Persistent microvascular hyperpermeability to plasma proteins is a characteristic feature of normal wound healing. Does the leaf essential oil of Ocimum gratissimum heal wounds by promoting this feature? Evan’s blue dye (20mg/kg body weight) in normal saline was administered intravenously through marginal ear vein of experimental rabbits (n=5). Each animal served as its own control. One hour after Evan’s blue dye administration, 0.1ml each of Ocimum oil, histamine dihydrochloride (30µg/ml) and normal saline were randomly administered by intra-dermal injection at the prepared sites on each of the animals. Increase in vascular permeability was assessed by dye effusion test. Analysis of the differences in vascular permeability between treatment groups showed that, Ocimum oil, in intensity and duration, was significantly (

Agbani, E. O.

Fakoya, F. A.

Iwalewa, E. O.

Orafidiya, 'Lara O.

ATHMSI Journals

 253Afr. J. Trad. CAM (2005) 2 (3): 253 - 258        Research Paper                                                              Afr. J. Traditional, Complementary and Alternative Medicines www.africanethnomedicines.net ISSN 0189-6016©2005          VASCULAR PERMEABILITY- INCREASING EFFECT OF THE LEAF ESSENTIAL OIL OF OCIMUM GRATISSIMUM LINN AS A MECHANISM FOR ITS WOUND HEALING PROPERTY.   ’Lara O. Orafidiya*a, F. A. Fakoyab, E. O. Agbanic and E. O Iwalewad aDepartment Of Pharmaceutics, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria, bDepartment Of Anatomy and Cell Biology, Faculty of Basic Medical Sciences, College Of Health     Sciences, Obafemi Awolowo University, Ile-Ife, Nigeria cDepartment of Clinical and Administrative Pharmacy, Faculty of Pharmacy, Niger Delta University, Wilberforce Island, Nigeria.dDepartment of Pharmacology, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria. *E-mail  laraoraf@yahoo.com  Abstract Persistent microvascular hyperpermeability to plasma proteins is a characteristic feature of normal wound healing. Does the leaf essential oil of Ocimum gratissimum heal wounds by promoting this feature? Evan’s blue dye (20mg/kg body weight) in normal saline was administered intravenously through marginal ear vein of experimental rabbits (n=5). Each animal served as its own control. One hour after Evan’s blue dye administration, 0.1ml each of Ocimum oil, histamine dihydrochloride (30µg/ml) and normal saline were randomly administered by intra-dermal injection at the prepared sites on each of the animals. Increase in vascular permeability was assessed by dye effusion test. Analysis of the differences in vascular permeability between treatment groups showed that, Ocimum oil, in intensity and duration, was significantly (p<0.05) more effective in increasing cutaneous capillary permeability over a 24h period after treatment. The ability of Ocimum oil in increasing vascular permeability may be one of the factors that contribute to its wound healing property.  Key words: Ocimum gratissimum, vascular permeability, mechanism of action.  Introduction  Wounds occurs as a result of physical injuries that break and expose the skin epidermal and dermal layers, causing a damage to the blood vessels and leading eventually to bleeding. Wound healing is a complex sequence of events intiated by the stimulus of injury to tissues. These events involve four main processes: (a) coagulation, (b)inflammation and debridement of wound, (c)epithelia repairs and (d)tissue remodeling and colleagen deposition (Rashed et al 2003). It is known that   254Afr. J. Trad. CAM (2005) 2 (3): 253 - 258   any agent which accelerates one or two of the above events is a promoter of wound healing (Abu-Al-Basal 2001). Medicinal plant extracts, decoctions and concotions have been in use traditionally to treat various skin lesions(burns and wounds), and these plants have demonstrated wound healing properties in various animal models (Suguna et al., 1996., Chithra et al 1998., Bale and Sheikh 2000a, b., Abu-Al-Basal 2001, Rashed et al 2003 ). Formulations of the leaf essential oil of Ocimum gratissimum Linn., Family Lamiaceae, (Ocimum oil) have been made in a variety of bases as topical antiseptics for use in the treatment of minor wounds, boils and pimples (Orafidiya et al, 2001) basically for its established antimicrobial properties (El-Said et al, 1969; Begum et al, 1993). Ocimum oil contains mainly thymol, (48.1%), p-cymene, (12.5%), and 40 other constituents in trace amounts (Martins et al, 1999).. The oil significantly resolves the inflammatory lesions of Acne vulgaris (Orafidiya et al, 2002, 2004a), promotes the inflammatory and proliferative phases of wound healing and possesses proven efficacy against a variety of clinical isolates and type strains of micro-organisms including wound contaminants (Orafidiya et al, 2003). Being a potential wound healing agent, we sought to investigate possible mechanisms for its wound healing property.  Material and methods Plant material and preparation of Ocimum oil.  The leaves of O. gratissimum were collected and identified as earlier reported (Orafidiya et al, 2003, 2004a, b, c) Ocimum oil was extracted from the fresh leaves by hydro-distillation using the British Pharmacopoeia method. The oil was then stored in glass containers in the refrigerator until needed.  Experimental animals  The “principles of laboratory animal care” [NIH Publication No. 85-23] were followed in this study. Five adult albino rabbits (average weight 4.4kg) were used for the experiment. They were procured from the laboratory animal resource section of the College of Health Sciences, Obafemi Awolowo University Ile-Ife. They were acclimatized for 10 days before commencement of the experiment and maintained on rabbit pellets (Ladokun Feeds, Ibadan, Nigeria) and water ad libitum.  Preparation of animal skin sites  Skin sites were prepared 24h prior to administration of the test samples by clipping the hairs on the flanks, extending toward the dorsal region of the animals. All the sites were depilated with Royal Crown® Depilatory Shaving powder (J. Strickland & Co. Memphis. TN 38106 USA) which was applied for 5 min and rinsed off subsequently with fresh water. Three skin sites of approximately 4cm2 each (on either side) were demarcated on each rabbit for the application of Ocimum oil, control (normal saline) and reference agent (histamine dihydrochloride, 30µg/ml).      255Afr. J. Trad. CAM (2005) 2 (3): 253 - 258  Animal treatment and evaluation  Evan’s blue dye (20mg/kg body weight) in normal saline was administered intravenously through the marginal ear vein of the rabbit. Each animal served as its own control. One hour after Evan’s blue dye administration, 0.1ml each of Ocimum oil, histamine dihydrochloride (30µg/ml) and normal saline were randomly administered by intra-dermal (i.d.) injection at the prepared sites in each of the 5 rabbits. Increased vascular permeability was assessed by dye effusion and observations were recorded at 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 18 and 24hr after i.d. injections of the test and control agents. The intensity of the reaction was graded grossly as 1=negligible, 2=mild, 3=moderate and 4=marked; according to the method of Miles and Whihelm (1955) and expressed as average increase in vascular permeability.  Statistical analysis  Group data difference was evaluated by a modified t-test [n<20] (Gardiner, et al, 1998). Statistical analysis was done using the SPSS version 11.0 software program package (SPSS Inc., Chicago, IL). Test for statistical significance was by paired sample student t-test and one- way analysis of variance at 95% confidence interval.  Results  The vascular permeability-increasing (VPI) effect of Ocimum oil was grossly significant (p<0.01) relative to reference and control agents from 0.5h onwards after i.d. injection (Figure 1). Ocimum oil VPI activity reached moderate levels (3.2±0.84) within the first 1h, became marked (3.82±0.22) first at about 3h, and was maintained over a 6h period (3h→9h) after i.d. injection of Ocimum oil (Figure 1). VPI activity of the oil declined very slowly; mild values (2.07±0.64) were recorded grossly between 10h and 18h. Activity was negligible and comparable to histamine (p=0.07) 22h after oil administration (Figure 1). Normal saline showed no VPI activity after administration; sites appeared as when untreated throughout the 24h test period.  VPI activity of histamine was mild (3h→4h) and negligible (0.5h→2h, 6h→24h) during the test period; maximum activity (1.6±0.41) was recorded at 3h after i.d. injection (Figure 1). One-way analysis (ANOVA) of the differences in vascular permeability between treatment groups showed that, Ocimum oil, in intensity and duration, was significantly (p<0.05, Std. Error= 0.14-0.15) most effective in increasing cutaneous capillary permeability over a 24h period after administration.   Discussion Vascular hyperpermeability is a characteristic feature of wound healing that has been found to continue at wound sites for a considerable time after the cessation of overt bleeding (Brown et al, 1988; Breuing et al, 1992). Thus, blood vessels continue to leak plasma proteins for several days after the initiating trauma has ceased and even for a short time after a complete epidermal covering has been restored. The result is persistent extravasations of fibrinogen and ongoing deposition of provisional fibrin gel matrix, events that favor the continued induction of granulation tissue. (Clark et al, 1988; Van De Water et al 1992).   256Afr. J. Trad. CAM (2005) 2 (3): 253 - 258      Figure 1   Vascular permeability-increasing effect of the leaf essential oil of Ocimum gratissimum Linn**** ****** ** *********00.511.522.533.544.50 5 10 15 20 25 30Time  (h) ** variation significant at p<0.01 and * at p<0.05 between group) Average increase in vascular permeabilty Normal saline +^ Histamine +  ̂Ocimum oil +  ̂ +^ (n=5; 1=negligible,  2=mild, 3=moderate, 4=marked).     This ascertion is buttressed when Evan’s blue dye, injected intravenously , forms coloured complex with serum albumin, which leaks out into the extracellular space only when vascular capillary permeability is sufficiently increased to allow the passage of albumin molecules leading to blue discoloration of the skin (Carpenter and Lynn, 1991). The area and intensity of the blue coloration is proportional to increase in vascular permeability within limits  (Spector, 1958). The results of this study showed that, Ocimum oil, in intensity and duration, was significantly (p<0.05) more effective in increasing cutaneous capillary permeability over a 24h period after administration (Figure 1). Mechanism for the initiation and maintenance of persistent vascular hyperpermeability in healing wounds has been reported to be due to a vascular permeability factor (VPF) (Clauss et al, 1990). VPF is a selective mitogen for endothelial cells and a reported  chemotactic for monocytes (Clauss et al, 1990) that enhances the permeability of local venules and small veins with a potency some 50,000 times that of histamine (Senger et al, 1990; Keck et al, 1989).  Brown et al, (1992) reported that VPF and its mRNA are expressed in normal keratinocytes, both in situ and in tissue culture, and that VPF expression is markedly increased in the activated, migrating keratinocytes involved in wound healing. Hints that Ocimum oil invokes inflammatory responses and promotes the proliferation of matrix keratinocytes (which are physiological events that occur in wound healing) were    257 Afr. J. Trad. CAM (2005) 2 (3): 253 - 258   given in recent studies investigating the toxicity and the effects of the oil in cyclophosphamide induced hair loss (Orafidiya et al, 2004b; 2004c).  In conclusion, this study has demonstrated the ability of Ocimum oil in increasing vascular permeability, a possible additional feature to its wound healing property.  References  1. Abu-Al-Basal, M. (2001) The Influence of Some Local Medicinal Plant Extracts on Skin Wound Healing Activity; Evaluated by Histological and Ultra-Structural Studies. Ph.D. Thesis, University of Jordan, Amman, Jordan. 2. Bale, S.Hj., and Sheikh, K.A. ( 2000a) The wound healing properties of Channa striatus-cetrimide cream-tensile strength measurement. J.  Ethnopharmacol.71:  93-100.  3. Bale, S.Hj., and Sheikh, K.A. ( 2000b) The wound healing properties of Channa striatus-cetrimide cream-wound contraction and glycosaminoglycan measurement J. Ethnopharmacol. 73: 15-30. 4. Begum, J., Yusuf, M., Chowdhury, U., and Wahab, M.A.  (1993) Studies on essential oils for their antibacterial and antifungal properties. Part 1. Preliminary screening of 35 essential oils. Banglasdesh  J. Sci. Ind. Res. 28: 25-34. 5. British Pharmacopoeia, (1988) A137-A138, Her Majesty Stationary Office, London. 6. Breuing, K., E. Eriksson, L. Paul, and D.R. Miller. (1992). Healing of partial thickness porcine skin wounds in a liquid environment. J. Surg. Res. 52: 50. 7. Brown, L.F., L. Van De Water, V.S. Harvey, andH.F. Dvorak. (1988). Fibrinogen influx and accumulation of cross-linked fibrin in healing wounds and in tumor stroma . Am. J. Pathol. 130: 455 . 8. Brown, L.F., Yeo, K.T., Berse, B., Yeo, T., Senger, D.R., Dvorak, H.F., and Van De Water. L. (1992) Expression of Vascular Permeability Factor (Vascular Endothelial Growth Factor) by Epidermal Keratinocytes during Wound Healing. J. Exp. Med. 176: 1375-1379 9. Carpenter S.E and Lynn B. (1981) Vascular and sensory responses of the human skin to mild injury after topical treatment with capsaicin. Br J Pharmacol  73: 755-8 10. Chithra, P., Sajithlal, G.B., Chandrakasan, G. (1998) Influence of Aloe vera on collagen characteristics in healing dermal wounds in rats. Mol.  Cellular  Biochem. 181: 71-76. 11. Clark, R.A .F., and Henson, P.M. (1988) . The Molecular and Cellular Biology of Wound Repair. Plenum Publishing Corporation, New York. 12. Clauss, M., Gerlach, M., Gerlach, H., Brett, J., Wang, F., Familletti, P.C.,  Pan, Y.-C.E., Olander, J.V., Connolly, D.T. and Stern, D. (1990). Vascular permeability factor : a tumor-derived polypeptide that induces endothelial cell and monocyte procoagulant activity, and promotes monocyte migration. J. Exp. Med. 172: 1535 . 13. El-Said, F., Sofowora, E.A., Malcolm, S.A. and Hofer, A (1969). An investigation into the efficacy of Ocimum gratissimum as used in Nigerian native medicine. Planta Medica  17: 195-200.  258Afr. J. Trad. CAM (2005) 2 (3): 253 - 258  14. Gardiner, W.P. and  Gettinby, G.: (Edits) (1998) Experimental design techniques in statistical practise- A practical software base approach.pp 23-26, 1st edition. Horwood pubishing. Chinchester.  15. Keck, P.J ., Hauser, S.D., Krivi, G., Sanzo, K., Warren, T ., Feder, J. and Connolly, D.T. (1989). Vascular permeability factor, an endothelial cell mitogen related to PDGF. Science  246: 1309. 16. Martins, A.P., Salgueiro, L.R., Vila, R., et al (1999) Composition of the essential oils of Ocimum canum, O. gratissimum and O. minimum. Planta Med. 65: 187-189.  17. Miles, A.A. and Whilhelm D.L. (1955) Enzyme like globulins from reproducing the vascular phenomena of inflammation. 1. An active permeability factor and its inhibition in guinea pig serum. Br J Exptl Pathol  36: 71-81 18. Orafidiya, L.O., Oyedele, A.O., Shittu A.O., and Elujoba, A.A (2001)  The formulation of an effective topical antibacterial product containing Ocimum gratissimum leaf essential oil. Inter.  J. Pharmaceu.  224: 177- 183. 19. Orafidiya, L.O., Agbani, E.O., Oyedele, A.O., Babalola, O.O., and  Onayemi, O (2002)  Preliminary clinical tests on topical preparations of Ocimum gratissimum Linn leaf essential oil for the treatment of acne vulgaris. Clin Drug Invest  22:  313-319. 20. Orafidiya, L.O., Agbani, E.O., Abereoje, O.A., Awe, T., Abudu, A., and Fakoya, F. A. (2003). An investigation into the wound healing properties of the essential oil of Ocimum gratissimum Linn. J. Wound Care. 12 : 331-334  21. Orafidiya, L.O., Agbani, E.O., Oyedele, A.O., Babalola, O.O., and  Onayemi. O. (2004a).  Effect of aloe vera gel on the anti acne properties of the essential oil of Ocimum gratissimum Linn leaf.- a preliminary clinical investigation.  Inter. J. Aromather. 14: 15-21. 22. Orafidiya, L.O., Agbani, E.O., Iwalewa, E.O., Adelusola, K.A., and Oyedapo, O.O. (2004b). Studies on the acute and sub-chronic toxicity of the essential oil of Ocimum gratissimum Linn leaf. Phytomedicine 11: 71-76. 23. Orafidiya, L.O., Agbani, E.O., Adelusola, K.A., Iwalewa, E.O., Adebanji O.A., Adediran, E.A.F and Agbani, N.T.(2004c). A study of the effect of the essential oil of Ocimum gratissimum Linn on Cyclophosphamide induced inhibited hair growth Inter. J. Aromather. .14:  119-128  24. Rashed, A.N., Afifi, F.U., Disi, A.M.(2003) Simple evaluation of the wound healing activity of a crude extract of Portulaca oleracea  L. (growing in Jordan) in Mus musculus JVI-1 J. Ethnopharmacol. 88: 131-136. 25. Senger, D.R., Connolly, D.T., Van De Water, L., Feder, J. and Dvorak, H.F. (1990). Purification and NHZ-terminal amino acid sequence of guinea pig tumor-secreted vascular permeability factor. Cancer Res. 50: 1774. 26. Spector, W.G. (1958) Substances which affect capillary permeability. Pharmac. Rev. 10: 475-505. 27. Suguna, L., Sivakumar, P., Chandrakasan, G. (1996). Effects of Centella asiatica extract on dermal wound healing in rats. Ind. J.Exp.Biol. 34: 1208-1211. 28. Van De Water, L., ffrench-Constant, C., and Brown. L. (1992). Fibronectin expression during cutaneous wound healing. In: Fetal Wound Healing. Adzick, N.S., and Longaker, M.J. editors. Elsevier Science Publishing Co., Inc., New York . 281-301.   

English

VASCULAR PERMEABILITY- INCREASING EFFECT OF THE LEAF ESSENTIAL OIL OF OCIMUM GRATISSIMUM LINN AS A MECHANISM FOR ITS WOUND HEALING PROPERTY.

https://journals.athmsi.org/index.php/ajtcam/article/download/29/39/

VASCULAR PERMEABILITY- INCREASING EFFECT OF THE LEAF ESSENTIAL OIL OF OCIMUM GRATISSIMUM LINN AS A MECHANISM FOR ITS WOUND HEALING PROPERTY.

Abstract

Similar works

Full text

Available Versions

ATHMSI Journals