EVALUATION OF THE EFFICACY OF SOME BEE ... - Annals of RSCB

Annals of RSCB

Vol. XVII, Issue 1/2012

EVALUATION OF THE EFFICACY OF SOME BEE PRODUCTS UPON THE CHEMICALLY INDUCED CUTANEOUS ERYTHEMA IN WISTAR RATS C. V. Andriţoiu1 , Anca Irina Prisăcaru2, Corina Andriescu3, I. M. Popa1 GHEORGHE ASACHI TECHNICAL UNIVERSITY OF IAŞI, ROMANIA: DEPARTMENT OF PHYSICAL CHEMISTRY, FACULTY OF CHEMICAL ENGINEERING AND ENVIRONMENTAL PROTECTION; 2DEPARTMENT OF NATURAL AND SYNTHETIC POLYMERS; 3LABORATORY OF PATHOLOGICAL ANATOMY, SF. PARASCHEVA CLINICAL HOSPITAL OF INFECTIOUS DISEASES, IAŞI, ROMANIA 1

Summary The present experiment evaluates the efficacy of the topical treatment with propolis, honey and Royal Jelly of chemically induced erythema in Wistar rats. Clinical and histopathological results reveal the benefic effect of the above mentioned bee products upon erythema, with complete healing of the lesion after seven days of treatment, and with evident clinical improvements even from the third day of treatment. In conclusion, the bee products tested in this experiment may be used with good results in the treatment of cutaneous erythema, as it is a simple, efficient and biocompatible therapy. Key words: cutaneous erythema, propolis, honey, Royal Jelly [email protected]

Introduction The discovery of natural compounds able to stimulate the regeneration of the tissues gained great importance in the last years, in the sense of developing new nontoxic formulations. Previous studies reveal that propolis is efficient in wound-healing (Burdock et al., 1998; Castro, 2001). The main compounds from propolis that are responsible for tissue repair are represented by the fatty acids derivatives, β-steroids, vitamins and mineral salts (Burdock et al., 1998; Castro, 2001), aromatic acids and phenols, cinammic acid and cinammic alcohol derivatives, benzaldehyde derivatives, terpene compounds with antiinflammatory action, flavones, flavonols (βbisabolol, for example) with decongestant effect and tonic effect upon capillaries (Merică, 2003), flavonoids (with bactericidal and antiviral properties) (Havsteen, 2002). Propolis proved to have broad-spectrum antimicrobial activity, antifungic activity, tissue repair effect, and

anesthetic action (Merică, 2003). This nontoxic bee product is efficient in the treatment of severe lesions, including the purulent and non-purulent cutaneous lesions (Castro 2001). Regarding honey, its composition explains its traditional use in cosmetics. Besides the content of phytohormonal and bioactivating factors, the cosmetical value of honey consists in its emollient qualities and in its ability to generate, through osmosis, a higher blood flow towards the cutaneous tissue, thus improving its nutrition. Honey is also a traditional ingredient of creams, offering them emollient and hydration properties (Piana, 1989). Honey has antiseptic and preserving effects, being included in different pharmaceutical formulations such as lotions, creams, cosmetic facial masks, and anti-wrinkle products (Merică, 2003). Topically applied Royal Jelly (RJ) increases the wound repair process in the case of burns and superficial wounds, as 258

Annals of RSCB


well as in the treatment of seborrhea and acne (Piana, 1989). The main chemical constituents are: water, glucides (mainly glucose and fructose), protides (albumin, globulin, and a number of amino acids), lipids (fatty acids, sterols, phsopholipids), vitamins (B1, B2, B6, vitamin B5, biotin, and vitamin C), minerals, hormones ( with effect upon aged skin, with superiour efficiency after the application of natural estrogens in comparison with the synthetic ones) and oligoelements (boron, mangan, copper, zinc, iron, molybdenum, chlorine, fluorine, iodine, cobalt etc.), acting in very small amounts as activators and modifiers of the skin tissue, as energy transporters, and catalysts of the skin metabolism (Cosmovici et al., 1980). As a cutaneous bioactivator, RJ has been introduced in numerous cosmetic preparations, stimulating the cellular metabolism and normalizing the secretion of the sebaceous glands, limiting the skin aging process by tonifying the skin, and improving the hydration and elasticity of skin (Piana, 1989). The present experiment aims to evaluate the efficacy of some bee products (propolis, honey, RJ) in the topical treatment of chemically induced erythema in laboratory animals.

methods (first, the hair was cut with a scissor; then an epilation cream was applied, according to the indications on the label of the product; consequently, the epilated area was cleaned with normal saline solution); • the chemical burn was induced by washing the two paravertebral areas of each rat with a 85% aqueous solution of lactic acid; the affected areas were left for 24 hours, without cleaning them with normal saline solution, in order to obtain the chemical burn; • after 24 hours, the laboratory animals were topically treated with the above mentioned bee products for 7 days, until complete healing of the wound; during these 7 days, macroscopic and clinical examinations were achieved; • In the end, a specimen sample of tissue removed from the healed skin of all rats was taken with a 3 mm biopsy punch in order to be analyzed by histopathological examination. The collected samples were fixed in 10% buffered formalin for at least 24 h, progressively dehydrated in solutions containing an increasing percentage of ethanol (70, 80, 95, and 100%, v/v), embedded in paraffin under vacuum, sectioned at 5 µm thickness, deparaffinized, and stained with hematoxylineosin (HE). The bee products were purchased from Stupina LLC, Romania.

Material and methods In order to demonstrate the woundhealing effects of bee products (propolis, honey, and RJ), the following experimental model of chemical burn was achieved: • female rats, Wistar strain, with a body weight comprised between 220-250 g, have been divided into 4 groups: the reference group (group 1, non-treated), propolis group (group 2, treated with propolis), honey group (group 3, treated with honey), RJ group (group 4, treated with royal jelly); • the dorsal surface of the animals was shaved by mechanical and chemical

Results Macroscopic evaluation The macroscopic evaluation of epidermal lesions for all the 3 experimental treated groups demonstrated the efficacy of the treatment with bee products. Improvements have been clearly observed even from the third day of treatment, while complete remission of erythema was obtained after 7 days of treatment (table I).

259

Annals of RSCB


Table I. Macroscopic examination EXPERIMENTAL GROUPS DAYS OF EXPERIMENT

Nontreated group

Propolis group

Honey group

RJ group

DAY 0

DAY 2

DAY 3

DAY 4

DAY 5

DAY 7

remarked in all the 3 treated groups, suggesting recent regeneration. The basal membrane of the epidermis revealed rarely and isolated by a slight accentuation (non-treated group). The superficial dermis shows a minimal isolated densification by collagenization for the non-treated group, while for the other three treated experimental groups the normal aspect of the dermis predominates (table III). The lymphocytes are minimal, in normal range, with slight differences among the 4 groups. The existent lymphocytes are disposed either diffused in the superficial dermis, or perivascular (table II). A discrete edema is remarked in the superficial dermis (table IV). The aspects of congestion are minimal to moderate (table II). The small capillaries are dilated, filled with normal erythrocytes. Haemorrhage through erythro diapedesis is present only isolated, only for the non-treated group. The muscle fragments attached to cutaneous fragments do not reveal any changes. The cutaneous appendages appear to be normal.

Histopathological examination The histopathological examination reveal that the epidermis of the 3 treated groups appears regenerated, with minimal differences among them. The epidermis is intact, rectilinium, with orthokeratosis, representated by 1-2 cell layers (non-treated group), that alternate with areas made up of 2-3 cell layers: basal layer, spinous layer, granular or cornified layer (non-treated group and honey group). Rarely, 3-4 layers are present (propolis group). In the areas with 1-2 layers, the cells are small, basophilic, with hyperchrome nuclei having a slightly increased volume and frequently with an irregular contour – aspects that certify the recent regeneration of the epithelium. In areas with 2-3 layers, there can be seen vacuolar degeneration and aspects of “koilocytes” (large cells with small nuclei, moderately hyperchrome, with perinuclear halo). There are also present large pale vesiculous nuclei, with finely dispersed chromatin and abundant cytoplasm. These cytonuclear aspects are 260

Annals of RSCB


Table II. Histopathological examination MICROGRAPHS Non-treated group (group 1)

parakeratotic focus (HEx200)

epidermis and dermis (HEx100)

recently regenerated epidermis (HEx100)

dermis and hypodermis with congestion (HEx100) Propolis group (group 2)

dermis with discrete horizontalization of the collagen fibers (HEx100) Honey group (group 3)

epidermis and discrete edema in the dermis (HEx100)

stasis and edema at the dermishypodermis limit (HEx200)

regenerated epithelium with 3- 4 cell layers (HEx200)

superficial dermis with discrete horizontalization of the collagen fibers (HEx200)

RJ group (group 4)

recently regenerated epidermis and dermis (RJ group - HEx200)

regenerated epithelium with 2-3 cell layers (RJ group - HEx400)

261

regenerated epithelium and perivascular fibroblasts (RJ group - HEx400)

Annals of RSCB


Table III. Collagenization of the dermis

Groups

Collagenization Superficial dermis Reticular dermis Non-treated group ++ +/Propolis group +/Honey group +/RJ group +/(evaluation scale of collagenization: - absent, +/-discrete, + moderate, ++ severe)

Table IV. Intensity of edema at the end of the treatment Groups Edema Superficial dermis Reticular dermis Non-treated group + + Propolis group + Honey group + RJ group + (evaluation scale of edema: - absent, +/-discrete edema, + moderate edema, ++ severe edema)

Discussions (Natarajan et al., 1996; Yoshioka et al., 1979). CAPE hinders the increase of the acute phase reactants, indicating the fact that it can be used with good results in the treatment of cutaneous lesions (Koltuksuz et al., 2001). Sehn and colleagues noticed that the inclusion of propolis in an ointment base influenced the healing process and the keratinocytes proliferation, in comparison with the control group, while the propagation of propolis was dependednt to the wound-healing stages (Sehn et al., 2009). The use of honey in this experiment is based on more reasons: the high amounts of glucose from honey are freely released at the level of the skin into the interstitial liquid of dermis and epidermis, and then gets into cells. The use of glucose at the level of skin, appreciated by its molecules marked with a radioactive tracer, is of 0,2 µmol/min/mg human skin (dermis and epidermis). This value can be compared to that of the in vivo active muscular tissue (Freinkel, 2002). More than a half of the glucose used by the skin takes place at the level of epidermis, being higher in the basal layer than in the other layers (Alecu, 2006). Numerous glucides, among which a great variety of hexoses and sialic acid, are found in the subcellular structure of keratinocytes

The selection of the bee products tested in the present experiment has been rigorously analyzed. The use of propolis, honey, and RJ in the topical treatment of chemically induced cutaneous erythema was achieved taking into consideration the following aspects: first, the intention to test natural compounds that are easily obtained and have perfect applicability in dermatology and cosmetics; second, we wanted to evaluate the effect of every single bee product, in order to include them, in the future, in skin care formulations. Propolis, due to the caffeic acid phenethyl ester (CAPE), accelerates the wound-healing process and decreases the oxidative stress after the incision induced lesion (Serarslan et al., 2007). It has antibacterial action, immunomodulating effect, suppresses the lipid peroxidation and stimulates the metabolism (Natarajan et al., 1996; Williams et al., 2004; Awale et al., 2005). The fast development of the regeneration epithelium consequently to the lesions treated with CAPE was revealed by histopathological examination, as this compound is able to improve the woundhealing because of its antioxidant and antiinflammatory properties 262

Annals of RSCB


from all the layers. Thus, glucides and their derivatives are present into the structure of cell organelles, of plasmatic membrane and desmosomes, and as well as in the composition of the cell walls (Walsh and Chapman, 1991). There has been also demonstrated that oligosaccharides protect the desmosomes from the activity of proteolytic enzymes, being at the same time a prevention element from the premature desquamation (Cherepanova et al., 2002). All these extremely important mechanisms led us to the usage of honey in the present experiment, having as result the remition of the cutaneous erythema for the honey group. Furthermore, glycoproteins are compounds found in many structures of the skin: plasmatic membrane, desmosomes, basal membrane, lamellar bodies, Golgi apparatus of the keratinocytes, even in melanosomes. A series of glycolipids are attached to the surface of the epidermal cells. In general, these glycoproteins contain glucides with 6 atoms of carbon: manose, galactose, or N-acetyl glucosamine. Among glucides, fructose is found only in the granular layer, while N-acetyl glucosamine is present in spinous and granular layer (Freinkel, 2001). Honey stimulates the increase of the repair tissue, improves the healing and produces the debridement (Oryan and Zaker, 1998). There could be observed that topical use of honey was efficient in the treatment of seborrhoeic dermatitis and dandruff (AL-Waili, 2001). In cosmetology, RJ is used in formulations in concentrations of 1-3% in order to stimulate the epidermis and to improve its nutrition, to attenuate the wrinkles and to offer its shiny aspect. Allergic reactions are possible to take place, mainly cutaneous eruptions (Merica, 2003). The amounts of RJ that has been used in the present experiment did not produce any of the allergic events; furthermore, RJ acted as a therapy agent upon the cutaneous lactic acid induced erythema. Moreover, the prolonged application of the ointments containing estrogens, that are to be found in

RJ, on an atrophic tissue, after the initial stage of progressive repair leads to the trophicity of the dermis (Braha, 1998). The estrogens seem to act upon the skin in order to maintain its trophicity by the following mechanisms: the increase of the skin thickness, the increase of vascularisation, the increase of collagen quantity and quality, the regulation of the pilous follicles and sebaceous glands (Cowley et al., 1997). Moreover, an active metabolism of estradiol has been revealed at the level of keratinocytes (Alten and Kissel, 1998). RJ stimulates every conjunctive tissue, improving the nutrition of the cell. RJ is recommended in the treatment of seborrhoeic skin, with wrinkles, dehydrated, as well as in the treatment of prematurely aged skin, as it contains amino acids, water-soluble vitamins, fat-soluble vitamins, glucides, mineral slats (Cosmovici et al., 1980). In the present experiment, important results have been obtained, both from the clinical point of view (showing improvements even from the third day of treatment, while complete remission of erythema was obtained after 7 days of treatment) and histopathological point of view, thus revealing the efficacy of the tested bee products.

Conclusions The bee products tested in the present experiment (propolis, honey, Royal Jelly) proved to be efficient in the treatment of cutaneous erythema, offering us the future possibility to include them in new innovating formulations of cosmeceuticals.

Acknowledgments This paper was supported by the project PERFORM-ERA "Postdoctoral Performance for Integration in the European Research Area" (ID-57649), financed by the European Social Fund and the Romanian Government. 263

Annals of RSCB


References

Koltuksuz, U.; Mutuş, H.M.; Kutlu, R.; Ozyurt, H.; Cetin, S.; Karaman, A.; Gürbüz, N.; Akyol, O.; Aydin, N.E.: Effects of caffeic acid phenethyl ester and epidermal growth factor on the development of caustic esophageal stricture in rats. J. Pediatr. Surg., 36, 1504–1509, 2001. Merică, E.: Tehnologia produselor cosmetice, p. 448-451, 2003, Ed. Kolos, Iaşi. Natarajan, K.; Gingh, S.; Burke, T.R.; Grunberger D.; Aggarwal, B.B.: Caffeic acid phenethyl ester is a potent and specific inhibitor of activation of nuclear transcription NF-j B. Proc. Natl. Acad. Sci., 93, 9090– 9095, 1996. Oryan, A., Zaker, R.: Effects of topical application of honey on cutaneous wound healing in rabbits. Zentralb. Veterinarmed. A., 45, 181–188, 1998. Piana, G.P.: Albinele şi produsele apicole. In: Produsele stupului, hrană, sănătate, frumuseţe, pp. 153-158, 1989, Ed. Apimondiam. Sehn, E.; Hernandes, L.; Franco, S.L.; Gonçalves, C.C.M.; Baesso, M.L.: Dynamics of reepithelialisation and penetration rate of a bee propolis formulation during cutaneous wounds healing, Anal. Chim. Acta, 635, 115– 120, 2009. Serarslan, G.; Altuğ, E.; Kontas, T.; Atik E.; Avcit, G.: Caffeic acid phenetyl ester accelerates cutaneous wound healing in a rat model and decreases oxidative stress, Clin. Exp. Dermatol., 32, 709–715, 2007. Walsh, A.; Chapman, S.J.: Sugars protect desmosome and cornearome glycoproteins from proteolysis, Arch. Dermatol. Res., 283, 3, 174-179, 1991. Williams, R.J.; Spencer, J.P.; Rice-Evans, C.: Flavonoids: antioxidants or signalling molecules?. Free Rad. Biol. Med., 36, 7, 838-849, 2004. Yoshioka, T.; Kawada, K.; Shimada, T.; Mori, M.: Lipid peroxidation in maternal and cord blood and protective mechanism against activated-oxygen toxicity in the blood. Am. J. Obstet. Gynecol., 135, 372–376, 1979.

Alecu, M.: Patologia moleculară a pielii, pp. 56-64, 2006, Ed. Medicală, Bucureşti. AL-Waili, N.: Therapeutic and prophylactic effects of crude honey on chronic seborrheic dermatitis and dandruff. Eur. J. Med. Res., 6, 306–308, 2001. Awale, S.; Shrestha, S.P.; Tezuka, Y.; Ueda, J.Y., Matsushige, K., Kadota, S.: Neoflavonoids and related constituents from Nepalese propolis and their nitric oxide production inhibitory activity. J. Nat. Prod., 68, 6, 858-64, 2005. Braha, S.L.: Elemente de dermatologie şi cosmetologie, p. 212-213, 1998, Ed. Cermi, Iaşi. Burdock, G.A.: Review of the biological properties and toxicity of bee propolis (propolis). Food Chem Toxicol., 36, 4, 347363, 1998. Cherepanova, O.A.; Kalmykova, N.V.; Koronkina, I.V.; Aré, A.F.; Gorelik, Iu.V.; Pinaev, .P.: Differences in the character of interaction of normal and transformed human keratinocytes with laminin isoforms (Russian). Tsitologiia, 44, 2, 151-8, 2002. Cosmovici, L., Samborschi, E., Zisu, L.: Cosmetica. Ştiinţă – artă – frumuseţe, pp. 146, 148, 1980, Ed. Medicală, Bucureşti. Cowley, S.M.; Hoare, S.; Mosselman, S.; Parker, M.G.: Estrogen receptor α and β from heterodimers on DNA. J. Steroid. Biochem., 272, 19858-19862, 1997. De Castro, S.L.: Propolis: Biological and pharmacological activities. Therapeutic uses of this bee-product. Ann. Rev. Biomed. Sci., 3, 49-83, 2001. Freinkel R.K.: Carbohydrate metabolism of epidermis. In: Biology of the skin, chap. 14, pp. 452-454, 2001. Edited by R.K. Freinkel and D. Woodley, Parthenon Publishing Group, London. Freinkel, R.K.: Metabolism of the skin. In: Biology of the skin, chap. 11, pp. 191-199, 2001. Edited by R.K. Freinkel and D. Woodley, Parthenon Publishing Group, London. Havsteen, B.H.: The biochemistry and medical significance of the flavonoids. Pharmacol. Therapeut., 96, 67-202, 2002.

264