantly the proliferation rate, the wound heal- the 1st, 2nd and 3rd sampling periods. ing potential of the cells, as well as the expression of all Fig. 12 shows the therapeutic role -mean 6 standard protein-markers tested, compared to the known centri- deviation- of DG extracts on the human keratinocyte fuged extract. 35 cell line HaCaT after H 2O2 induced oxidative stress, [0044] For the first time, the bioactive potential of the wherein treatments after oxidative stress were per- glandular extract from the proximal part of the digestive formed with 1/10 diluted DG extracts derived from tract of Cornu aspersum was identified at all levels of this snails during said three sampling periods. development. Indeed, this extract appears to induce sig- nificantly the proliferation rate, the migration and wound Description 40 healing potential, as well as the expression of all protein- markers tested. Among all extracts tested, only this spe- [0039] With this invention, an improved method is de- cific glandular extract of the invention demonstrated a veloped for a snail extract acquisition process, respective significant therapeutic role on cells under H 2O2 induced the most commonly used method until now. In this re- oxidative stress in vitro. The bioactive potential and ther- spect, a non stressful method was tested in order to ob- 45 apeutic role of this glandular extract was yet apparent in tain clear pedal mucus from snails crawling on glass sur- extremely low concentrations. As skin ageing, skin ex- faces in comparison to snail centrifugation which may posure to various damaging environmental factors, or result in snail mortality. A drying and subsequent re-sus- even skin burns could result in oxidative stress of human pension of the snail extract, comparatively to extract cen- epithelial cells, this glandular extract could be further trifugation, was tested on both snail extracts obtained by 50 used not only in cosmetology but also as a pharmaceu- crawling snails, but also centrifuged ones. The effect of tical product. specific glandular extracts namely, albumen gland of the [0045] As to the snail extract acquisition, the secretion reproductive system and proximal part of the digestive of the mollusk Cornu aspersum is a natural product that tract of the snails on regenerative properties of the Ha- bears regenerative properties when applied topically on CaT human keratinocyte cell line was further evaluated. 55 skin. An initial step in this experimental procedure was The effect of the specific extracts on the HaCaT cell line to apply the extraction method used to date as presented was evaluated by estimating the proliferation rate, the in said US - 5538740 on adult snails. migration and wound healing potential, as well as the [0046] Fig. 1 shows a schematic representation of ex-

7 13 EP 3 124 030 A1 14 perimental design, wherein C stands for centrifuged as to cell proliferation (a), HaCaT cells (CLS order snails, W for ’walking’ snails, cf for centrifuged snail ex- No. 300493) are a human keratinocyte cell line used tracts, dry for dried snail extract. The experimental pro- in scientific research. They are utilized for their high cedure was repeated on three sampling seasons: GB for capacity to differentiate and proliferate in vitro. Cells October, period of reproductive activity, A for February- 5 were grown in T-75 flasks using Dulbecco’s modified hibernation, and resp. E for May, activity period. Eagle’s medium containing 10% (v/v) FBS (fetal bo- [0047] A non stressful method of snail extract acquisi- vine serum) and 5% penicillin/streptomycin (1/1). tion was tested. According to this method, snails were Cells were incubated at 37°C in an atmosphere con- allowed to crawl or ’walk’ on a set of inert plates under taining 5% CO2. HaCaT cells cultured on 6-well or optimum conditions, being defined as e.g. high humidity 10 12-well Petri dishes were treated with different con- and 20°C on the e.g. glass plates for about 2 hours. Clear centrations of snail extracts at 0, 25, 50 and 100 pedal mucus was carefully removed from said glass mg/ml during several time points: 2, 4, 6 and 8 days. plates and placed into glass petri dishes. Further treat- In order to estimate cell proliferation and cell viability, ment of the snail extracts obtained either by said snail culture samples were transferred to a Neubauer centrifugation C, or snail crawling W-’walking’ consists 15 chamber counter and were examined under a light of (a) centrifugation cf and (b) drying and subsequent microscope using the trypan blue exclusion method. resuspension of said dried snail extract dry shown in Fig. 1. Specifically, cf extract was obtained after two succes- [0051] As to cell migration (b) brings wound healing, sive centrifugations at 5000 rpm for 10 minutes. The final HaCaT cells cultured on 6-well Petri dishes were treated supernatant was then separated and filtered through a 20 with said concentrations of snail extracts at 0, 25, 50 and microporous filter having a pore size of 0,22m m. Dry 100 mg/ml during different several time points of 2, 4, 6 extract was obtained by placing the snail extracts into and 8 days. In vitro wound-healing assays were initially glass petri dishes into a chamber at 40°C for 24h. The performed as previously described (Brieva et al 2008). dried snail extract was further resuspended in aqueous Briefly, HaCaT were seeded in T6-well culture dishes at solution, typically with pH 7,4 and filtered through a mi- 25 a density of 3·10 5 cells per well. Confluent cell monolay- croporous filter having a pore size of 0,22 mm. ers were then gently scratched with a pipette tip across [0048] Furthermore two specific glandular extracts the entire diameter of the dish. Next, cells were rinsed were tested for their effect on the HaCaT human kerati- with medium to remove all cellular debris, and treatment nocyte cell line. For this purpose, tissue samples were with snail extracts was applied to three wells, whereas isolated from: the albumen gland AL with the embedded 30 the rest of the wells served as control. Cultures were FPSC from the reproductive system, and the proximal observed immediately after wounding and 24 h and 48 part of the digestive tract and gland DG in a number of h later, and phase-contrast pictures were taken of the snails wherein n=30. Tissue samples were homogenized wounded area using a high-resolution microscopic cam- and centrifuged at 5000 rpms for 10 min. The supernatant era connected to the microscope. Quantification of mi- of these samples were used as bioactive substances in 35 grated cells was measured in the healed area. HaCaT cell cultures visible in Fig. 1. All samples of snail [0052] The effect of snail extracts on wound healing extracts were stored at -25° C until use. capability and cell migration potential of HaCaT cells [0049] Based on the annual life cycle of Cornu asper- could not be comparatively studied, due to the fact that sum and in order to evaluate the optimum sampling sea- cell migration was rapid within 24 h in all treatments, with son, snail extracts were acquired at three sampling sea- 40 or without snail extracts. Therefore, it was tried to in- sons: (a) October GB - period of reproductive activity, (b) crease the wound and the time of healing. Specifically, February A-hibernation, and (c) May E-activity period. cell monolayers in p60 Petri dishes were gently scratched The toxicity of each sample was assayed by the trypan usinga cellscraper with diameter 1,5 cmacross theentire blue exclusion method and the highest effect/toxicity ratio diameter of the dish. According to the results previously was defined in order to employ the specific concentration 45 obtained from cell proliferation, the effect of specific con- for the in vitro assays on HaCaT cell line. centrations was tested at a time period of 8 days. In order [0050] With regard to the materials and methods in- to avoid the accumulation of toxic substances in the cell volved, the evaluation of the effect of specific snail ex- medium during this prolonged time period, every two tracts on the regenerative properties of HaCaT cells, and days the medium was replaced with new, whereas snail the determination of the optimum extract in terms of tis- 50 extracts were also renewed. Quantification of migrated sue specific, time and dosage dependent treatment, was cells was measured in the healed area by high-resolution tested in four levels: (a) the induction of cell proliferation ; camera connected to the stereoscope (Zeiss 2000-C) (b) the stimulation of cell migration ;(c) the protection using the AxioVision Rel. 4.7 software. and/or healing role under oxidative stress and(d) the [0053] As to protective/healing role under oxidative increase of the expression of ECD (cell-cell adhesion 55 stress (c), according to the oxidative stress model (Liu molecule), β-catenin (survival molecule),β 1-integrin et al 2012), treatment with different concentrations of (cell-substrate adhesion molecule). Each level is set out H2O2 ranging from 0,1 to 1 mM for different time periods in detail below: of 0, 0,5, 1, 2, 4 hours on HaCaT cells was tested in order

8 15 EP 3 124 030 A1 16 to establish the IC50 for H 2O2, that is treatment (concen- ylamide slab gels and transferred electrophoretically on- tration and time of effect) which results in 50% mortality to nitrocellulose membranes (0.45 mm, Schleicher and in HaCaT cells. Cell viability was documented by the MTT Schuell, Keene N. H. 03431, USA). Non-specific binding assay (Merlin et al 1992). Cells were treated with the sites on the membranes were blocked with 5% (w/v) non- 5 abovementioned concentrations of H2O2 and 0,5 ng/ml fat milk in TBST (Tris Buffered Saline-Twin 20) (20 mM of MTT solution was added. Cells were then incubated Tris-HCl, pH 7,5, 137 mM NaCl, 0,1% (v/v) Tween 20) at 37°C for 2-3 h and the resulting formazan crystals were for 30 min at room temperature. Subsequently, the mem- dissolved by the addition of Dimethyl sulfoxide (DMSO). branes were incubated overnight with the appropriate pri- Absorbance was measured at 540 nm. IC50 was estab- mary antibodies. Antibodies used were as follows: Anti- 10 lished with 0,75 mM H2O2 for 2 hours. In order to deter- E-cadherin, clone DE CMA-1, Anti-β-Catenin and Anti- mine whether snail extracts perform a protective and/or Integrin B1 (Merck). After washing in TBST (3x5 min), a healing effect on cells that undergo oxidative stress, the blots were incubated with horseradish peroxidase- two different approaches were applied: at first the pro- linked secondary antibodies, polyclonal goat anti-mouse tective role of snail extract including, treatment with the immunoglobulins and polyclonal goat anti-rabbit immu- optimum snail extract respective concentration and time 15 noglobulins (DAKO, High Wycombe, Buckinghamshire, of effect; Induction of oxidative stress for IC50 (0,75 mM UK), washed again in TBST (3x5min), and the bands

H2O2 for 2 hours) and Documentation of cell viability by were detected by enhanced chemiluminescence (Cell the MTT assay, resp. Signaling, Beverly, MA, USA) with exposure to Fuji Med- [0054] The other approach about the healing role of ical X-ray films. Films were quantified by laser-scanning snail extract includes induction of oxidative stress for20 densitometry (GelPro Analyzer Software, Media Cyber- IC50 (0,75mM H 2O2 for 2 hours); removal of cell medium, netics) and blots were plotted using SigmaPlot 10.0. cell washing, and replacement with new cell medium; The results of the tests set out above are as follows: treatment with the optimum snail extract (concentration firstly, improving the snail extract processing: the dried and time of effect); and documentation of cell viability by snail extract increased regenerative properties of HaCaT the MTT assay. 25 cells versus the centrifuged one. [0055] As to expression of E-cadherin (cell-cell adhe- [0058] However, according to the results of this inven- sion), β-catenin (survival molecule) and integrin- β1 (cell- tion, snail extracts obtained either by centrifugation (C) substrate adhesion) (d): during the first sampling season or by snail crawling on glass plates (W), both exhibited in October the expression of the particular protein-indi- a significant positive effect at the regenerative properties cators in the cells after the effect of all extract samples 30 of the cells in vitro, it was the processing of the snail was studied. All the extracts were tested irrespective of extract that is dried instead of the centrifuged snail ex- the way they were obtained either C, W, AL and DG and tract, that further increased significantly: the proliferation further treated cf and dry in all concentrations at 25, 50 rate a shown in Fig. 2, the migration and wound healing and 100 mgr/ml and time periods 2, 4, 6 and 8 days used. potential as shown in Fig. 4, as well as the expression of Judging from the cell proliferation results of the first sam- 35 protein-markers of cell survival, cell-cell and cell-sub- pling season, the extracts obtained from snails during strate adhesion as shown in Fig. 5 of HaCaT cells. the two following sampling seasons were tested only at [0059] Indeed, Fig. 2 shows the proliferation rate of the concentrations and time periods that gave the opti- HaCaT cell line under the effect of snail extracts obtained mum effect. Specifically, the effect of dried extract dry at from live adult snails during their reproductive period 50 and 100 mgr/ml after 6 and 8 days and the effect of 40 (GB). CGB and WGB refer to centrifuged and ’walking’ digestive tract and gland DG at 12,5 and 25 mgr/ml after snails, respectively; CF to centrifuged snail extract, dry 2 and 4 days, were tested. to dried snail extract. Numbers represent treatment con- [0056] Cells were homogenized in cold lysis buffer (20 centrations in mgr/ml. mM β-glycerophosphate, 50 mM NaF, 2mM EDTA, 20 [0060] The proliferation rate of HaCaT cells appeared mM Hepes ((4-(2-hydroxyethyl)-1-piperazineethanesul- 45 to be positively influenced under the effect of snail ex- fonic acid), 0,2 mM Na 3VO4, 10 mM benzamidine, pH 7, tracts obtained from live adult snails during the first sam- containing 200 mM leupeptin, 10 mM trans-epoxy succi- pling season, reproductive period-GB. In general, the nyl-L-leucylamido-(4-guanidino) butane, 5 mM DTT proliferation rate increased two days after treatment, fol- (dithiothreitol), 300 mM phenyl methyl sulfonyl fluoride lowing a decrease close to the proliferation rate of the (PMSF) and 1% v/v Triton X-100), and extracted on ice 50 control group, while any statistically significant effect of for 30 min. Samples were centrifuged (10,000 g, 10 min, treatment was observed after 6 and 8 days (CGB CF 25, 4°C) and the supernatants were boiled with 0,33 volumes CGB dry 100, WGB dry 50 & 100). Noticeably, the same of SDS/PAGE sample buffer [330 mM Tris-HCl, 13% v/v sample of snail extract, either CGB visible in Fig. 2-A,C glycerol, 133 mM DTT, 10% w/v SDS (Sodium Dodecyl or WGB in Fig. 2-B,D, induced significantly cell prolifer- Sulfate), 0,2% w/v bromophenol blue]. Protein concen- 55 ation when dried, while this positive influence was not tration was determined using the BioRad protein assay. observed when the snail extract was centrifuged. [0057] Equivalent amounts of protein (100 mg) were [0061] On the contrary, as shown in Fig. 3, all snail separated on 10% (w/v) acrylamide, 0,275% (w/v) bisacr- extracts obtained during the second sampling season in

9 17 EP 3 124 030 A1 18

February-A from hibernating snails, demonstrated no markers of cell survival, cell-cell and cell-substrate ad- significant positive effect on cell proliferation rate. Indeed hesion of HaCaT cells were in line with cell proliferation Fig. 3shows theproliferation rate of HaCaT cell line under and wound healing results. Extracts from centrifuged the effect of snail extracts obtained from live adult snails snails being further treated with centrifugation (CGB cf) during hibernation (A). CA and WA refer to centrifuged 5 from said 1st sampling period, had no effect on the ex- and ’walking’ snails, respectively. CF stands for centri- pression of E-cadherin at all concentrations tested as fuged snail extract, resp. dry for dried snail extract. Num- shown in Fig. 5-A. On the other hand, the effect of the bers represent treatment concentrations (mgr/ml). same extract further treated by drying and resuspension [0062] In order to corroborate these results, snail ex- (CGB dry) resulted in a 2-fold increase in the expression tracts of the second sampling season were used in treat- 10 levels of E-cadherin at the concentration of 100 mgr/ml ments testing levels of protein expression (E-cadherin, on the 8th day of the effect compared to the control. The β-catenin, integrin-β1), while they were not used as po- same results were obtained regarding β-catenin. On the tential bioactive substances in estimating ’wound’ heal- other hand, the expression of integrin-β1 remained un- ing, or their healing/protective role in induced oxidative changed under the effect of all bioactive substances. stress experiments in HaCaT cells. 15 [0068] Extracts from crawling snails being further treat- [0063] The results of cell proliferation obtained from ed by centrifugation (WGB cf), had no effect in the ex- extracts from active snails of the third sampling season pression levels of E-cadherin at all concentrations as May-E corroborated the results obtained so far. Dried shown in Fig. 5-B. On the other hand, the effect of the snail extracts again increased significantly cell prolifera- same extract being further treated by drying and resus- tion. Specifically, snail extract from centrifuged snails 20 pension (WGB dry), resulted in a 1,7-fold increase of E- (CEdry) increased slightly cell proliferation rate (10%) at cadherin expression levels compared to the control, at 50 mg/ml concentration, while it had no significant effect the concentrations of 50 and 100 mgr/ml on the 6th and at the highest concentration. Snail extract from crawling 8th day of the effect. WGB dry effect resulted also to a snails (WEdry), also had a high effect on proliferation statistically significant increase (1,4-fold compared to the rate (50%) at the concentration of 100 mgr/ml. All signif- 25 control) in the expression levels of β-catenin at the con- icant effects were observed 8 days after treatment. centration of 25 mgr/ml on the 8th day of the effect. In- [0064] Fig. 4 shows the percentage of Wound healing tegrin-β1 levels increased 2 fold compared to the control, (mean6 standard deviation) potential of the human ke- only under the effect of WGB dry 100 on the 8th day of ratinocyte cell line HaCaT after treatment with snail ex- effect. tracts obtained from live adult snails during A. the repro- 30 [0069] Fig. 6 shows the relative expression of E-cad- ductive period-October -CGB and WGB refer to centri- herin, β-catenin and integrin-β1 in the human keratinoc- fuged and walking snails, respectively- andB. during ytes cell line HaCaT after the effect of extract from cen- their activity period-May -CE and WE refer to centrifuged trifuged (CA dry) and crawling (WA dry) snails from the and ’walking’ snails, respectively- CF stands for centri- 2nd sampling period in February-A further being treated fuged snail extract, and dry for dried snail extract resp. 35 by drying. Numbers represent treatment concentrations Numbers represent treatment concentrations mgr/ml. in mgr/ml. [0065] Cell migration of HaCaT cell line was signifi- [0070] Concerning the effect of extracts obtained from cantly increased after treatment with snail extracts that snails of the 2nd sampling period in February A, only the were mainly dried. Specifically, wound healing potential optimum time and dosage dependent treatments based of this cell line was significantly higher ranging from40 on the results from the 1 st sampling in October-GB were 34,7% to 48,3%, compared to the control (28,8% wound applied: concentrations of 50 and 100 mgr/ml of extract healing) when treated mainly with dried snail extracts ob- further treated by drying after 6 and 8 days of effect. No tained from snails during their reproductive period in Oc- change was observed in the expression levels of E-cad- tober-GB as shown in Fig. 4-A. Similar results were ob- herin, β-catenin and integrin-β1 at all treatments tested. tained from dried extracts from active snails of the third 45 Specifically, under the effect of both used concentrations sampling season in May- E shown in Fig.4-B with a higher of 50 and 100 mgr/ml, no particular changes were ob- ’wound’ healing potential of CEdry50 (42,8%) and served in the expression of the 3 examined proteins on WEdry100 (34,5%) compared to the control (28,8%). the 6th and 8th day of the effect. [0066] Fig. 5 shows the relative expression of E-cad- [0071] Fig. 7 shows the relative expression of E-cad- herin, β-catenin and integrin-β1 in the human keratinoc- 50 herin, β-catenin and integrin-β1 in the human keratinoc- ytes cell line HaCaT after the effect of extract from A. ytes cell line HaCaT after the effect of mucus from cen- centrifuged snails from said 1st sampling period further trifuged (CE dry) and crawling (WE dry) snails from said being treated by centrifugation (CGB CF) or drying (CGB 3rd sampling period in May further being treated by dry- dry), and B. from crawling snails from said 1st sampling ing. Numbers represent treatment concentrations in period further being treated by centrifugation (WGB CF) 55 mgr/ml. or drying (WGB dry). Numbers represent treatment con- [0072] Concerning the effect of extracts obtained from centrations in mgr/ml. snails of the 3 rd sampling period in May-E when the snails [0067] The results regarding the expression of protein- were active, increased expression levels of the examined

10 19 EP 3 124 030 A1 20 proteins mostlyon the 8 thday of theeffect were observed. tions in mgr/ml. Specifically, the CE dry effect caused an increase in the Following the latter result, only diluted (1/10) DG extracts expression levels of E-cadherin and β-catenin of 1,7-fold were tested from snails during the two following sampling compared to the control in both examined concentrations periods DGA-February-hibernation and DGE-May-activ- of 50 and 100 mgr/ml. Integrin-β1 levels increased 1,8- 5 ity and for all other tests thereafter performed (wound fold compared to the control at the concentration of 50 healing, protein expression, oxidative stress). Addition- mgr/ml on the 8 th day of the effect. WE dry effect resulted ally a more diluted treatment (12,5 mgr/ml) was tested. in increased β-catenin and integrin-β1 levels of 1,5 and The DGA extract influenced negatively the proliferation 1,7-fold respectively compared to the control at the con- rate of HaCaT cellsas shown in Fig. 9-A. On the contrary, centration of 100 mgr/ml on the 8 th day of the effect. The 10 the DGE extract demonstrated a positive effect on cell same concentration of the extract WE dry, resulted in a proliferation rate 4 days after treatment in both concen- 1,5-fold increase in the expression levels of E-cadherin trations of 12,5 and 25 mgr/ml of the initial diluted extract onthe 8 thday of the effect.The concentration of 50 mgr/ml used as shown in Fig. 9-B. caused a 1,3-fold increase of the particular protein com- [0077] Fig. 10 shows the percentage of wound healing pared to the control levels, also on the 8 th dayof the effect. 15 (mean6 standard deviation) potential of the human ke- [0073] The extract from the proximal part of the diges- ratinocyte cell line HaCaT after treatment with snail ex- tive tract and gland increases the regenerative properties tracts obtained from the albumen (AL) and digestive tract of HaCaT cells and plays a therapeutic role under strong and gland DG of A. the reproductive period in October, chemical oxidative stress. For the first time the bioactive and B. their period of activity in May. Numbers represent ability of the glandular extract from the proximal part of 20 treatment concentrations in mgr/ml. the digestive tract and gland DG of Cornu aspersum was [0078] Considering the wound healing capability of Ha- identified in all four levels of thisin vitro process. This CaT cells, a positive effect was evident on the cells after extract appeared to induce significantly the proliferation treatment with the DG extract, while no positive effect rate, the migration and wound healing potential, as well was revealed after treatment with theAL extract both as the expression of protein-markers of cell survival, cell- 25 obtained from snails during the reproductive season as cell and cell-substrate adhesion on HaCaT cells. Most shown in Fig. 10-A. Moreover, the AL extract when com- importantly, among all the extracts tested, only this one bined to theDG extract (AL50/DG25) inhibited said demonstrated a therapeutic role under induced oxidative wound healing effect of the latter extract (DG25) on Ha- stress against H2O2. CaT cells as shown in Fig. 10-A. Therefore, the AL extract [0074] Fig. 8 shows the proliferation rate of HaCaT cell 30 was not used as a bioactive substance during the two line under the effect of the albumen AL and digestive following sampling seasons of February and May. In line tract and gland (DG) extract obtained from snails during with the previous results a positive effect was also dem- their reproductive period. Numbers represent treatment onstrated by the DG extract when obtained during the concentrations in mgr/ml. third sampling season (DGE) at a smaller concentration [0075] Regarding proliferation rate, an initial treatment 35 12,5 mgr/ml as shown in Fig. 10-B. with specific snail extracts from the albumen AL and the [0079] Fig. 11 shows the relative expression of E-cad- proximal part of the digestive tract and gland (DG) dem- herin, β-catenin and integrin-β1 in the human keratinoc- onstrated a negative effect on cell proliferation rate as ytes cell line HaCaT after the effect of DG extract from shown in Fig. 8-A,B. Due to the fact that this experimental A. the 1st sampling period in October-DGB. the 2nd one procedure was tested for the first time, and given that the 40 in February-DGA, and C. the 3 rd one in May- DGE. Num- albumen gland provides nutrients to the fertilized ova, bers represent treatment concentrations in mgr/ml. while the digestive gland has been proposed to play a [0080] Considering protein expression levels, DG ex- fundamental role in shell regeneration, lower concentra- tract from snails of said first sampling period of reproduc- tions of these snail extracts were tested. Thus, these spe- tive activity in October applied on HaCaT cells increased cific extracts were diluted by a factor 1/10 and were fur- 45 the expression levels of E-cadherin and β-catenin (1,5 ther tested as shown in Fig. 8-C,D. TheAL extract and 1,8-fold compared to the control) two days after treat- showed a positive effect 2 and 6 days after treatment, ment at the concentration of 25 mgr/ml. The expression although it was not statistically significant as shown in level of integrin-β1 increased 1,4-fold compared to the Fig. 8-C. On the other hand, the DG extract resulted in control four days after treatment at the same concentra- a statistically significant increase in cell proliferation rate 50 tion. The concentration of 12,5 mgr/ml had no effect on two days after treatment as shown in Fig. 8-D. Moreover, the expression levels of the examined proteins as shown the combined effect (1/1) of these two glandular extracts in Fig. 11-A. AUDG did not appear to influence positively cell prolifer- [0081] The effect of the DG extract from snails of said ation rate. second sampling period of hibernation-DGA in February, [0076] Fig. 9 shows the proliferation rate of HaCaT cell 55 had no effect on the expression levels of E-cadherin, β- line under the effect of the DG extract diluted by a factor catenin and integrin- β1. Specifically, both concentrations 1/10 obtained from snails during A. hibernation DGA, B. of 12,5 and 25 mgr/ml had no effect on the expression activity DGE. Numbers represent treatment concentra- levels of the examined proteins on the 2 nd and 4th day of

11 21 EP 3 124 030 A1 22 the effect as shown in Fig. 11-B. sequent resuspension of the snail extract, was tested on [0082] Contrarily to the above obtained results, the ef- both snail extracts obtained either by centrifuged or fect of DG extract from active snails of said third sampling crawlingsnails. Dried snailextracts increased significant- period of activity period-DGE in May resulted in a statis- ly the proliferation rate, the migration and wound healing tically significant increase of the expression levels of the 5 potential of HaCaT cells, as well as the expression of examined proteins mostly four days after treatment at protein-markers of cell survival, cell-cell and cell-sub- both concentrations. Moreover, two days after treatment, strate adhesion in vitro, compared to centrifuged ex- the expression levels of E-cadherin and β-catenin were tracts. According to these results, although snail mucus statistically significantly increased. Specifically, E-cad- obtained either by centrifugation or by snail crawling on herin expression levels were increased 1,4-fold com- 10 glass plates, exhibited a significant positive effect in the pared to the control levels on the 2 nd day of effect at the regenerative properties of the cells in vitro, further treat- concentration of 25 mgr/ml and on the 2 nd and 4th day of ment of the snail extract, that is dried versus centrifuged effect at both concentrations used of 12,5 and 25 mgr/ml. snail extract increased significantly the proliferation rate, β-catenin exhibited a similar pattern of expression under the migration and wound healing potential, as well as the the same effects and the same time course, with its levels 15 expression of protein-markers of cell survival, cell-cell being 2-fold increased compared to the control. On the and cell-substrate adhesion. This is presently attributed contrary, integrin- β1 levels increased by 3-fold compared to the presence of more effective growth factors in terms to control levels only on the 4th day of the effect at both of composition or concentration and/or antimicrobial sub- concentrations as shown in Fig. 11-C. stances in dried snail mucus, compared to the superna- [0083] Fig. 12 shows the therapeutic role (mean620 tant of centrifuged snail mucus. standard deviation) of DG extracts on the human kerat- [0087] A second part of the invention concerns the inocyte cell line HaCaT after 2 HO2 induced oxidative evaluation of the effect of specific glandular extracts al- stress 0,75 mM H2O2 for 2h. Treatments after oxidative bumen gland of the reproductive system, proximal part stress were performed with 1/10 diluted DG extracts de- of the digestive tract of the snails on regenerative prop- rived from snails during the three sampling periods in 25 erties of the HaCaT human keratinocyte cell line. For the DG25-October, DGA25-February, DGE25-May. first time, the bioactive potential of the glandular extract [0084] For the first time, the extract of the proximal part from the proximal part of the digestive tract of Cornu as- of the digestive tract and gland has been recognized as persum was identified at all levels of this process. This a potential therapeutic factor for HaCaT cells under H 2O2 extract appeared to induce significantly the proliferation induced oxidative stress. Specifically, extracts obtained 30 rate, the migration and wound healing potential, as well from snails during all sampling seasons DG in October- as the expression of all protein-markers tested. Among reproductive activity, DGA in February-hibernation, DGE all extracts tested, only this specific glandular extract ob- in May-activation increased significantly cell viability after tained by snails of all sampling seasons, i.e.October-re- H2O2 induced oxidative stress at the rate of 0,75 mM for productive activity, February-hibernation, May-activa- 2h. The capability of HaCaT cells to overcome this strong 35 tion, demonstrated a significant therapeutic role on cells oxidative stress, which led to IC50, i.e. with 50% cell mor- under H2O2 induced oxidative stress in vitro. Noticeably, tality, was apparent after the up to 30-fold increase of although this extract obtained from hibernating snails did cell viability and proliferation two days after treatment not appear to influence cell proliferation or migration, it with the DG extracts as shown in Fig. 12. was effective after strong chemical oxidative stress as a [0085] The experimental results above yield the follow- 40 therapeutic factor. This initial paradox observation is ing considerations: regenerative properties of the human presently attributed to the physiological role of this gland keratinocyte cell line HaCaT appear to be positively in- during hibernation as it could maintain an antioxidant role fluenced by factors mainly in dried snail extracts, on the in snails with low metabolic rates. In addition, the thera- one hand, and in snail extracts of the proximal part of the peutic role of this extract obtained from active snails is digestive tract and gland, on the other hand. 45 presently explained by the presence of regenerative fac- [0086] Until now, the most commonly used method in tors in its glandular secretions as regeneration of epithe- snail extract acquisition consists of two steps, a first cen- lial cells is mandatory. The bioactive potential and ther- trifugation of the snails in order to obtain the extract and apeutic role of this glandular extract was apparent yet in a further centrifugation of the snail extract. The superna- extremely low concentrations. As skin ageing, skin ex- tant of the second centrifugation is used in cosmetology. 50 posure to various damaging environmental factors, or In this development, an attempt was made to develop even skin burns could result in oxidative stress of human alternative methodology for both steps of the already epithelial cells, this glandular extract can be further used used process of snail extract acquisition and evaluate not only in cosmetology but also as a pharmaceutical the effect ofthe bioactivesubstances in theobtained snail product. extracts on regenerative properties of the HaCaT human 55 [0088] Based on the annual life cycle of Cornu asper- keratinocyte cell line. Specifically (i) a non stressful meth- sum and in order to evaluate the optimum sampling sea- od, was tested in order to obtain clear pedal mucus from son, snail extracts were acquired at three sampling sea- snails crawling on glass surfaces, and (ii) drying and sub- sons: October-period of reproductive activity, February-

12 23 EP 3 124 030 A1 24 hibernation, and May- activity period. According to the 2003;51(338):211-8. results obtained, the optimum sampling season for snail extracts was mainly during reproductive activity of snails Brieva A, Philips N, Tejedor R, Guerrero A, Pivel (October), and also on May when snails were active. On J.P., Alonso-Lebrero J.L., Gonzalez S. Molecular the other hand, snail extracts obtained from snails during 5 Basis for the Regenerative Properties of a Secretion hibernation did not exhibit regenerative properties, which of the Mollusk Cryptomphalus aspersa. Skin Phar- could be explained by the decrease in metabolic rate of macol Physiol 2008;21:15-22. snails during hibernation (Guppy and Withers, 1999). Re- generative properties of snail extracts obtained during Brooks SA, Leathern AJ. Prediction of lymph node reproductive activity are presently attributed to the pres- 10 involvement in breast cancer by detection of altered ence of growth factors and antimicrobial substances in glycosylation in the primary tumour. Lancet snail mucus composition. It is known that Cornu asper- 1991;38:71-4. sum performs multiple mating with different mating part- ners during a reproductive season. During prolonged Calow P (1978) Why some metazoan mucus secre- mating events ranging from 2-8 hours, (Koemtzopoulos 15 tions are more susceptible to microbial attack than & Staikou 2007, Garefalaki 2010), snails are exposed to others (1978) The Americal Naturalist, 149-152. infections as they do not move and expose parts of their reproductive system to the environment. Moreover, dart Cottrell JM, Henderson IF, Pickett JA, Wright DJ shooting during copulation could potentially drive the ev- (1993) Evidence for glycosaminoglycans as a major olution of a wound healing mechanism in this species. 20 component of trail mucus from the terrestrial This mechanism could act through the release of regen- Arion ater L. Comparative Biochemistry and Physi- erative factors during the reproductive period. In general, ology, 104B, 455-468. snail mucus produced by snails during activity reproduc- tive or not is crucial in defense and locomotion of gastro- Cranga F, Cranga Y. L’: Zoologie, Symbol- pods that are exposed to the external environment (Den- 25 ique, Imaginaire, Médecine et Gastronomie. du Bien ny 1980, Davies & Hawkins 1998). Public, Dijon, 1991.

References Davies MS, Hawkins SJ (1998) Mucus from marine molluscs. Advances in marine biology, 34, 2-71. Ac- [0089] 30 ademic Press.

Ansart A., Vernon P., Daguzan J. 2002. Elements of DennyMW (1980) Locomotion: thecost ofgastropod cold hardiness in a littoral population of the land snail crawling. Science 208: 1288-1290. Helix aspersa (Gastropoda: ). Journal of Comparative Physiology B, 172, 619-625. 35 Denny MW (1989) Invertebrate mucous secretions: functional alternatives to vertebrate paradigms. In: Ashcroft GS, Horan MA, Ferguson MW (1995)The Symposium XLIII of the Society for Experimental Bi- effects of ageing on cutaneous wound healing in ology; Mucus and related Topics. (E. Chantler & NA mammals. J. Anat. 187(Pt 1) 1-26. Ratcliffe eds), 337-366. 40 Badiu DL, Balu AM, Barbes L, Luque R, Nita R, Radu Deyrup-Olsen I, Louie H, Martin AW, Luchtel DL M, Tanase E, Rosoiu N (2008). Physico-chemical (1992). Triggering by ATP of product release by mu- characterisation of lipids from Mytilus galloprovincia- cous granules of the land slug Ariolimax columbi- lis (L.) and Rapana venosa and their healing prop- anus. American Journal of Physiology,232,760-765. erties on skin burns. Lipids 43, 829-841. 45 Dittbrenner N, Lazzara R, Koehler HR, Mazzia C, Barnhart M.C. & McMahon BR (1987) Discontinuous Capowiez Y, Triebskorn R (2009) Heat tolerance in CO2 release and metabolic depression in dormant Mediterranean land snails: Histopathology after ex- land snails. Journal of Experimental Biology, 128: posure to different temperature regimes. Journal of 123-138. 50 Molluscan Studies (2009) 75: 9-18.

Baron-Barthelemy M. Mémoire sur les Preparations Dwek MV, Ross HA, Streets AJ et al. Helix pomatia à Base d’Hélicine Admises à I’Exposition Universelle agglutinin lectinbinding oligosaccharides of aggres- de 1855. Sirop et Bonbons Héliciés. De Bailly, Divry sive breast cancer. Int J Cancer 2001; 95:79-85. et Cie, Paris, 1855. 55 Fuchs SY, Ougolkov AV, Spiegelman VS, Minamoto Bonnemain B. Helix and drugs: snails for health care T (2005) Oncogenic beta-catenin signaling networks from Antiquity to these days. Rev Hist Pharm (Paris) in colorectal cancer. Cell Cycle 4, 1522-1539.

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Garefalaki ME, Triantafyllidis A, Abatzopoulos TJ, Merlin JL, Azzi S, Lignon D, Ramacci C, Zeghari N, Staikou A (2010) The outcome of sperm competition Guillemin F (1992). MTT assays allow quick and re- is affected by behavioural and anatomical reproduc- liable measurement of the response of human tu- tive traits in a simultaneously hermaphroditic land mour cells to photodynamic therapy. Eur. J. Cancer snail. J. Evol. Biol. 23: 966-976. 5 28A, 1452-1458.

Garefalaki ME (2010). Invention of intraspecific dif- PonsF, KoenigM, Michelot R et al. L’effetbronchore- ferentiation of mating behavior and sperm competi- laxant de l’hélicidine, un extrait d’Hélix pomatia, fait tion in the edible snail Helix aspersa. PHD thesis. intervenir une libération de prostaglandine E2. Department of Zoology, School of Biology, Aristotle 10 Pathol Biol 1999;47:73-80. University of Thessaloniki. Quevauviller A, Mainil J, Garcet S. Le mucus d’Hélix Guiller A. & Madec L. (2010) "Historical biogeogra- pomatia L. Preparation, composition, propriétés phy of the land snail Cornu aspersum: a new sce- thérapeutiques et pharmacodynamiques. Rev narioinferred fromhaplotype distributionin the West- 15 Pathol Gen Comp 1953;653:1514-38. ern Mediterranean basin. BMC Evolutionary Biology 2010; 10-18. Renwrantz L, Berliner U (1978) A galactose specific agglutinin, a blood-group H active polysaccharide, Herreid CF 1977. Metabolism of land snails ( and a trypsin inhibitor in albumin glands and eggs of lacteal) during dormancy, arousal and activity. Com- 20 arbustorum () Journal of Inverte- parative Biochemistry and Physiology, 56: 211-215. brate Pathology, 31, 171-179.

Iglesias-de la Cruz MC, Sanz-Rodriguez F,. Zamar- Runham NW, Laryea A (1968) Studies on the mat- ron A, Reyes E, E. Carrasco E, Gonzalez S, Juarranz uration of the reproductive system of Agrolimax re- A. Asecretion of themollusc Cryptomphalus aspersa 25 ticulates) (Pulmonata: Limacidae) Malacologia, 7, promotes proliferation, migration and survival of ke- 93-108. ratinocytes and dermal fibroblasts in vitro Interna- tional Journal of Cosmetic Science, 2012, 34, Skingsley DR, White AJ, Weston A (2000) Analysis 183-189 of pulmonate mucus by infrared spectroscopy. Jour- 30 nal of Molluscan Studies, 66, 363-371. Knudsen KA, Soler AP (2000) Cadherin mediated cell-cell interactions. Methods Mol. Biol.Staikou A 1999. Shell temperature, activity and re- 137,409-440. sistance to desiccation in the polymorphic land snail vindobonensis. Journal of Molluscan Stud- Koemtzopoulos Evripidis (2007). in 35 ies, 65: 171-184. the land gastropod Helix aspersa: mating behaviour and histological invention of the sperm storing organ. Sumner Yamada KM, Miyamoto S (1995) Integrin transmembrane signaling and cytoskeletal control. Koemtzopoulos E., Staikou A., 2007. Variation in Curr. Opin. Cell Biol. 7, 681-689. spermathecal morphology is independent of sperm 40 competition intensity in populations of the simulta- Tompa AS (1984) Land Snails (). neously hermaphroditic land snail Cornu aspersum. In: The : vol.7 Reproduction, (eds. Wilbur Zoology 110: 139-146. KM ed in chief, Tompa AS, Verdonk NH, van den Biggelaar JAM). pp 47-140, London. Academic Kuang S, Doran SA, Wilson RJA, Goss GG, Gold- 45 Press. berg JI (2002) Serotonergic sensory-motor neurons mediate a behavioral response to hypoxia in pond Tsoutsos D, Kakagia D, Tamparopoulos K. The ef- snail embryos. Journal of Neurobiology 52:73-83. ficacy of Helix aspersa Müller extract in the healing of partial thickness burns: a novel treatment for open Kumar S, Ghosh D, Biswas TK, Dutta U, Das P, Kun- 50 burn management protocols. J Dermatolog Treat. du S (2008) Spermatheca gland extract of snail (Tel- 2009;20(4):219-22. escopium telescopium) has wound healing potential: an an experimental invention in rabbits. International Webster L, Henderson R, Katz N, Ellis D. Charac- journal of Lower Extremity Wounds, 7,204-209. terization of confusion, an adverse event associated 55 with intrathecal ziconotide infusion in chronic pain Machin J. 1966. The evaporation of water from Helix patients. Pain Med 2001;2:253-4. aspersa IV. Lossfrom themantle of the inactivesnail. Journal of Experimental Biology, 45, 269-278. Withers P, Pedler S, Guppy M (1997) Physiological

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adjustments during aestivation by the Australian H2O2 induced oxidative stress is furthermore esti- land snail Rhagada tescorum (Mollusca: Pulmonata: mated, in order to evaluate the potential protective Camaenidae). Australian Journal of Zoology, 45: or therapeutic role of bioactive substances in snail 599-611. extracts. 5 Zouq NK, Keeble JA, Lindsay J. et al. (2009) FAK 5. Method according to one of the preceding claims, engages multiple pathways to maintain survival of characterized in that the extraction process, the fibroblasts and epithelia differential roles for paxillin products obtained after extraction and the conditions and p130Cas. J. Cell Sci. 122(Pt 3), 357-367. are optimised, particularly wherein said snails are 10 arranged to crawl or walk under optimum conditions of moisture, temperature and duration, which are de- Claims fined as a high humidity and room temperature of approx. 18° to 22°, particularly of about 20°C on said 1. Method for snail extract acquisition comprising at surface means, being selected from glass plates, for least two steps, characterized in that a first step for 15 about 2 hours, wherein further treatment of the snail obtaining the extract consists of (i) a non stressful extracts obtained either by snail centrifugation snail extract acquisition, wherein a batch of snails is (C-centrifugation), or snail crawling W (-’walking’) selected which are brought to move by crawling, re- consists of (a) centrifugation (cf) and (b) drying and sp. pedalling on a set of surface means notably made subsequent resuspension of the dried snail extract of inert material particularly glass plates measuring 20 (dry), particularly wherein said receptacles consist under a set of predetermined conditions including of petri dishes notably of glass, hygroscopic, temperature and duration, yielding a specifically wherein said (cf) extract is obtained after quantity of clear pedal mucus, which is removed from two successive centrifugations at a certain speed, said surface means and placed into receptacles, and notably of about 5000 rpm for a certain duration cor- which is followed by a further step, wherein said fur- 25 responding thereto, notably of about 10 minutes; ther step consists of (ii) drying with a subsequent more particularly wherein the final supernatant is resuspension step of the produced snail extract. then separated and filtered through a microporous filter having a pore size notably of about 0,22 mm; 2. Method according to claim 1, characterized in that yet more particularly wherein the Dry extract is ob- said further step actually consists of a centrifugation 30 tained by placing the snail extracts into a set of inert of the snail extract. preferably substantially smooth plates, particularly glass petri dishes into a chamber at approx. 40°C a 3. Method according to one of both preceding claims, certain duration corresponding thereto, notably of characterized in that a snail type is selected from about 24h; the gastropod family as Cornu aspersum, an extract 35 still more particularly wherein the dried snail extract is acquired from the proximal part of the digestive is further resuspended in aqueous solution, notably tract of the snails, wherein a fraction is extracted from with a pH of about 7,4, and filtered through a micro- said snail parts, wherein a snail extract is derived porous filter having said pore size of about 0,22 mm. from said fraction thereof. 40 6. Method according to one of the preceding claims, 4. Method according to one of the preceding claims, characterized in that said snail extract is derived characterized in that snail extracts are acquired from a segment of the proximal part of the digestive from specific glandular extracts, thereby including tract of the snails Cornu aspersum, both albumen gland of the reproductive system and particularly in that said snail extract is derived from proximal part of the digestive tract of the snails, 45 a fraction consisting of specific glandular extracts in particularly wherein the evaluation of the effect of extremely low concentrations, said specific extracts on regenerative properties of more particularly in that said snail extract is derived the HaCaT human keratinocyte cell line is carried from a fraction consisting of still furtherbody parts out; of Cornu aspersum thereby including DG extract more particularly wherein the effect of the specific 50 which is acquired only from the proximal part of the extracts on the HaCaT cell line is evaluated by esti- digestive tract; mating the proliferation rate, the migration and yet more particularly in that said snail extracts are wound healing potential, as well as the expression acquiredessentially atany of thethree samplingsea- of protein-markers of cell survival (β-catenin), cell- sons, which are determined either as cell adhesion (E-cadherin) and cell-substrate adhe- 55 sion (integrin-β1); (a) during a period of reproductive activity, es- yet more particularly wherein the response of the sentially circumscribed between the months of HaCaT cell line treated with snail extracts under September to November in the area of origin

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being the Mediterranean, resp. 11. Snail extract obtained according to a method as de- (b) during a period of hibernation between the fined in any one of the preceding claims, character- months of December to February and/or ized in that either the supernatant of a second cen- (c) during a period of activity between the trifugation of the initially derived snail extract -either months of March to May; 5 by snail centrifugation or by snail crawling- or the resuspended dried snail extract, or the supernatant still more particularly in that said snail extracts are of the homogenised parts of the digestive tract and acquired at the optimum sampling season for snail gland is used in cosmetology, particularly wherein extracts being any of both sampling seasons mainly the bioactive potential and therapeutic role of the during said period of reproductive activity of snails 10 aforementioned glandular extract is present in ex- (a), and also during said period of activity (c); tremely low concentrations, notably wherein regen- possibly in that said snail extractsare acquiredwhen erative properties of the human keratinocyte cell line snail mucus is produced by snails during activity, ei- HaCaT are positively influenced by factors in (A) ther reproductive or not. dried snail extracts and (B) in snail extracts of the 15 proximal part of the digestive tract and gland. 7. Method according to one of the preceding claims, characterized in that said bioactive potential of the 12. Snail extract obtained according to a method as de- glandular extract from the proximal part of the diges- fined in one of the preceding claims for use for pro- tive tract of Cornu aspersum is identified at all levels tecting an affected organ in a mammal, for the pre- of this evaluation process, wherein this extract in- 20 vention of said organ disease affecting said mam- duces, resp. increases significantly the proliferation mal, rate, the migration and wound healing potential, as particularly wherein the weight of said extract ob- well asthe expressionof all protein-markers involved tained ranges from 1% to 10% of live snail weight, for which it is used. more particularly wherein a certain quantity of said 25 extract, in particular about 1% of snail live weight, is 8. Method according to any one of the preceding obtained from snails having been allowed to crawl claims, characterized in that only said specificglan- or pedal for a certain time, typically two hours, on dular extract obtained from snails of all said sampling said glass plates, with in this case, subsequent mor- seasons (a), (b), resp. (c) is submitted to a chemical tality up to four days after snails’ use still being zero, oxidative stress, in particular a strong chemical oxi- 30 yet more particularly wherein a certain quantity of

dative stress, more particularly under H 2O2 induced said extract, in particular about 10% of snail live oxidative stress, yielding a significant therapeutic ef- weight, is obtained from snails subjected to two sub- fect on cells. sequentcentrifugations, typicallyat 2000 rpm for one minute. 9. Method according to any one of the preceding35 claims, characterizedin that it is started from a snail 13. Snail extract obtained according to a method as de- extract of Cornu aspersum of natural origin, or from fined in any one of claims 1 to 9 for treatment of said a cultured origin, particularly applied in a large scale organ disease, notably with regard to skin ageing, such as in cooperation with snail farms. skin exposure to various damaging environmental 40 factors, or even skin burns, particularly 10. Method according to any one of the preceding for use in a method of treatment of a said disease in claims, characterized in that the weight of the ex- a mammal, which comprises the step consisting in tract obtained ranges from 1% to 10%, of live snail the administration of an effective amount of said ex- weight, tract of Cornu aspersum thereto. particularly wherein a certain quantity of said extract, 45 in particular about 1% of snail live weight, is obtained 14. Use in cosmetology of the supernatant of a second from snails having crawled for a certain time, typically centrifugation of the initially derived snail extract, ei- two hours, on said glass plates, ther by snail centrifugation or by snail crawling, or more particularly wherein a certain quantity of said the resuspended dried snail extract, or the superna- extract, in particular about 10% of snail live weight, 50 tant of the homogenised parts of the digestive tract is obtained from snails subjected to two subsequent and gland of said second centrifugation step of the centrifugations, typically at 2000 rpm for one minute, snail extract in the method according to one of the yet more particularly wherein a further increase of claims 2 to 9, characterized by skin ageing, skin crawling time or centrifugation repeats are carried exposure to various damaging environmental fac- out, thereby yielding a still further increase in the55 tors, or even skin burns. quantity of the snail extracts obtained, especially in the latter case of commercial exploitation with said 15. Use of said glandular extract as defined in one of the snail farms. claims 11 to 13, characterized as a pharmaceutical

16 31 EP 3 124 030 A1 32 product, on the one hand, and/or use thereof, where- in the secretion of snails is used in the regeneration of burnt skin and in the management of open wounds, resp. use of the effect of the bioactive sub- stances in the obtained snail extracts on regenera- 5 tive properties of the HaCaT human keratinocyte cell line.

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REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description

• US 5538740 A [0012] [0045]

Non-patent literature cited in the description

• ANSART A. ; VERNON P. ; DAGUZAN J. Elements • CRANGA F; CRANGA Y. L’Escargot: Zoologie, of cold hardiness in a littoral population of the land Symbolique, Imaginaire. Médecine et Gastronomie. snail Helix aspersa (Gastropoda: Pulmonata). Jour- du Bien Public, Dijon, 1991 [0089] nal of Comparative Physiology B, 2002, vol. 172, • Mucus from marine molluscs.DAVIES MS ; 619-625 [0089] HAWKINS SJ. Advances in marine biology. Academ- • ASHCROFT GS ; HORAN MA ; FERGUSON MW. ic Press, 1998, vol. 34, 2-71 [0089] The effects of ageing on cutaneous wound healing • DENNY MW. Locomotion: the cost of gastropod in mammals. J. Anat., 1995, vol. 187, 1-26 [0089] crawling. Science, 1980, vol. 208, 1288-1290 [0089] • BADIU DL ; BALU AM ; BARBES L ; LUQUE R ; • Invertebrate mucous secretions: functional alterna- NITA R ; RADU M ; TANASE E ; ROSOIU N. Phys- tives to vertebrate paradigms. DENNY MW. Sympo- ico-chemical characterisation of lipids from Mytilus sium XLIII of the Society for Experimental Biology; galloprovincialis (L.) and Rapana venosa and their Mucus and related Topics. 1989, 337-366 [0089] healing properties on skin burns. Lipids, 2008, vol. • DEYRUP-OLSEN I ; LOUIE H ; MARTIN AW ; 43, 829-841 [0089] LUCHTEL DL. Triggering by ATP of product release • BARNHART M.C. ; MCMAHON BR. Discontinuous by mucous granules of the land slug Ariolimax co- CO2 release and metabolic depression in dormant lumbianus. American Journal of Physiology, 1992, land snails. Journal of Experimental Biology, 1987, vol. 232, 760-765 [0089] vol. 128, 123-138 [0089] • DITTBRENNER N ; LAZZARA R ; KOEHLER HR ; • BARON-BARTHELEMY M. Mémoire sur les Prepa- MAZZIA C ; CAPOWIEZ Y ; TRIEBSKORN R. Heat rations à Base d’Hélicine Admises à I’Exposition Uni- tolerance in Mediterranean land snails: Histopathol- verselle de 1855. Sirop et Bonbons Héliciés. De Bail- ogy after exposure to different temperature regimes. ly, Divry et Cie, Paris [0089] Journal of Molluscan Studies, 2009, vol. 75, 9-18 • BONNEMAIN B. Helix and drugs: snails for health [0089] care from Antiquity to these days.Rev Hist Pharm •DWEKMV; ROSS HA ; STREETS AJ et al. Helix (Paris), 2003, vol. 51 (338), 211-8 [0089] pomatia agglutinin lectinbinding oligosaccharides of • BRIEVA A ; PHILIPS N ; TEJEDOR R ; GUERRE- aggressive breast cancer. Int J Cancer, 2001, vol. 95, RO A ; PIVEL J.P. ; ALONSO-LEBRERO J.L. ; 79-85 [0089] GONZALEZ S. Molecular Basis for the Regenerative • FUCHS SY ; OUGOLKOV AV ; SPIEGELMAN VS ; Properties of a Secretion of the Mollusk Cryptomph- MINAMOTO T. Oncogenic beta-catenin signaling alus aspersa. Skin Pharmacol Physiol, 2008, vol. 21, networks in colorectal cancer. Cell Cycle, 2005, vol. 15-22 [0089] 4, 1522-1539 [0089] • BROOKS SA ; LEATHERN AJ. Prediction of lymph • GAREFALAKI ME ; TRIANTAFYLLIDIS A ; ABAT- node involvement in breast cancer by detection of ZOPOULOS TJ ; STAIKOU A. The outcome of altered glycosylation in the primary tumour. Lancet, sperm competition is affected by behavioural and an- 1991, vol. 38, 71-4 [0089] atomical reproductive traits in a simultaneously her- • CALOW P. Why some metazoan mucus secretions maphroditic land snail. J. Evol. Biol, 2010, vol. 23, are more susceptible to microbial attack than others 966-976 [0089] (1978). The Americal Naturalist, 1978, 149-152 • GAREFALAKI ME. Invention of intraspecific differ- [0089] entiation of mating behavior and sperm competition • COTTRELL JM ; HENDERSON IF ; PICKETT JA ; in the edible snail Helix aspersa. PHD thesis, 2010 WRIGHT DJ. Evidence for glycosaminoglycans as a [0089] major component of trail mucus from the terrestrial slug Arion ater L.Comparative Biochemistry and Physiology, 1993, vol. 104B, 455-468 [0089]

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• GUILLER A. ; MADEC L. istorical biogeography of • QUEVAUVILLER A ; MAINIL J ; GARCET S. Le mu- the land snail Cornu aspersum: a new scenario in- cus d’Hélix pomatia L. Preparation, composition, pro- ferred from haplotype distribution in the Western priétés thérapeutiques et pharmacodynamiques. Mediterranean basin. BMC Evolutionary Biology, Rev Pathol Gen Comp, 1953, vol. 653, 1514-38 2010, 10-18 [0089] [0089] • HERREID CF. Metabolism of land snails (Otala lac- • RENWRANTZL ; BERLINER U.A galactosespecific teal) during dormancy, arousal and activity. Compar- agglutinin, a blood-group H active polysaccharide, ative Biochemistry and Physiology, 1977, vol. 56, and a trypsin inhibitor in albumin glands and eggs of 211-215 [0089] (helicidae). Journal of Inverte- • IGLESIAS-DE LA CRUZ MC ; SANZ-RODRIGUEZ brate Pathology, 1978, vol. 31, 171-179 [0089] F; ZAMARRON A ; REYES E, E. ; CARRASCO E ; • RUNHAM NW ; LARYEA A. Studies on the matura- GONZALEZ S ; JUARRANZ A. A secretion of the tion of the reproductive system of Agrolimax reticu- mollusc Cryptomphalus aspersa promotes prolifera- lates) (Pulmonata: Limacidae). Malacologia, 1968, tion, migration and survival of keratinocytes and der- vol. 7, 93-108 [0089] mal fibroblasts in vitro. International Journal of Cos- • SKINGSLEYDR ; WHITE AJ ; WESTON A. Analysis metic Science, 2012, vol. 34, 183-189 [0089] of pulmonate mucus by infrared spectroscopy. Jour- • KNUDSEN KA ; SOLER AP. Cadherin mediated nal of Molluscan Studies, 2000, vol. 66, 363-371 cell-cell interactions. Methods Mol. Biol., 2000, vol. [0089] 137, 409-440 [0089] •STAIKOUA. Shell temperature, activity and resist- • Sexual selection in the land gastropod Helix aspersa: ance to desiccation in the polymorphic land snail Ce- mating behaviour and histological invention of the paea vindobonensis. Journal of Molluscan Studies, sperm storing organ. Koemtzopoulos Evripidis, 2007 1999, vol. 65, 171-184 [0089] [0089] • SUMNER YAMADA KM ;MIYAMOTO S. Integrin • KOEMTZOPOULOS E. ; STAIKOU A. Variation in transmembrane signaling and cytoskeletal control. spermathecal morphology is independent of sperm Curr. Opin. Cell Biol., 1995, vol. 7, 681-689 [0089] competition intensity in populations of the simultane- • Land Snails (Stylommatophora). TOMPA AS. The ously hermaphroditic land snail Cornu aspersum. Zo- Mollusca: vol.7 Reproduction. Academic Press, ology, 2007, vol. 110, 139-146 [0089] 1984, vol. 7, 47-140 [0089] • KUANG S ; DORAN SA ; WILSON RJA ; GOSS • TSOUTSOS D ; KAKAGIA D ; TAMPAROPOULOS GG ; GOLDBERG JI. Serotonergic sensory-motor K. The efficacy of Helix aspersa Müller extract in the neurons mediate a behavioral response to hypoxia healing of partial thickness burns: a novel treatment in pond snail embryos.Journal of Neurobiology, for open burn management protocols. J Dermatolog 2002, vol. 52, 73-83 [0089] Treat, 2009, vol. 20 (4), 219-22 [0089] • KUMAR S ; GHOSH D ; BISWAS TK ; DUTTA U ; • WEBSTER L ; HENDERSON R ; KATZ N ; ELLIS DAS P ; KUNDU S. Spermatheca gland extract of D. Characterization of confusion, an adverse event snail (Telescopium telescopium) has wound healing associated with intrathecal ziconotide infusion in potential: an an experimental invention in rabbits. In- chronic pain patients. Pain Med, 2001, vol. 2, 253-4 ternational journal of Lower Extremity Wounds, 2007, [0089] vol. 7, 204-209 [0089] • WITHERS P ; PEDLER S ; GUPPY M. Physiological • MACHIN J. The evaporation of water from Helix as- adjustments during aestivation by the Australian land persa IV. Loss from the mantle of the inactive snail. snail Rhagada tescorum (Mollusca: Pulmonata: Ca- Journal of Experimental Biology, 1966, vol. 45, maenidae). Australian Journal of Zoology, 1997, vol. 269-278 [0089] 45, 599-611 [0089] •MERLINJL; AZZI S ; LIGNON D ; RAMACCI C ; •ZOUQNK; KEEBLE JA ; LINDSAY J. et al. FAK ZEGHARI N ; GUILLEMIN F. MTT assays allow engages multiple pathways to maintain survival of fi- quick and reliable measurement of the response of broblasts and epithelia differential roles for paxillin human tumour cells to photodynamic therapy.Eur. and p130Cas. J. Cell Sci., 2009, vol. 122, 357-367 J. Cancer, 1992, vol. 28A, 1452-1458 [0089] [0089] •PONSF; KOENIG M ; MICHELOT R et al. L’effet bronchorelaxant de l’hélicidine, un extrait d’Hélix pomatia, fait intervenir une libération de prostaglan- dine E2. Pathol Biol, 1999, vol. 47, 73-80 [0089]

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