|Year : 2009 | Volume
| Issue : 2 | Page : 66-71
Dermal Wound Healing Effect of Pistacia Lentiscus Fruit's Fatty Oil.
N Boulebda1, A Belkhiri2, F Belfadel3, A Bensegueni4, L Bahri3
1 Laboratoire de Phytopharmacognosie et Pharmacochimie des Substances Naturelles. Urmedco, Faculté de Médecine, UM Constantine. Algérie - 25000, Algeria
2 Laboratoire de Phytopharmacognosie et Pharmacochimie des Substances Naturelles. Urmedco, Faculté de Médecine, UM Constantine. Algérie - 25000; Laboratoire de Pharmaco-Toxicologie, UM Constantine. Algérie - 25000, Algeria
3 Département de Biologie, Faculté des Sciences biologiques, UM Constantine, Algérie - 25000, Algeria
4 Département des sciences vétérinaires, faculté des Sciences biologiques, UM Constantine, Algérie – 25000, Algeria
|Date of Submission||28-Jan-2009|
|Date of Acceptance||16-Feb-2009|
|Date of Web Publication||2-Jan-2010|
Laboratoire de Phytopharmacognosie et Pharmacochimie des Substances Naturelles. Urmedco, Faculté de Médecine, UM Constantine. Algérie - 25000; Laboratoire de Pharmaco-Toxicologie, UM Constantine. Algérie - 25000
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Several natural products have been shown to accelerate wound healing process. The present study was undertaken to evaluate the effect of Pistacia lentiscus fruits fatty oil on cutaneous wound healing in rat, and to compare this effect to that of saponifiable and unsaponifiable oily fractions. Full-thickness excision wounds were made on the back of anesthetised rats. The fruit's oil and the two fractions were assessed together with a conventional drug, i.e. Madecassol® . Preparations were topically applied on the area of excised wounded once a day and assessed for a period of 26 days. During this period, wound area was measured and photographically documented. Wound contraction, expressed as percentage, was significantly (P<0.05) enhanced in the presence of Pistacia lentiscus oil, unsaponifiable oily fraction and Madecassol® treatments compared to the control, untreated animals. Furthermore, wound healing potentially effect was more pronounced in case of the oily unsaponifiable fraction-treated group compared to the others groups. Results clearly substantiate the healing potential effect on wound of a topic application of the Pistacia lentiscus fruits fatty oil and its unsaponifiable fraction.
Keywords: Pistacia lentiscus, dermal wound, oil, healing activity
|How to cite this article:|
Boulebda N, Belkhiri A, Belfadel F, Bensegueni A, Bahri L. Dermal Wound Healing Effect of Pistacia Lentiscus Fruit's Fatty Oil. Phcog Res 2009;1:66-71
|How to cite this URL:|
Boulebda N, Belkhiri A, Belfadel F, Bensegueni A, Bahri L. Dermal Wound Healing Effect of Pistacia Lentiscus Fruit's Fatty Oil. Phcog Res [serial online] 2009 [cited 2021 Jan 25];1:66-71. Available from: http://www.phcogres.com/text.asp?2009/1/2/66/58134
| Introduction|| |
Wound healing is a complex process that involves a series of biochemical and cellular reactions, beginning with homeostasis, repithelialisation, granulation tissue formation and remodelling of the extracellular matrix ,,, . Searches for safer and effective wound healing agents from medicinal plants have become more important areas of active research. According to traditional medicine, wounds have been treated topically with various medicinal herbs or their extracts since time immemorial. Many plants have been shown to possess therapeutic potential as promoters of wound healing. Aloe vera  , Centella asiatica  , Pterocarpus angolensis  , Channa striatus-cetrimide  , Datura alba  , Terminalia chebula  , Cinnamomum zeylanicum  , Pterocarpus santalinus  , Phellinus gilvus  , Napoleons imperialis, Ocimum gratissimum and Ageratum conyzoides , Butea monosperma  , Cassia fistula  , Plagiochasma appendiculatum  , Embelia ribes  , Sphaeranthus indicus  , Tragic involucrata  , Tephrosia purpurea  .
Pistacia lentiscus is a small tree very widespread in the whole of Mediterranean basin to the South-West of black Sea. Its use as a medicinal herb is known since antiquity  . The mastic obtained by incising the trunk contains 30% of resin, an essential oil (2%) and a bitter principle. The leaf contains flavonoids, an essential oil and tannins. The fruit is a small drupe, from which is expressed a fatty oil  .
Pistacia lentiscus is well known medicinal plant in the Mediterranean region. All parts of the plants posses medicinal uses. The uses of its roots, leaves, fruits and mastic have been reported in many traditional pharmacopoeias. However, medicinal virtues of the fatty fruit's oil are particularly known in North Africa, in the eastern region of Algeria to Tunisia. The fruit oil is used internally for respiratory allergies, externally to treat sore throats, and locally applied for wounds and burns , .
The aerial parts of Pistacia lentiscus L. has traditionally been used in the treatmant of hypertension and possesses stimulant and diuretic properties ,, . The leaves of the plant were found to have antimicrobial, antioxidant, hepatoprotective activities ,,,, . The resin obtained from mastic Pistacia lentiscus is known as antimicrobial, antioxidant, antiatherogenic, anti-ulcer, antiproliferatif agent ,,,,,, . Pharmacological evaluation of this species has also revealed an essential oil with an anti-inflammatory, antifungal, antibacterial and antioxidant activities ,,,, .
Despite its wide use in North Africa traditional medicine as a well known wound and burn healing traditional remedy, a little has been done on pharmacological evaluation of the fatty oil of Pistacia lentiscus fruits. This present study is the first report dealing with the in vivo experimental evaluation of the wound healing effect of Pistacia lentiscus fruit's oil.
| Material and Methods|| |
Plant materials and fatty oil expression
Pistacia lentiscus L. (Anacardiaceae) was selected as a potential wound healing popular remedy based on ethnopharmacological information, provided by the local communities. Fresh ripe fruits (1 kg) of Pistacia lentiscus L were collected in mid November 2006 from the locality of Mila (50 miles North West of Constantine, Algeria). Plant was properly identified and voucher specimen (n° PL1106) was deposited in pharmacognosy and botany laboratory of the faculty of medicine, Mentouri University of Constantine (Algeria). The freshly harvested fruits of Pistacia lentiscus were extracted according to traditional manner, by cold expression using a traditional screw press to deliver, after filtration, clear green yellowish oil (225 g).
Saponification of oil
An oily fraction (50 g) was added to 200 ml of alkaline alcoholic solution (KOH 0.5N) and heated for 1 hour in a reflux set. After cooling, alcohol was removed under reduced pressure using a rotavapor. 100 ml of distilled water was then added to the remaining residue and the aqueous solution was exhaustively extracted with freshly distilled diethyl ether (3x150 ml) to remove completely the organic fraction. The latter was dried over anhydrous sodium sulphate Na 2 SO 4 before total evaporation of the organic solvent to deliver 1.54 g of unsaponifiable fraction, denote thereafter (UNSAP). The remaining aqueous fraction was acidified to neutral pH and extracted with distilled diethyl ether (3x150 ml) to remove completely the free fatty acid containing saponifiable fraction (18.87 g) , referred to thereafter as (SAP).
Adult male albino Wistar rats weighting 300 ± 20 g were used. Animals were fed on a commercial pellet diet and water ad libitum. All rats were acclimatized to laboratory hygienic conditions 10 days before starting the experiment.
The animals were divided in six groups of five (05) animals each: group I: Control (untreated group, CT), group II: Madecassol-treated group (MAD), group III: Pistacia fruit's oil-treated rats (OIL), group IV: Saponifiable oily fraction-treated rats (SAP), group V: Unsaponifiable oily fraction-treated rats (UNSAP) and group VI: Paraffin oil-treated rats (PRF as Vehicle).
The study was carried out following the guidelines of the principals of Laboratory animal Care "Guide for the Care and Use of Laboratory Animals" (DHHS, 1985)
Excision wound model
Twenty four (24) hours before the beginning of the wound healing experiment, the dorsal skin of rats were (was) shaved. After 24 hours, all animals were anesthetized by intramuscular injection of Ketamin chlohydrate (15 mg/kg). A predetermined dorsal area (0.59 in x 0.39 in) was first sterilized (70% alcoholic solution) and was then inflicted by cutting away a full thickness of skin. The wounds were left undressed to the open environment and the animals were kept individually in separates cages.
Animal were treated with a daily topic administration of 15 mg/kg from each of Pistacia lentiscus oil (OIL), saponifiable (SAP), unsaponifiable fraction (UNSAP), Madecassol® (MAD) as reference healing drug ,, and paraffin oil as vehicle , . The used Pistacia based phytopreparations (OIL, SAP, UNSAP) were dissolved in paraffin oil (10 %, v/v) before testing. 50 ml of each tested products was applied slowly, using an insulin syringe, from the central point extending outside the wound area to ensure inclusion of the edges.
Measurement of wound area
Throughout the experimentation period (26 days), measures of wound area were made on days 0, 2, 6, 10, 14, 18, 22 and finally 26 days using a electronic digital calliper (Fukuoka Japan); precision 0,001 inch) and tracing wound margin using a transparent paper  . Percentage wound contraction was determined using the following formula  :
P.W.C. = [Healed area / Original wound area] x 100.
Wound areas were photographed documented using a professional AGFA780c camera equipped with macro zoom.
Paraffin liquid was purchased from Grifrer Barbezat, Pharmacien, Decines (Rhône), Madecasol® (powder 2%) from Ind. Farmaceutica, Nova Argentia, Ketamine hydrochloride injection from Bioniche, Canada. All chemicals were of analytical grade.
Results are expressed as mean ± SD. of at five animals in each group. The results were analyzed statistically using Student's t-test to identify the differences between groups. The data were considered significantly different at P < 0.05.
| Results and Discussion|| |
Healing was assessed by morphological evaluation of the wound at regular time intervals for a period of 26 days. Contraction of wound for treated and untreated animal groups were measured and results were expressed as percentage wound contraction (P.W.C.) and shown in [Table 1] and [Figure 1].
At day 2 after wounding, no significant wound healing effect was observed for treated and untreated groups. Values of PWC, observed at 6, 10, 14 and 18, clearly indicates that evolution of healing process is significantly (P<0.05) potentiated in case of UNSAPtreated group respectively (36.0, 53.5, 63.0, and 86.0 %) and at a lesser extend for OIL-(15.7, 30.4, 64.0 and 92.0 %) and MAD-(16.0, 19.5, 58.0 and 88.5) treated groups, when compared to control CT- (16.5, 14.5, 38.5 and 65.0 %) and paraffin oil PAR-(15.0, 20.0, 35.2, 74.6) groups.
Its worth noting, that at time interval of 6 to 10 days after wounding, the values of PWC no significantly varied in case of CONT- (from 16.5 to 14.5 %), MAD-(from 16.0 to 19.5%), SAP-(from 20.6 to 16.0%) and PAR-(15.0 to 20.0%) treated groups. However for the same period (6 to 10 days), PWC values was significantly upward for UNSAP-group (from 36.0 to 53.5%, p<0.01) and OIL-treated group (from 15.7 to 30.4%, p<0.05). Furthermore, daily visually observations indicates the presence of signs of infection around wounds area of CONT-, MAD-, SAP and PAR-treated rats, and absence of such infections for UNSAP- and OIL-treated groups.
Finally, from 22 to 26 days, PWC values evolution in different groups become no statistically different. Time for wound closure for different groups (treated and untreated rats) are comparable and all showed effective wound healing process (96% - 99.8 %) after 26 days treatment.
Results of area measurements, expressed as PWC, showed in [Table 1] and [Figure 1] clearly indicate a wound healing potentiating effect in Pistacia lentiscus fruit's oil (OIL) and its unsaponifiable fractions (UNSAP) treated rats. However, this effect seems not clearly established in case of oily saponifiable (SAP) fraction.
Healing potentiating effect of UNSAP-treated animals is distinct and more pronounced, from 6 to 10 days after wounding, contrasting to the rest of groups where the healing process was distinctly slowed, as confirmed by visually observation of wound animals. Inspection of wound area confirmed the presence of signs of infection in most animals, except those treated with fruit's oil and oily unsaponifiable fraction. Probably this observation is due to the presence of potential anti-microbial effect of oily unsaponifiable fraction of the Pistacia lentiscus fruits. This may justify the light observed advantage of the oily unsaponifiable fraction with regard to the Madecassol as reference healing agent. Antimicrobial effect is reported from leaves, mastic gum and essential oil of Pistacia lentiscus ,,, . No report of study in literature is concerned by antimicrobial effect of the fatty oil or its unsaponifiable fraction.
Morphological evaluation indicates that healing potentiating effect of oily unsaponifiable fractions was specifically observed in period from 6 to 18 days after wounding.
According to previous studies, this period (6 to 18 days) might represent the proliferative phase of the healing process ,, . This step might be affected by phytochemicals from the UNSAP-fraction. Preliminary phytochemical investigations of the latter fraction has revealed the well-know unsaponifiable containing compound, such as tocopherols and phytosterols. Many biological and pharmacological activities have been associated with such compounds. In particularly, phytosterols have been previously found to exhibit anti-inflammatory and anti-oxydant activities , and tocopherols (e.g. vitamine E) are major anti-oxydant agents in cellular membranes and plasmatic lipoproteins and have hypolipimiant and immunostimulant activities , . Its worth asking whether such compounds are implicated in the wound healing process observed particularly for the UNSAP-fraction treated rat.
| Conclusion|| |
It may therefore be concluded that under present working condition Pistacia lentiscus fruit's oil and particularly its unsaponifiable fraction have been determined as active healing agent. This potentiating effect is probably associated with UNSAP fraction containing compounds. Studies are underway to establish nature of the bioactive photochemicals associated with the observed antimicrobial and healing effects and the mechanism involved.
| References|| |
|1.||P. Evans. The healing process at cellular level: a review. Physio therapy 66: 256-259 (1980). |
|2.||M. Calvin. Cutaneous wound repair. Wounds 10: 12-32 (1998). |
|3.||A.J. Singer and R.A. Clark. Cutaneous wound healing. N. Engl.J. Med 341(10): 738-746 (1999). |
|4.||S. Enoch and D. John Leaper. Basic science of wound healing. Surgrery 23(2):37-42. (2005). |
|5.||S.W. Choi, B.W. Son, Y.S. Son, Y.I. Park, S.K. Lee and M.H. Chung. The wound healing effect of a glycoprotein fraction isolated from Aloe vera. British Journal of Dermatology 145: 535-545 (2001). |
|6.||L. Suguna, P. Sivakumar and G. Chandrakasan. Effects of Centella asiatica extract on dermal wound healing in rats. Indian J Exp Biol 34: 1208-1211 (1996). |
|7.||Hutchings, A.H. Scott, G. Lewis and A. Cunningham, Zulu Medicinal plants : An inventory, (University of Natal Press, Pietermaritzburg, 1996). |
|8.||S. Hj Baie and K.A. Sheikh. The wound healing properties of Channa striatus-cetrimide cream-wound contraction and glycosaminoglycan measurement. Journal of Ethnopharmacology 73: 15-30 (2000). |
|9.||K.S. Priya, A. Gnanamani, N. Radhakrichnan and M. Babu. Healing potential of Datura alba on burn wounds in albino rats. Journal of Ethnipharmacology 83: 193-199 (2002). |
|10.||L. Suguna, S. Singh, P. Sivakumar, P. Sampath and G. Chandrakasan. Influence of Terminalia chebulla on dermal wound healing in rats. Phytother Research 16: 227-231 (2002). |
|11.||J.V. Kamath, A.C. Rana and A.R. Chowdhury. Pro-healing effect of Cinnamomum zeylanicum bark. Phytotherapy research 17: 970-972 (2003). |
|12.||T.K. Biswas, L.N. Maity and B. Mukherjee. Wound healing potential of Pterocarpus santalinnus Linn.: a pharmacological evaluation. The Internatinal Journal of Lower Extremity Wounds 3: 143-150 (2004). |
|13.||J.S. Bae, K.H. Jang and H.K. Jin,. Polysaccharides isolated from Phellinus gilvus enhances dermal wound healing in streptozotocin-induced diabetic rats. J. Vet. Sci. 6: 161-164 (2005). |
|14.||K.F. Chah, C.A. Eze, C.E. Emuelosi and C.O. Esimone Antibacterial and wound healing properties of methanolic extracts of some Nigerian medicinal plants. Journal of Ethnopharmacology 104: 164-167 (2006). |
|15.||M. Sumitra, P. Manikandan and L. Suguna. Effect of Butea monosperma on dermal wound healing in rats. The International Journal of Biochemistry & Cell Biology 37: 566-573 (2005). |
|16.||M.S. Kumar, R. Sripriya, H.V. Raghavan and P.K. Sehgal. Wound healing potential of Cassia fistula on infected Albino rat Model. Journal of Surgical Research 131: 283-289 (2006). |
|17.||M. Singh, R. Govindarajan, V. Nath, A.K. Singh Rawat and S. Mehrotra. Antimicrobial, wound healing and antioxidant activity of Plagiochasma appendiculatum Lehm. Et Lind. Journal of Ethnopharmacology 107: 67-72 (2006). |
|18.||H.M. Kumara Swamy, V. Krishna, K. Shankarmurthy, B. Abdul Rahiman, K.L. Mankani, K.M. Mahadevan, B.G. Harish and H. Raja Naika. Wound healing activity of embelin isolated from the ethanol extract of leaves of Embelia ribes Burn. Journal of Ethnopharmacology 109: 529-534 (2007). |
|19.||F. Sadaf, R. Saleem, M. Ahmed, A. Syed Iqbal and L. Navaidul-Zafar. Healing potential of cream containing extract of Sphaeranthus indicus on dermal wounds in Guinea pigs. Journal of Ethnopharmacology 107: 161-163 (2006). |
|20.||R. Perumal Samy, P. Gopalakrishnakone, M. Sarumathi and S. Ignacimuthu. Wound healing potential of Tragia involucrate extract in rtas. Fitoterapia 77: 300-302 (2006). |
|21.||S. Lodhi, R. Singh Pawer, A. Pal Jain and A.K. Singhai. Wound healing potential of Tephrosia purpurea (Linn.) Pers. In rats. Journal of Ethnopharmacology 108: 204-210 (2006). |
|22.||D. Palevitch and Z. Yaniv. Medicinal plants of the Holy Land, (Modan Publishing House, Tel Aviv, Israel, 2000). |
|23.||L. Boulos, Medicinal plants of North Africa, (Reference Publications Inc. , Michigan, 1983) 143. |
|24.||K. Boukef and H. R.Souissi. Contribution ΰ l'etude des plantes medicinales en medecine populaire en Tunisie. Rev. Soc. Pharm. Tunisie. 2(3): 34- 35. (1982). |
|25.||R.Y. Bentley and H. Trimen, Medicinal plants, (J. and A Churchill, London, 1980) 68. |
|26.||Villar, M.J. Sanz and M. Payo. Hypotensive effect of Pistacia lentiscus L. Int J Crude Drug Res 25: 1-3. (1987). |
|27.||M.J. Sanz, M.C. Terencio and M. Paya. Isolation and hypotensive activity of a polymeric procyanidin fraction from Pistacia lentiscus L. Pharmazie 47: 466-471 (1992). |
|28.||M.S. Ali-Shtayeh, R.M.R. Yaghmour, Y.R. Faidi, K.A.L. Salem and M.A. Al-Nuri. Antimicrobial activity of 20 plants used in folkloric medicine in the palestinian area. Journal of Ethnopharmacology 60: 265-271 (1998). |
|29.||S. Janakat and H. Al-Merie. Evaluation of hepatoprotective effect of Pistacia lentiscus, Phillyrea latifolia and Nicotiana glauca. Journal of Ethnopharmacology 83: 135-138 (2002). |
|30.||S. Kordali, A. Cakir, H. Zengin, M.E. Duru. Antifungal activities of the leaves of three Pistacia species grown in turkey. Fitoterapia 74: 164-167 (2003). |
|31.||P. Ljubuncic, H. Song, U. Cogan, H. Azaizeh and A. Bomzon. The effects of aqueous extracts prepared from the leaves of Pistacia lentiscus in experimental liver disease. Journal ofEthnopharmacology 100: 198-204 (2005). |
|32.||N. Benhammou, F.A. Bekkara and T.K. Panovska. Antioxidant and antimicrobial avtivities of the Pistacia lentiscus and Pistacia atlantica extracts. African Journal of Pharmacy and Pharmacology 2: 022-028 (2008). |
|33.||A.H.Y. Abdel-Rahman and A.M.Y. Soad. Mastic as antioxidant. Journal of the American Oil Chemists Society 52: 423-429 (1975). |
|34.||P. Marone, L. Bono, E. Leone, S. Bona, E. Carretto and L. Perversi. Bactericidal activity of Pistacia lentiscus mastic gum against Helicobacter pylori. Journal of Chemotherapy 13: 611-614 (2001) |
|35.||G.V.Z. Dedoussis, A.C. Kaliora, S. Psarras, A. Chiou, A. Mylona, N.G. Papadopoulos and N.K. Andrikopoulos. Antiatherogenic effect of Pistacia lentiscus via GSH restoration and downregulation of CD36 mRNA expression. Atherosclerosis 174: 293-303 (2004). |
|36.||M. J. Al-Habbal, Z. Al-Habbal and F.U. Huwez. A doubleblind controlled clinical trial of mastic and placebo in the treatment of duodenal ulcer. Clin. Exp. Pharmacol. Physiol. 11:541-544 (1984). |
|37.||M. S. Al-Said, A. M. Ageel, N. S. Parmar and M. Tarik. Evaluation of mastic a crude drug obtained from Pistacia lentiscus for gastric and duodenal anti-ulcer activity. Journal of Ethnopharmacology 15: 271-278 (1986). |
|38.||K.V. Balan, J. Prince, Z. Han, K. Dimas, M. Cladaras, G.H. Wyche, N.M. Sitaras and P. Pantazis. Antiproliferative activity and induction of apoptosis in human colon cancer cels treated in vitro with constituents of a product derived from Pistacia lentiscus L. var. Chia. Phytomedicine 14: 263-172 (2007). |
|39.||A.J.N. Prichard. The use of essentiaal oils to treat snoring. Phytotherapy Research 18: 696-699 (2004). |
|40.||E.M. Giner-Larza, S. Manez, M.C. Recio, R. Giner-Pons, J.M. Prieto and M. Cerda-Nicolas. Oleanolic acid, a 3-oxotriterpene from Pistacia, inhibits leukotriene synthesis and has antiinflammatory activity. European Journal of Pharmacology 428: 137-143 (2001). |
|41.||M.E. Duru, A. Cakir, S. Kordali, H. Zengin, M. Harmadar, S. Izumi and T. Hirata. Chemical composition and antifungal properties of essential oils of three Pistacia species. Fitoterapia 74: 170-176 (2003). |
|42.||L. Iauk, S. Ragusa, A. Rapisarda, S. Franco and V.M. Nicolosi. In vitro antimicrobial activity of Pistacia lentiscus L. extracts: preliminary report. Journal of Chemotherapy 8: 207-209 (1996). |
|43.||P. Magiatis, E. Melliou, A.L. Skaltsounis, I.B. Chinou and S. Mitaku. Chemical composition and antimicrobial activity of the essential oils of Pistacia lentiscus var.chia. Planta Med 65:749-751 (1999). |
|44.||C. Gardeli, V. Papageorgiou, A. Mallouchos, K. Theodosis and M. Komaitis. Essential oil composition of Pistacia lentiscus L. and Myrtus communis L.: Evaluation of antioxidant capacity of methalonic extracts. Food Chemistry 107: 1120-1130 (2008). |
|45.||J.P. Pointel, H. Boccalon, M. Cloarec, J.M. Ledevehat. Titrated extract of Centella asiatica (TECA) in the treatment of venous insufficiency of the lower limbs. Angiology 38: 46-50 (1987). |
|46.||Poizot and D.C.R. Dumez. Modification of the kinetics of healing after iterative exeresis in the rat. Action of a triterpenoid and its derivatives on the duration of healing. Acad. Sci. [D] 286: 789-792 (1978). |
|47.||J.C. Lawrence. The effect of asiaticoside on guinea pig skin. J. Invest. Dermatol. 49: 95-96. (1967). |
|48.||P. Martin. Wound healing-aiming for perfect skin regeneration. Science 276 (5309): 75-81. (1997). |
|49.||C. Corbiθre. Comparaison de l'effet anti-proliferatif de trios steroids vegetaux (diosgenine, hecogenine, tigogenine) sur la lignee 1547 d'osteosarcome humain. Implication de la mitochondrie et de la cyclooxygenase-2 dans l'appoptose induite par la diosgenine sur les lignees 1547, HEp-2 (laryngocarcinome) et M4Beu (melanome). Thθse de doctorat, Universite de Limoge, Limoges, France. 2003. |
|50.||J. Moreno. Effect of olive minor components on oxidative stress and arachidonic acid mobilization and metabolism by macrophages RAW 264.7. Free Radical Biology and Medicine 35, 1073-1081 (2003). |
|51.||M. Chavance, B. Herbert, C. Fournier, C.Janot and G.Vernhes Vitamin status, immunity and infections in an elderly population. Eur. J. Clin. Nutr. 43: 825-837 (1989). |
|52.||Y. Wang, D.C. Hwang, B. Licing and R.R. Watson. Nutritional status and immune responses in mice with murine AIDS are normalizd by vitamin E supplementation. J. Nutr 124, 2024-2032 (1994). |