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    Fresh juice of Cocos nucifera was directly used while its aqueous kernel extract (AKCN) was obtained after 72 h soaking of 1:2 (w / v) fresh kernel in chloroform:methanol. The extracts, in the concentrations / strengths of 10, 50 and 100%, were used in the anti-pyretic and antiinflammatory studies. The latter producing a more effective effects in all assays used. This finding has scientifically supported the folkloric used of C.

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    Fresh juice of Cocos nucifera was directly used while its aqueous kernel extract (AKCN) was obtained after 72 h soaking of 1:2 (w / v) fresh kernel in chloroform:methanol. The extracts, in the concentrations / strengths of 10, 50 and 100%, were used in the anti-pyretic and antiinflammatory studies. The latter producing a more effective effects in all assays used. This finding has scientifically supported the folkloric used of C.

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    61 views11 pages

    516 526

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    Adi Indra Wijaya
    Fresh juice of Cocos nucifera was directly used while its aqueous kernel extract (AKCN) was obtained after 72 h soaking of 1:2 (w / v) fresh kernel in chloroform:methanol. The extracts, in the concentrations / strengths of 10, 50 and 100%, were used in the anti-pyretic and antiinflammatory studies. The latter producing a more effective effects in all assays used. This finding has scientifically supported the folkloric used of C.

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    Sournal of Pharmacology and Texivology 1 (6): 516+ ISSN 1816-196% © Academie Joumnals Ine, USA ‘The Anti-inflammatory, Anti-pyretic and Wound Healing Activities of Cocos nucifera (MATAG Types) Fresh Juice and Kernel Extract in Experimental Animals °°Z.A, Zakaria, ‘| Reezal, “AM. Mat kis, MAN. Somehit, "MLR, Sulaiman, °A.HLL Marmin, '. Sidek, ‘SH. Hui, ‘MHA, Rahim and L. Abdul Rabrman "Faculty of Biotechnology and Life Sciences, *School of Medical Science Industry, Universit Industri Selangor, Jalan Zirkon A7/A, Seksyen 7. 440000 Shah Alam, Selangor, Malaysia "Department of Biomedical Sciences, Faculty of Medicine and Health Science, ‘Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia Abstract: The present study was catied out to evaluate the potential of © meifora as antipyretic, ant-inflammatory and wound healing agents. The fiesh juice of C: mctfera (FICN) was directly used while its aqueous kemel extract (AKCN) was obtained after ‘TP soaking of 1:2 (Ww) ies keane in 21 (vv) ehloeofoenemethanol, The extracts, in the coneentrationsstrenaths of 10, 50 and 100%, were used in the anti-pyretic and anti inflammatory studies while these inthe concentration of 100% wers used only in the Wound healing study. The fresh juice and aqueous kemel extuct of C. muctfra exhibited ‘Sgnilant (p<0.05) ant-inflammatory and antipyretie activities and promote wound healing with the fatter producing a more effective effects in all assays used, This finding has scientifically supported the folkloric used of C. reife in the weatment of inflammation, pyrexia and wound. Key words: Cocos muevfra, ati-inflanunatory, antipyretic, wound heal Introduction Covos reifora(C. mucsfora), known in Malay 0s ‘kelapa’, belongs to the fuily of Pama in Areccacae over snditis originated from Malaysia, Polynesia and Southem Asia andis now prolific Jn South Armerica, India and he Pacific Island (Ahmad and Raji, 1993), The juice and kemel (meat) of C. mueifera have been used in Malays folk medicine for numberof ailments such as to relieve fever, stomach upset, headaches, diathea and dysentery (Ahunad and Raji, 1993; Muss, 1998). The {ce has adequate natural minerals and high quality proteins, which are valuable for growth and repair ‘of tc bly (Pehonsch etal, 2000), Medical reseatet has discovered thatthe coconut cil obtained from ‘the kernel consists of monolauri and laurie acid, which helps the immune system in a beneficial ‘manner. The monolaurie acids were use by the body to produce high levels of antisierobial activity (Mid-American Murketing Coep, 2004). Laure acid was the busie of monolaurn and was part of the chemical constituents of sodium Lay! sulfate that has been discovered to promotes health and used in adjunct treatment of viral diseases, Italso found to be of the active chemicals in controlling Human. Immunodeficiency Virus (HIV) disease (Dayrit, 2004). Revent study by Alanis ea. (2005) = Seo of Biecnlogy nd Life Scenes, Uiversi nd Sana, Sala Zion AA Seen 7, 4600 Shah Alay Selangor. alsa “Te oo 225400 Fax. 08-SS137999 316 J. Pharmacol Toxicol, 1 (6): 516-526, 2006 thas demonstrated that the C. mutfora aqueous and methanol extract possess antibacterial properties against the Escericia col, Shigella lene, Shigella sonnei ox Salmonella spp. with the latter being more active than the former. Although C meifera hus been use for & long time as herbal medicine in Malaysia, no Pharmacological studies have been reported on the antiinflammatory, antipyretic and wound healing activites ofthis plant. Although there is one report on C: nucifera antinociceptive and antioxidant activities (Alviane ea, 2004), the sample used was different from the one usd inthe prsent study. ‘The present study was carried out to elucidate the potential anti-inflammatory, antipyretic and wound ‘healing activities of the aqueous extracts of C, reife juice and kernel in animal meds, ‘Materials and Methods Plans Materials The this of Cocos nucifera (Palmee) were collected from Pusat Pembnguan Komodit, Tele BBharu Hutan Mlintang, Perak It was identified to be from MATAG types (a hybrid ecebination of Malayan Dwarf andthe Tagmaran Tall coeomt tee), Two materials from the fats ice and kernal) ‘were tested for its ant-inflammatory, antipyretic and wound healing activity, Preparation ofthe Juice of. rueifera ‘The tosh juices fC, nuetfore (FICN) wsed in the study was obtained price to the experiments, ‘This isto retain most of its organoleptics and nutritional characteristics. Three concentrations were ‘being tested: 100% concentration strength (pure juice) which was taken directly from the fait and, 10 and 50% concentrations!strengths of FICN that was prepared by citing the pure juice in distilled ‘water (DH,O) with the ratio of 1 (¥¥). Preparation of Extract ofthe Keel of C nucifera ‘The aquecus Kem extract of C. muesfera kernal (AKCN) were obtained using the chloroftern ‘methanol (CM) solution mixture (2:1) (viv) as described by Zakaria (2001) but with slight smodiicaions. Brietly, Une keel were macerated widh CM in a rao of 1:2 (wv) for overnight (24). ‘After 24h, it was filtered and the supernatant were collected and left for another 30 min to finish the process. Two layers ean be see: the aquaous supernatant on the upper layer and the CM supernatant ‘onthe lower layer. The aqueous supernatant also called as the stock solutions were collected and ‘evaporated at 70°C fo remove the excess methanol residue. The product from the evaporation considered as the 100% concentratons/srenglhs ofthe AKCN, Chemicals ‘The following chemieals were used: acetylsulieylic acid (ASA) (Bayer eo. Ltd, Malaysia), blorofemn, methanol and ethyl acetate (AR grade, Metek KGaA, Germany), ewer's yeast (AR grade, Sigma Chem, Co, St Louis, USA), sodium chloride and formaldehyde (AR grade, Carlo Erba, (Germany), ariflavine (Bayer Co, Lic, Malaysia). All solutions were prepured immediately pir 10 the experiments Animals ‘All animals used in this smy were obtained from the Animal House, Paclty of Veterinary, ‘Universiti Putra Malaysia (UPMD, The Sprague dawley rats with mean weight of 162 g were used in J. Pharmacol Toxicol, 1 (6): 516-526, 2006 all ofthe experiments except for the wound healing test, in which Bo/b-C mice with weight ranging fiom 25-30 g were used The animal were housed in cages under laboratory standard conditions ina ‘temperature 22°C)-contolled room, with fee access to food and water, Aniiinflammasory Aetiity ‘The anti-inflammation study was carried cut according to the method described by Chakraborty eral, (2004) with slight mocificaions. The Sprague dewley rats wore treated with intraperitoneal (ip) injection of DH,O, ASA (200 mg kg~), FICN (10, 50 and 100% concentration) and AKON, 30 min prior to 5% formalin sub plantar administration on the right hind paw of te rats. ‘The paw thickness resulting fom edema eaused by formeln was measured in all groups for 6 at every 30 min intervals, Aniipyretie Activity ‘The antipyretic activity of the FICN and AKCN was measured by slightly modifying the method deseribed. by Reanmongkol etal. (2002). Sprague davwley tats were fasted overnight with ‘water and food before the experiment. The rectal temperature ofthe rat wes measured using digital themmometsr (Temp Teller modsl OST; China) before treatment (BT) and 0,1, 2,3, 4, 5, 6and 7h after treatment (ip) with DH,O, ASA (200 mg kg), FICN (10, $0 and 100% concentrations) and AKCN (16, 50 ane 100% concentrations). The tnewer’s yeas (2 kg) (BY) which act as the pyrexia inducer were given ip afer 30 min of the test solutions administration Wound Healing Activity ‘The wound healing activity of JFCN and AKCN was earied ont according to the method described by Reczal (2003). A single surgical incision of about ? em wasinglictd atthe dorsal sufiee (of the Balb-C mice. The incision was weated daly by applying topically with eeiflavine, JECN (100%) and AKCN (100%) for a period of rine days. Three mice fiom each group groups were sundownly selected and suificed aller 24 of teutment as well as on the 3, 6th and Sth day for the callction ofthe skin fom the wound site for histological study. The skins collected were Kept in 10% Formalin toersuze itis well preserved forthe histological procedure, Evaluation of the wound healing activity was based on the designate values developed by Sperone al. 2002) but modified by Reezal (2003). The evaluation fer wound healing process ws made by observation ofthe cicatrizing activity, indicated by te formation ofthe granulation tissue and the conversion to fibrous mass called scar and the antiinflammatory activity of both estucts on the skin sample, The length, colour and inflammatory aspoots of the area around the wound were also examined, The inflammatory response, ‘Which isthe normal acute reaction ofthe tissue after any injury Was also closely observed. Statistical Analysis ‘The results are presented as Meant+Standard Estor of Mean (SEM), The Analysis of Variance (ANOVA) test was used to analyze and compare the data, with p<0.05 asthe limit of significance. Results Effects of C.mucifera on Anti-inflammatory Response in Rats ‘As can be seen fom the Fig. 1 the formalin-induced inflammatory edema was significanly (0.05) suppressed by 20 mg kg ASA throughout the experiment, starting 20 min aftr the latter J Pharmacol Toxicol, I (6): 516-526, 2006 —e pijoscnan + mnoicnan TE ASAGomg a) -cRGN TE ASAGO gE -CRON TE Ios sewterne TE tow arcane TE wenarenae Tee macenscnon 02s 100m HCNCRON 0259 Se 10S AKCNTEREN z z Zo Bo oss Sous. : i i ga Son I fous a a o| eas 4s 6 Saco “Tiere Fig 1 The ainlarumtery profes of dient concratons of feshtce (uel) and agus kemel exact (pans of metfo “ono + Ho Te ASA (100 mgs") ec acute? 40) tonsien = Toss Axen Se som rN He some aKeN so] —o- loon rex Fae ome aon 2 g” i Fo be i i 6 2s ™ whe Bro Las 4567 Bota s 4s 67 reine) iene) Fig, 2: The ant-pyretic profiles of different concentrations of ftesh uice (panel a) and aqueous kernel extract (panel b) of C ruerfera administration. Concomitantly, both extacts were also found to exhibit significant (p<0.05) anti- inflammatory activity with the FICN producing a more effective effect. In addition, both extracts at the concentration of 100% produced total recovery’ from inflammatory edema as can be seen after 1300 min oftheir administration, Phare, Tesco, 2 (0: $16-526, 2006 (oh: Geanettive isos: Epidlumy Je lllacenabicn cells ‘Gt Grannitrn este f= Sea Fig X@) The AKCN-pesed mice, st dy 3 oftiespermets, showed proprasive wud Heeling seseily ath viahie sea leraticn ad eiarapaticn ols Wel develop! paeultien ‘ert conmectire iewuee were yavonl ae the hoaling proce tae place, Evolfative oe (Githelalition wee observed under the forned sab (Chetan; F: Epitheliom, Zo: illenaion ells, Se: Scab “Fig 509) Atomoed sin relies were ebsirved en the AKCMnztod mice at day 6 of the rns, rho placaliaicn wer mers proba cn tre mice Inertia {scan be seen in sundance atthe waa ata togshor eth vs station hich iniicaes aavanoeheling proces Bi and pecs te scab appearatobeslouging off theson 520 J. Pharmacol. Texicol,} (6): $16-526, 2006 Epithelium; le: Inflammation cells, Se: Seaby St; Scar tissue Fig. (¢): Well-developed organization of scar tissue and new thick lining of epithelium cells were ‘beerved a the end ofthe experiment, Inflammation cells have been reduced the desis layer stats to shape. The scab appeared to be separated from the skin surface signifying ‘a comprehensive healing process Effects of C.nuctfera on Yeastinduced Fever in Rats ‘The antipyretic effects of FICN and AKCN assessed using the BY-induced assay were shown in panel a and b of Fig. 2, respectively. The BY-induced pyrexia was observed after 240 min of ts administration and contest increase util the end of observation, Interestingly, the ASA. was found to Significantly (¢<0.05) suppress the rat's Dody temperature forthe frst 3.1 during which the BY Aid not yet produce its pyretic eect and this hypothermic inducing activity was sill observed during, thenext 5 when BY stast to exhibit its pyretic effect. As can be seen tom the graph, between 4h 1o8 where pyetic activity was clearly observed, the FICN extracts was found to be more effective imredcing fever in rat when compared o its counterpart (AKCN). In addition, both extrats, in the concentration of 50%, were also found to cause hypothermia in rats during the fist 3 after BY ‘edministration. On the ether hand, the 100% concentration FICN did net show any activity during the same range of time while the AKCN allo exhibit significant (p-0.05) hypothermic activity Effects of C. nucifera on Wound Healing Activiy in Mice Forthe skin sections ofthe untreated group (negative contro), severe necrosis and hemorrhages ‘on the surfice of wound were observed on the 3d day ofthe experiment. Large number of infiltrating, eulrophils can be seen below he necrotic area and the growth of granulation dssue observed The scab also appeared a this period of study with the non-proliferive thickening of epithelium cells on the edge of te wound area. On the oth day of the experiment, severe necrosis and hemotties with slow growth of grannlation tissue can sill be seen on the surface of wound. A. large number of neutrophils canbe sen infiltrating the wound area At this dation of stay, the epithelium cells have lhickened due to epithelial regeneration and can be seen under the scab. On the th day of teatment, the presence of large numberof infiltrating macrophages and moderate amount of granulation tissue indicated delayed and incomplete wound heating sa J. Pharmacol Toxicol, 1 (6): 516-526, 2006 FFor the aeriflavine-treated group, the healing process stated to develope after 3rd day of ‘testment as evidence fiom moderate ncross and hemonages onthe surface of wound. Furthermore, ‘the presence of inflating neutrophils was found to be accompanied by a small number of infiltrating macrophages below the necrotic area andthe growth of grannation tissue was observed at this period ‘of study. The formation of scab and thickening of the epithelium cells can also be seen at this time of ‘tuly, These lesions indicated good growth of healing tissue, On the oth day ofthe experiment, the healing process has developed extensively as evidence from little necrosis and hemorthages on the surface of wound, Furthermore, a large numberof inflating macrophages below the necrotic area and extensive growth of the granulation tissue was also observed. On the 9th day of the experiment, extensive deposition f collagen fibers, elongated fbroeytes an fw capillaries canbe clealy observed ‘There was complete absence of hair follicles in healed area and kertinization was also noticed on completely grown epidermis suggesting complete healing of wounds. For the group treat with FICN, the sections of skin wound on the 3rd day oftreatment revealed ‘amore progressive healing indicated bythe extensive formation of granulation tissue with the migrstion ‘of nlammation cells tothe wound sit, The thickening of epithelium cells was visible at the ge of ‘the wound that hus already closed because ofthe scab formation. On the 6th day of treatment, the sranlaton fissue began to mature and appeared to form a fibrous connective tssus withthe presence ‘ofmonosytes observed, The inflammation calls showed less redness asthe wound healing progressed, ‘The sal hs started to slough ff from the skin asthe epithelium cells cover almost halFof the wound area, On the 9th day of the experiment, the sections of skin wound have demonstrated « more progressive healing characterized by extensive proliferation of brows conncctve isu, which lead tothe formation of fibrous mass or sear and te presence of. small mumiber of menoeytes, The mumber ‘of inflamunation cells began to decrease and the scab almost sloughed off Kerainization was also ‘observed on completely grown epidermis indicating complete healing of wounds Po the group tested with AKCN, the setions of skin wound esult showed thatthe granulation tissue was yet to form. However, the epthcium cells on the edge of the wound were found to thicken and the migration ofinammation cals like neutrophils at the wound area Was visible. Furthermore, the AKCN treated group also appsare with closed wound and formation of the scab (Fig. 3a), On the ‘th day of the experiment, the section of skin wound teated with AKCN exhilited the formation of granulation issue athe wound area (Fig. 3b). The epithelial regeneration has developed under the scab, ‘which lined up by the epithelium cells to form a new layer of epidermis, The indammation calls like ‘macrophages. and monocytes can be seen accumulated atthe wound area, ARer 9th day of ‘ucotment, the skin sections treated with AKCN revealed the formation of extensive fibrous mass, longated fibrocytes antl deposition of collagen fibers while the ilamed cells appeared less red than before (Fig, 3c). At this time, the seab ofthe wound has sloughed off from the shin and a new layer ‘of epidermis forme and completly covering the healed area. Kerainization on the completely grown epidermis was also noticed indicating complete healing of wounds, Discussion C. meifra is one ofthe most valuable plants to man and was used asa primary source of food, sinks and even medication, It has boen used as a traditional medicine by maay cultures (Cano and Velpato, 2004; Alanis et al. 2005), including in Asian counties, for hundreds of yeas to treat a wide ange ofillnstes (Aumad and Raj, 1992; Masa, 1998), For example, the fit of C. muifera contains the coconut water (ive) and coconut meat (ken) was Very fameus in the Malays felklore medicine J. Pharmacol Toxicol, 1 (6): 516-526, 2006 forits ability to eieve fever (Yahay, 2005). In adtion, Alleyne of af (2005) have also reported on the C. mucifera juice extract ability to control hypertension in a clinical trial cartied out at the University of the Wost Inti, St Augustine Trinidad and Tobago, West Indies, Previous studies have also demonstrated that the aquens extract of sk fiber of C. mucifer possessed peripherally and

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