PRELIMINARY STUDIES REGARDING PRELIMINARY STUDIES REGARDING

THE EFFECT OF COLLAGENIC GELS THE EFFECT OF COLLAGENIC GELS

WITH MARINE ALGAE EXTRACTS WITH MARINE ALGAE EXTRACTS

IN THE TREATMENT OF INTRAORAL HERPES SIMPLEX IN THE TREATMENT OF INTRAORAL HERPES SIMPLEX

CoordCoordiinatornatorss,,

ProfProf. Mariana PĂCURAR . Mariana PĂCURAR AssocAssoc. . ProfProf. . Monica MONEAMonica MONEA

Author: BECHIR Edwin Sever

Co-author: Farah CURT-MOLA

BackgroundBackground: :

The treatment of intraoral herpes simplex can also be performed The treatment of intraoral herpes simplex can also be performed

by alternative methods. by alternative methods.

ObjectiveObjective: :

The The aimaim of our researches was to determinate the healing of our researches was to determinate the healing

properties of products obtained from collagenic gel extracts of turbot properties of products obtained from collagenic gel extracts of turbot

marine fish skin, combined with the marine alga marine fish skin, combined with the marine alga Ceramium rubrum 5% Ceramium rubrum 5%

and 10%and 10%, in the treatment of intraoral herpes simplex., in the treatment of intraoral herpes simplex.

Collagen is now the base of a dynamic industrial development of various Collagen is now the base of a dynamic industrial development of various products applied in medicine, pharmacy, cosmetics, a.s.o products applied in medicine, pharmacy, cosmetics, a.s.o [SETNESCU R., [SETNESCU R., MARINESCU V., SETNESCU T., JIPA S., ALBU M., DRAGAN G.: Oxidation of Lyophilized Collagen in MARINESCU V., SETNESCU T., JIPA S., ALBU M., DRAGAN G.: Oxidation of Lyophilized Collagen in

Various Conditions, Rev. Chim. (Bucharest), 62, No. 7, 2011, p 731-735]Various Conditions, Rev. Chim. (Bucharest), 62, No. 7, 2011, p 731-735] ..

Although to date, 29 different types of collagen have been identified, type I Although to date, 29 different types of collagen have been identified, type I collagen is the most abundant and still the best studied collagen is the most abundant and still the best studied [ALBU MG, TITORENCU I, [ALBU MG, TITORENCU I, GHICA MV, Collagen-Based Drug Delivery Systems for Tissue Engineering, Biomaterials Applications for GHICA MV, Collagen-Based Drug Delivery Systems for Tissue Engineering, Biomaterials Applications for

Nanomedicine, Prof. Rosario Pignatello (2011 Ed.), ISBN: 978-953-307-661-4, InTech,]Nanomedicine, Prof. Rosario Pignatello (2011 Ed.), ISBN: 978-953-307-661-4, InTech,] ..

In the present, various methods are available for the extraction of collagen In the present, various methods are available for the extraction of collagen from the skin of many species of fish from the skin of many species of fish [[HEMA G.S., SHYNI K., MATHEW S., ANANDAN R., HEMA G.S., SHYNI K., MATHEW S., ANANDAN R., NINAN G., LAKSHMANAN P.T.: A simple method for isolation of fish skin collagen- biochemical characteri-NINAN G., LAKSHMANAN P.T.: A simple method for isolation of fish skin collagen- biochemical characteri-zation of skin collgagen extracted from Albacore Tuna (zation of skin collgagen extracted from Albacore Tuna (Thunnus AlalungaThunnus Alalunga), Dog Shark (), Dog Shark (Scoliodon Scoliodon SorrakowahSorrakowah) and Rohu () and Rohu (Labeo Rohita),Labeo Rohita), Annals of Biological Research 2013, 4(1):271-8; BEROUMAND A.: Annals of Biological Research 2013, 4(1):271-8; BEROUMAND A.: Comparative Study Between Different Methods of Collagen Extraction from Fish and its Properties, World Comparative Study Between Different Methods of Collagen Extraction from Fish and its Properties, World Applied Sciences J. 2012, 16 (3): 316-319; Applied Sciences J. 2012, 16 (3): 316-319; ZHANG F., WANG A., LI Z., HE S., SHAO L.: Preparation and ZHANG F., WANG A., LI Z., HE S., SHAO L.: Preparation and

Characterisation of Collagen from Freshwater Fish Scales, Characterisation of Collagen from Freshwater Fish Scales, Food and Nutrition SciencesFood and Nutrition Sciences, 2011, 2, 818-823], 2011, 2, 818-823]..

The development of numerous techniques, with notable progress from the The development of numerous techniques, with notable progress from the technological point of view, allows today the achievement of high standard technological point of view, allows today the achievement of high standard collagenic gel extracts from marine fish skin, combined with marine algae.collagenic gel extracts from marine fish skin, combined with marine algae.

INTRODUCINTRODUCTIONTION

The treatments with natural substances/mixtures of the soft tissue diseases in The treatments with natural substances/mixtures of the soft tissue diseases in the oro-facial area present great interest in dentistry. The anti-inflammatory the oro-facial area present great interest in dentistry. The anti-inflammatory ingredients obtained from natural resources, are well known, and they can be ingredients obtained from natural resources, are well known, and they can be used today in the regenerative therapy of the affected soft tissues used today in the regenerative therapy of the affected soft tissues [BONNER P.: [BONNER P.: Diagnosing Oral Lesions. Dentistry Today,Dentistry Today, 2000, 19(6); SCULLY C.: The oral cavity and lips, In Burns T., Breathnach S., Cox 2000, 19(6); SCULLY C.: The oral cavity and lips, In Burns T., Breathnach S., Cox N., Griffiths C. editors. N., Griffiths C. editors. Rook’s Textbook of DermatologyRook’s Textbook of Dermatology, 7th ed., 2004, Oxford: Blackwell Publishing, p. 43-46, Editor(s): T. , 7th ed., 2004, Oxford: Blackwell Publishing, p. 43-46, Editor(s): T. Burns, St. Breathnach, N. Cox, Ch. Griffiths; EVERSOLE L.R.: Immunopathology of oral mucosal ulcerative, desquamative, and Burns, St. Breathnach, N. Cox, Ch. Griffiths; EVERSOLE L.R.: Immunopathology of oral mucosal ulcerative, desquamative, and

bullous diseases: selective review of the literature. bullous diseases: selective review of the literature. Oral Surg. Oral Med., Oral PatholOral Surg. Oral Med., Oral Pathol., 1994, 77:555–571].., 1994, 77:555–571].

These anti-inflammatory ingredients can be obtained from natural resources These anti-inflammatory ingredients can be obtained from natural resources such as marine algaesuch as marine algae [BLUNT J.W., COPP B.R., MUNRO M.H.G., NORTHCOTE P.T., PRINSEP M.R.: Marine [BLUNT J.W., COPP B.R., MUNRO M.H.G., NORTHCOTE P.T., PRINSEP M.R.: Marine

natural productsnatural products Nat. Prod. Rep. Nat. Prod. Rep., 2006, 23:26-78]., 2006, 23:26-78].

At the same time, it is proved that marine algae have an anti-inflammatory At the same time, it is proved that marine algae have an anti-inflammatory action after the infection with some gram negative and gram positive germs action after the infection with some gram negative and gram positive germs [MAYER A.M.S., HAMANN M.T.: Marine Pharmacology in 2000: Marine Compounds with Antibacterial, Anticoagulant, [MAYER A.M.S., HAMANN M.T.: Marine Pharmacology in 2000: Marine Compounds with Antibacterial, Anticoagulant, Antifungal, Anti-inflammatory, Antimalarial, Antiplatelet, Antituberculosis, and Antiviral Activities; Affecting the Cardiovascular, Antifungal, Anti-inflammatory, Antimalarial, Antiplatelet, Antituberculosis, and Antiviral Activities; Affecting the Cardiovascular, Immune, and Nervous Systems and Other Miscellaneous Mechanisms of Action, Immune, and Nervous Systems and Other Miscellaneous Mechanisms of Action, Marine Biotechnology, 2004, , 2004, Vol. 6, No. 1].].

The structural modifications of the marine biomass were performed by the The structural modifications of the marine biomass were performed by the biotechnological processes of extraction, and the production of type I hydrolysed biotechnological processes of extraction, and the production of type I hydrolysed fibrillar collagen, by lyophilisation technique fibrillar collagen, by lyophilisation technique [FRIESS W.: Collagen-Biomaterial for drug delivery, [FRIESS W.: Collagen-Biomaterial for drug delivery, Eur J Eur J

Phare BiopharmPhare Biopharm, 1998, 45: 113-136; GUZMÁN S., GATO A., CALLEJA J. M.: Antiinflammatory, analgesic and free radical , 1998, 45: 113-136; GUZMÁN S., GATO A., CALLEJA J. M.: Antiinflammatory, analgesic and free radical scavenging activities of the marine microalgae scavenging activities of the marine microalgae Chlorella stigmatophoraChlorella stigmatophora and and Phaeodactylum tricornutum, Phaeodactylum tricornutum, Phytotherapy Research, , 2001, 2001, Vol. 15, Issue 3: 224-230; : 224-230; NAGAI T., IZUMI M., ISHII M.:NAGAI T., IZUMI M., ISHII M.: Fish scale collagen. Fish scale collagen. Preparation and partial characterizationPreparation and partial characterization International Journal of Food Science & Technology , , 2004, 2004, Vol. 39 Issue 3: 239 – 244; SIRBU R., NEGREANU-PIRJOL T., : 239 – 244; SIRBU R., NEGREANU-PIRJOL T., LECA M., BECHIR A., MARIS M., MARIS D. Rheological Characterisation of Collagen Gels from Marine Resources of Black Sea LECA M., BECHIR A., MARIS M., MARIS D. Rheological Characterisation of Collagen Gels from Marine Resources of Black Sea and Chlohexidine Salt for using in Dental Medicine, World Academy of Science, Engineering and Technology Conference, 2008, and Chlohexidine Salt for using in Dental Medicine, World Academy of Science, Engineering and Technology Conference, 2008, 2(7), 1101 - 1107, 2(7), 1101 - 1107, http://waset.org/publications/4252, Vol:19, S:191, Vol:19, S:191].].

Herpes simplex viruses (human herpesviruses 1 and 2) commonly cause Herpes simplex viruses (human herpesviruses 1 and 2) commonly cause

recurrent infection affecting the skin, mouth, lips, eyes, and genitals. recurrent infection affecting the skin, mouth, lips, eyes, and genitals.

Mucocutaneous infections cause clusters of small painful vesicles on an Mucocutaneous infections cause clusters of small painful vesicles on an

erythematous base erythematous base [[

http://www.merckmanuals.com/professional/infectiousdiseases/herpesviruses/ herpes_simplex_virus_hsv_infections.html

]]. .

Most often, HSV-1 causes gingivostomatitis, herpes labialis, and herpes Most often, HSV-1 causes gingivostomatitis, herpes labialis, and herpes

keratitis keratitis [EASTERN, J.S. (2011) [EASTERN, J.S. (2011) Dermatologic manifestations of herpes simplex.Dermatologic manifestations of herpes simplex. eMedicine eMedicineWebMDWebMD. .

www.emedicine.medscape.com]www.emedicine.medscape.com]. .

Viral shedding occurs from the lesions but it can occur even when lesions are Viral shedding occurs from the lesions but it can occur even when lesions are

not apparent not apparent [[TOVARU S, PARLATESCU I, TOVARU M, CIONCA L : TOVARU S, PARLATESCU I, TOVARU M, CIONCA L :

Primary herpetic gingivostomatitis in children and adults, Quintessence Int. 2009 Feb;40(2):119-24Quintessence Int. 2009 Feb;40(2):119-24]]. .

After the initial infection, HSV remains dormant in nerve ganglia, from which it After the initial infection, HSV remains dormant in nerve ganglia, from which it

can periodically emerge, causing symptoms. Recurrent herpetic eruptions are can periodically emerge, causing symptoms. Recurrent herpetic eruptions are

precipitated by overexposure to sunlight, febrile illnesses, physical or emotional precipitated by overexposure to sunlight, febrile illnesses, physical or emotional

stress, immunosuppression, or unknown stimuli stress, immunosuppression, or unknown stimuli [HARMENBERG, J.; ÖBERG, B.; [HARMENBERG, J.; ÖBERG, B.;

SPRUANCE, S. (2010). "Prevention of Ulcerative Lesions by Episodic Treatment of Recurrent Herpes SPRUANCE, S. (2010). "Prevention of Ulcerative Lesions by Episodic Treatment of Recurrent Herpes

Labialis: A Literature Review". Labialis: A Literature Review". Acta Dermato VenereologicaActa Dermato Venereologica 9090 (2): 122–130; (2): 122–130;

http://en.wikipedia.org/wiki/Herpes_labialishttp://en.wikipedia.org/wiki/Herpes_labialis]]. .

The first objectiveThe first objective of our research was represented by the manufacturing of our research was represented by the manufacturing

operations of the collagenic gels, of the algae extracts and of the mixture, in operations of the collagenic gels, of the algae extracts and of the mixture, in

reference with their physical and chemical properties, such as their pH, viscosity reference with their physical and chemical properties, such as their pH, viscosity

and organoleptical properties, of each component in part and of the mixtures.and organoleptical properties, of each component in part and of the mixtures.

The collagen gels used in this study were extracted from turbot skinThe collagen gels used in this study were extracted from turbot skin

(Scophthalmus rhombus).(Scophthalmus rhombus).

MATERIALS AND METHODSMATERIALS AND METHODS

The aspect in nature of the turbotThe aspect in nature of the turbot (Scophthalmus rhombus) (Scophthalmus rhombus) marine fishes marine fishes

Our method of extracting the collagen from marine fish skin (collagen which Our method of extracting the collagen from marine fish skin (collagen which

was used ulteriorly to prepare pharmaceutical formulations including marine was used ulteriorly to prepare pharmaceutical formulations including marine

algae), consisted of the dissection of fish muscles, cutting the skin into small algae), consisted of the dissection of fish muscles, cutting the skin into small

pieces, and the suspension, in approximately 10 volumes of 0.5M acetic acid, at a pieces, and the suspension, in approximately 10 volumes of 0.5M acetic acid, at a

temperature of 0-5temperature of 0-5ooC (because the denaturizing temperature of the collagen is C (because the denaturizing temperature of the collagen is

low, the procedure was performed below 5low, the procedure was performed below 5ooC). The suspension was filtered and C). The suspension was filtered and

the collagen was collected by centrifugation.the collagen was collected by centrifugation.

The marine algae, selected for our study and for the preparation of the new The marine algae, selected for our study and for the preparation of the new

pharmaceutical formulation for the treatment of the intraoral herpes lesions, was pharmaceutical formulation for the treatment of the intraoral herpes lesions, was

the extract of seaweed the extract of seaweed Ceramium rubrum.Ceramium rubrum.

The aspect in nature of The aspect in nature of Ceramium rubrum,Ceramium rubrum, the marine algae used in our researchesthe marine algae used in our researches

The extraction of active ingredients from marine alga The extraction of active ingredients from marine alga CeramiumCeramium rubrumrubrum was was

achieved by using water solution of ethyl-alcohol with a concentration of 70% by achieved by using water solution of ethyl-alcohol with a concentration of 70% by

weight (g alcohol/g solution). weight (g alcohol/g solution).

The ratio of algae powder / hydro-alcoholic solution was of ¼ (25 g algae for The ratio of algae powder / hydro-alcoholic solution was of ¼ (25 g algae for

100 g of mixture). 100 g of mixture).

The second objectiveThe second objective of the research was the determination of the of the research was the determination of the

tolerability and healing properties of two products, obtained from the collagenic gel tolerability and healing properties of two products, obtained from the collagenic gel

extract from turbot skin, combined with 5% and 10% concentration of extract from turbot skin, combined with 5% and 10% concentration of CeramiumCeramium

rubrumrubrum marine alga extract. marine alga extract.

The aspect of the collagenic gels with The aspect of the collagenic gels with CeramiumCeramium rubrumrubrum marine alga extract marine alga extract

used in our researchesused in our researches

The trial was conducted on 42 patients (17 women and 25 men, aged The trial was conducted on 42 patients (17 women and 25 men, aged between 11-46 years), affected by intraoral herpes simplex (graph 1). between 11-46 years), affected by intraoral herpes simplex (graph 1).

The patients were divided into three groups: The patients were divided into three groups:

- - the first groupthe first group of 15 patients (6 female and 9 male) were treated with of 15 patients (6 female and 9 male) were treated with collagenic gel containing collagenic gel containing CeramiumCeramium rubrumrubrum 5%; 5%;

- - the second groupthe second group of 15 patients (6 female and 9 male) were treated with of 15 patients (6 female and 9 male) were treated with collagenic gel containing collagenic gel containing CeramiumCeramium rubrumrubrum 10%; 10%;

- - the third groupthe third group of 12 patients (5 female and 7 male) represented the control of 12 patients (5 female and 7 male) represented the control group.group.

The distribution of the patients in groups, by gender and by the used gels, is The distribution of the patients in groups, by gender and by the used gels, is presented in graph 2.presented in graph 2.

The distribution of patients by The distribution of patients by age and gender age and gender

17

25

3

6 6

8

5

8

3 3

0

5

10

15

20

25

amount 10 19 20-29 30-39 40-49

female male

15

15

12

6 6

5

9

9

7

0

2

4

6

8

10

12

14

16

total female male

group 1 group 2 group 3

The distribution of patients in groupsThe distribution of patients in groups

In our study, we performed clinical evaluations, determining the required In our study, we performed clinical evaluations, determining the required

number of applications and the needed time for healing, for every gel in part, number of applications and the needed time for healing, for every gel in part,

respectively the remission period of intraoral herpes lesions.respectively the remission period of intraoral herpes lesions.

The application of the gels was performed on the affected area two times The application of the gels was performed on the affected area two times

daily, for 5 days.daily, for 5 days.

The evolution of the clinical symptomatology, after the first application of The evolution of the clinical symptomatology, after the first application of

the gel, was evaluated daily, for 7 days, in order to ascertain the healing process, the gel, was evaluated daily, for 7 days, in order to ascertain the healing process,

both objectively (the healing state of the herpes lesions and the remission period both objectively (the healing state of the herpes lesions and the remission period

of the disease), and subjectively (the receding degree and time of the pain). of the disease), and subjectively (the receding degree and time of the pain).

All the patients were kept under observation for one year and a half.All the patients were kept under observation for one year and a half.

The aspect of an intraoral

herpes lesion

RESULTSRESULTS

The evolution of the clinical symptomatology in the patients with intraoral The evolution of the clinical symptomatology in the patients with intraoral herpes treated with the collagenic gel with seaweed extracts is visualized in the herpes treated with the collagenic gel with seaweed extracts is visualized in the following table:following table:

The obtained results showed that the most beneficial healing action was The obtained results showed that the most beneficial healing action was obtained with the collagenic gel containing 10obtained with the collagenic gel containing 10% Ceramium Rubrum% Ceramium Rubrum..

Objective / Subjective Objective / Subjective Assessment / no. of patients Assessment / no. of patients

11 22 33 44 55 66 77 88

Collagenic gel Collagenic gel

Ceramium RubrumCeramium Rubrum 5%5%

15 patients 15 patients

EvolutionEvolution GoodGood 55 77 88 99 1111 1414 1414 1313

UnsatisfactoryUnsatisfactory 1010 88 77 66 44 11 11 22

PainPain GoodGood 33 55 66 88 1010 1414 1515

UnsatisfactoryUnsatisfactory 1212 1010 99 77 55 11 --

Collagenic gel Collagenic gel

Ceramium RubrumCeramium Rubrum 10%10%

15 patients15 patients

EvolutionEvolution GoodGood 77 88 1111 1212 1414 1515 1515 1414

UnsatisfactoryUnsatisfactory 88 77 44 33 11 -- -- 11

PainPain GoodGood 66 77 99 1212 1515 1515 1515

UnsatisfactoryUnsatisfactory 99 88 66 33 -- -- --

Witness lotWitness lot

12 patients 12 patients

EvolutionEvolution GoodGood 11 22 55 66 66 88 99 99

UnsatisfactoryUnsatisfactory 1111 1010 77 66 66 44 33 33

PainPain GoodGood -- 11 33 55 66 77 1010

UnsatisfactoryUnsatisfactory 1212 1111 99 77 66 55 22

DISCUSSIONSDISCUSSIONSThe collagen extract from marine fish skin presents thermal stability at The collagen extract from marine fish skin presents thermal stability at

elevated temperatures elevated temperatures [SETNESCU R., MARINESCU V., SETNESCU T., JIPA S., ALBU [SETNESCU R., MARINESCU V., SETNESCU T., JIPA S., ALBU M., DRAGAN G.: Oxidation of Lyophilized Collagen in Various Conditions, Rev. Chim. M., DRAGAN G.: Oxidation of Lyophilized Collagen in Various Conditions, Rev. Chim.

(Bucharest), 62, No. 7, 2011, p 731-735](Bucharest), 62, No. 7, 2011, p 731-735]..

After the researches of Dubber D. and Tilmann H., methanol extracts of After the researches of Dubber D. and Tilmann H., methanol extracts of Ceramium rubrumCeramium rubrum (at 10 mg dry weight per ml), and hexane extracts of (at 10 mg dry weight per ml), and hexane extracts of Laminaria digitata (Laminaria digitata (at 31 mg dry weight per ml), evoked strong antibacterial at 31 mg dry weight per ml), evoked strong antibacterial activities and inhibited almost all tested bacteria activities and inhibited almost all tested bacteria [DUBBER D., TILMANN H., Extracts of [DUBBER D., TILMANN H., Extracts of Ceramium rubrum, Mastocarpus stellatus and Laminaria digitata inhibit growth of marine and Ceramium rubrum, Mastocarpus stellatus and Laminaria digitata inhibit growth of marine and fish pathogenic bacteria at ecologically realistic concentrations, fish pathogenic bacteria at ecologically realistic concentrations, Aquaculture, Aquaculture, 2008, Vol. 274, 2008, Vol. 274,

Issues 2–4: 196-200]Issues 2–4: 196-200]. .

The red alga The red alga Ceramium rubrumCeramium rubrum is well-known as an agar source, and it is well-known as an agar source, and it can be used to obtain chlorophyll, the green pigment being an useful therapeutic can be used to obtain chlorophyll, the green pigment being an useful therapeutic agent agent [SAVA D., ROTARU-STANCIC M., DOROFTEI E., ARCUS M., Pharmaceutical Importance of some [SAVA D., ROTARU-STANCIC M., DOROFTEI E., ARCUS M., Pharmaceutical Importance of some Multicellular Red Algae Species from the Romanian Black Sea Shore, December 2009, Multicellular Red Algae Species from the Romanian Black Sea Shore, December 2009, Analele Societatii Analele Societatii

Nationale de Biologie CelularaNationale de Biologie Celulara;2009, Vol. 14 Issue 2, p 297];2009, Vol. 14 Issue 2, p 297]..

In the alga In the alga Ceramium rubrumCeramium rubrum b-carotene and retinol are present and this b-carotene and retinol are present and this is, probably, one of the reasons why the mixture of collagenic gel containing 10% is, probably, one of the reasons why the mixture of collagenic gel containing 10% Ceramium Rubrum Ceramium Rubrum alga induced a more beneficial effect on intraoral herpes alga induced a more beneficial effect on intraoral herpes lesions than in the lesions treated with gel containing 5% lesions than in the lesions treated with gel containing 5% Ceramium RubrumCeramium Rubrum..

• The reduction of the intraoral herpes simplex symptomatology has been The reduction of the intraoral herpes simplex symptomatology has been

observed from the second assessment in all of the patients. observed from the second assessment in all of the patients.

• The therapeutic effect of the collagenic gel containing 10% The therapeutic effect of the collagenic gel containing 10% Ceramium Ceramium

rubrum rubrum extract was quicker than the effect of the gel containing 5% extract was quicker than the effect of the gel containing 5%

Ceramium rubrumCeramium rubrum. .

• The recurrence of intraoral herpes simplex disease in the patients treated The recurrence of intraoral herpes simplex disease in the patients treated

with collagenic gel containing 10% with collagenic gel containing 10% Ceramium rubrumCeramium rubrum extract was lower than extract was lower than

in patients treated with the other gel.in patients treated with the other gel.

• The resources offered The resources offered by the Black Sea biosystem can be used, with good by the Black Sea biosystem can be used, with good

results, to reduce the healing time and the recurrence of intraoral herpes results, to reduce the healing time and the recurrence of intraoral herpes

simplex disease. simplex disease.

CONCLUSIONSCONCLUSIONS

Acknowledgement:Acknowledgement:

A part of the financial support for this research A part of the financial support for this research belong to CEEX 188 / 2006-2008belong to CEEX 188 / 2006-2008

1.1. SETNESCU R., MARINESCU V., SETNESCU T., JIPA S., ALBU M., DRAGAN G.: Oxidation of Lyophilized Collagen in Various Conditions, Rev. Chim. (Bucharest), 62, SETNESCU R., MARINESCU V., SETNESCU T., JIPA S., ALBU M., DRAGAN G.: Oxidation of Lyophilized Collagen in Various Conditions, Rev. Chim. (Bucharest), 62, No. 7, 2011, p 731-735;No. 7, 2011, p 731-735;

2.2. ALBU MG, TITORENCU I, GHICA MV, Collagen-Based Drug Delivery Systems for Tissue Engineering, Biomaterials Applications for Nanomedicine, Prof. Rosario ALBU MG, TITORENCU I, GHICA MV, Collagen-Based Drug Delivery Systems for Tissue Engineering, Biomaterials Applications for Nanomedicine, Prof. Rosario Pignatello (2011 Ed.);Pignatello (2011 Ed.);

3.3. HEMA G.S., SHYNI K., MATHEW S., ANANDAN R., NINAN G., LAKSHMANAN P.T.: A simple method for isolation of fish skin collagen- biochemical characteri-zation of HEMA G.S., SHYNI K., MATHEW S., ANANDAN R., NINAN G., LAKSHMANAN P.T.: A simple method for isolation of fish skin collagen- biochemical characteri-zation of skin collgagen extracted from Albacore Tuna (skin collgagen extracted from Albacore Tuna (Thunnus AlalungaThunnus Alalunga), Dog Shark (), Dog Shark (Scoliodon SorrakowahScoliodon Sorrakowah) and Rohu () and Rohu (Labeo Rohita),Labeo Rohita), Annals of Biological Research 2013, Annals of Biological Research 2013, 4(1):271-8; 4(1):271-8;

4.4. BEROUMAND A.: Comparative Study Between Different Methods of Collagen Extraction from Fish and its Properties, World Applied Sciences J. 2012, 16 (3): 316-319; BEROUMAND A.: Comparative Study Between Different Methods of Collagen Extraction from Fish and its Properties, World Applied Sciences J. 2012, 16 (3): 316-319; 5.5. ZHANG F., WANG A., LI Z., HE S., SHAO L.: Preparation and Characterisation of Collagen from Freshwater Fish Scales, ZHANG F., WANG A., LI Z., HE S., SHAO L.: Preparation and Characterisation of Collagen from Freshwater Fish Scales, Food and Nutrition SciencesFood and Nutrition Sciences, 2011, 2, 818-823;, 2011, 2, 818-823;6.6. BONNER P.: BONNER P.: Diagnosing Oral Lesions. Dentistry Today,Dentistry Today, 2000, 19(6); SCULLY C.: The oral cavity and lips, In Burns T., Breathnach S., Cox N., Griffiths C. editors. 2000, 19(6); SCULLY C.: The oral cavity and lips, In Burns T., Breathnach S., Cox N., Griffiths C. editors. Rook’s Rook’s

Textbook of DermatologyTextbook of Dermatology, 7th ed., 2004, Oxford: Blackwell Publishing, p. 43-46, Editor(s): T. Burns, St. Breathnach, N. Cox, Ch. Griffiths; , 7th ed., 2004, Oxford: Blackwell Publishing, p. 43-46, Editor(s): T. Burns, St. Breathnach, N. Cox, Ch. Griffiths; 7.7. EVERSOLE L.R.: Immunopathology of oral mucosal ulcerative, desquamative, and bullous diseases: selective review of the literature. EVERSOLE L.R.: Immunopathology of oral mucosal ulcerative, desquamative, and bullous diseases: selective review of the literature. Oral Surg. Oral Med., Oral PatholOral Surg. Oral Med., Oral Pathol ., .,

1994, 77:555–571;1994, 77:555–571;8.8. BLUNT J.W., COPP B.R., MUNRO M.H.G., NORTHCOTE P.T., PRINSEP M.R.: Marine natural productsBLUNT J.W., COPP B.R., MUNRO M.H.G., NORTHCOTE P.T., PRINSEP M.R.: Marine natural products Nat. Prod. Rep. Nat. Prod. Rep., 2006, 23:26-78;, 2006, 23:26-78;9.9. MAYER A.M.S., HAMANN M.T.: Marine Pharmacology in 2000: Marine Compounds with Antibacterial, Anticoagulant, Antifungal, Anti-inflammatory, Antimalarial, MAYER A.M.S., HAMANN M.T.: Marine Pharmacology in 2000: Marine Compounds with Antibacterial, Anticoagulant, Antifungal, Anti-inflammatory, Antimalarial,

Antiplatelet, Antituberculosis, and Antiviral Activities; Affecting the Cardiovascular, Immune, and Nervous Systems and Other Miscellaneous Mechanisms of Action, Antiplatelet, Antituberculosis, and Antiviral Activities; Affecting the Cardiovascular, Immune, and Nervous Systems and Other Miscellaneous Mechanisms of Action, Marine Biotechnology, 2004, , 2004, Vol. 6, No. 1

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