The antioxidant and antitussive properties of Gongronema Latifolium  
used locally by Nigerian poultry farmers for the treatment of fowl
cough was investigated. The phytochemical results revealed the presence
of saponins (0.69mg/100g), tannins (0.0416mg/100g), alkaloids
(0.046mg/100g) and flavonoids (0.016mg/100g). Triterpenes and cardiac
glycosides were also present in detectable concentrations. In vivo
studies on the efficacy of the plant leaves in treatments against fowl
cough in Hubbard broilers gave positive results. The leaf extract
significantly reduced the mortality rate of the broilers by 25% within
3 weeks of treatment and by 40% in 6 weeks of administration, when the
broilers were 13 weeks old. The reduction in mortality coincided with
reductions in the microbial loads in the trachea of the sick 7-week old
broilers. The number of aerobic bacteria was reduced from 36 x 102
cfu/ml to 8 x 102 cfu/ml of viscera suspension within 3 weeks of
administration. Also the number of pleuro-pneumonia like organisms
(PPLO) was reduced from to 12 x 102 cfu/ml to 4 x 102 cfu/ml of viscera
suspension within 2 weeks, and totally eliminated within 3 weeks of
treatment. The in vivo antibacterial potency of the plant extract may
be ascribed to the presence of antioxidative compounds like saponins,
alkaloids, tannins, triterpenes and cardiac glycosides in the plant
leaves

Akpan, E.J.

Ebong, G.A.

Essien, J.P.

Bioline International

 * Corresponding author:  Essien, J P JASEM ISSN 1119-8362 All rights reserved   J. Appl. Sci. Environ. Manage. December, 2007 Vol. 11(4) 47 - 50 Full-text Available Online at  www.bioline.org.br/ja  Antioxidant and Antitussive Properties of Gongronema latifolium Leaves Used Locally for the Treatment of Fowl Cough in Nigeria  1ESSIEN, J P; 2EBONG, G A; 3AKPAN, E J 1Department of Microbiology, 2Department of Chemistry,  3Department of Biochemistry University of Uyo, P. M. B. 1017, Uyo, Nigeria Email: jomato652003@yahoo.com  ABSTRACT: The antioxidant and antitussive properties of Gongronema latifolium used locally by Nigerian poultry farmers for the treatment of fowl cough was investigated. The phytochemical results revealed the presence of saponins (0.69mg/100g), tannins (0.0416mg/100g), alkaloids (0.046mg/100g) and flavonoids (0.016mg/100g). Triterpenes and cardiac glycosides were also present in detectable concentrations. In vivo studies on the efficacy of the plant leaves in treatments against fowl cough in Hubbard broilers gave positive results. The leaf extract significantly reduced the mortality rate of the broilers by 25% within 3 weeks of treatment and by 40% in 6 weeks of administration, when the broilers were 13 weeks old. The reduction in mortality coincided with reductions in the microbial loads in the trachea of the sick 7-week old broilers. The number of aerobic bacteria was reduced from 36 x 102 cfu/ml to 8 x 102 cfu/ml of viscera suspension within 3 weeks of administration. Also the number of pleuro-pneumonia like organisms (PPLO) was reduced from to 12 x 102 cfu/ml to 4 x 102 cfu/ml of viscera suspension within 2 weeks, and totally eliminated within 3 weeks of treatment. The in vivo antibacterial potency of the plant extract may be ascribed to the presence of antioxidative compounds like saponins, alkaloids, tannins, triterpenes and cardiac glycosides in the plant leaves. @JASEM  Gongronema latifolium (Asclepiadiaceae) is a wild climber widely distributed in the southeastern states of Nigeria. Apart from being used as bitter spice or flavouring agent in many traditional Nigerian dishes (Anaso and Onochie 1999) the plant leaves has been found very efficacious as an antidiarrhoea and antitussive (Sofowora 1982 and Iwu 1993), which confirmed the speculations made by local poultry farmers that uses it for the treatment of common respiratory diseases (CRDs) associated with broilers and laying birds in the tropics. CRD in poultry is caused mainly by a pleuro-pneumonia like organisms, Mycoplasma (Jasper 1990) although other bacteria such as the β- haemolytic streptococci, Pseudomonas and Staphylococcus aureus have also been associated with tussis in animals (Thomas 1979 and Paul et al., 1982). This work was carried out to investigate the antioxidants and antitussive properties of G. latifolium.  MATERIALS AND METHODS Collection and Preparation of Leaf Samples: Leaf samples of G. latifolium were collected from mature fruiting climbers in the tropical rainforest of the Niger Delta. The leaves were placed in a flat basket and sun dried for 72 hours under a mean diurnal temperature of 33.2 + 10C, to avoid the escape of volatile components by oven-drying. The dry leaves were then ground with a Christy-Norris hammer mill to pass through a 1mm sieve to obtained a fine powder which was stored in a clean dry airtight glass bottle at ambient temperature until analysed.  Extraction and Phytochemical Analysis of Leaf Samples: Preliminary phytochemical analysis of the petroleum ether extract of the leaves was carried out according to the methods outlined by Harbone (1973) and Trease and Evans (1989). Both petroleum ether and aqueous extracts were prepared. About 500g of the fine sample powder was successively extracted with petroleum ether at room temperature. The extract was concentrated under pressure to yield 40g of petroleum ether extract. In preparing the aqueous extract about 150g of the dry sample powder was boiled in 1.5L distilled water for 1½ h. The resultant mixture was then decanted and filtered through a Whatman filter paper No.1. The filtrate was evaporated to dryness on a hot plate at an initial temperature of 1000C and the dry powder obtained was suspended in 10ml distilled water, stirred and refiltered. The concentration of the extract was determined in grams equivalent of the dried leaves per ml and the solutions were then stored at 40C until ready for use.  Screening for cardiac glycosides, phlobatannins, tannins and triterpenes were done by picric acid, hydrochloric acid, ferric chloride and sulphuric acid tests respectively. The saponnis was detected by ammonia test, the alkaloids by Keller – Williams and Dragendroffs tests, while the polyphenols was detected by the ferric chloride and potassium iodide tests. Quantitative measurement of total crude sapogenins was by the gravimetric method of Brain et al (1968). The Keller-Killiani test for 2-deoxy sugars (AOAC 1975) was used for the assay of the cardiac glycosides, with degoxin as standard.  Determination of Antitussive Properties of G. latifolium leaves: Sixty 7 week-old cough infested Hubbard broilers weighing 1.5g were weight-matched and carefully picked from a stock of 800 birds. The birds were divided into three groups (A, B, C) of 20 broilers. Each bird was caged separately under standard poultry farm conditions and fed with standard diets and water ad libitum. Two sets (group A and B) of the sick birds were Antioxidant and Antitussive Properties of Gongronema latifolium * Corresponding author:  Essien, J. P. 48treated with the aqueous extract of G. latifolium leaves administered orally through the drinking water. The third set (group C) of sick birds which served as the control were not treated with the leave extract. The mortality rates of treated birds in group A and those in group C were determined weekly for 1½ month. Survivors of the tussis attack were examined every week for weight loss or gain. Treatments were analysed for significant differences using Chi-square analysis for mortality and linear contrast analysis of variance for weight loss (Sokal and Rolf, 1969). The second set of treated birds (group B) served as samples for bacteriological studies.  Bacteriological Analysis of Sick Bird’s Trachea: The in vivo effect of the extract treatment on the bacteria associated with tussis in poultry was also examined. During the bacteriological analysis the sick birds were killed and the neck cut off and dissected under total asepsis to obtain the trachea. The trachea itself was cut open to obtained the viscera. Suspensions of the viscera were prepared in sterile distilled water and diluted once to obtain 102 dilution levels. The bacterial load and quality of the sick birds trachea were determined by the pour and spread plate techniques (FDA, 1984) using Bacto-nutrient agar (NA) and Bacto-pleuro-pneummonia like organisms (PPLO) agar. The NA was used for enumeration and isolation of aerobic bacteria while PPLO agar was used for the enumeration and isolation of Mycoplasma. Inoculated agar plates were incubated at 370C for 48 hours. Afterwards the colonies found on the agar plates were enumerated. Representative colonies of the pathogens were isolated, purified and identified by their cultural, morphological and biochemical properties as described by Cowan (1985).  RESULTS AND DISCUSSION The results of the phytochemical analyses presented in Table 1 revealed the presence in detectable concentrations, of saponins, tannins, alkaloids, terpenes and cardiac glycosides in the petroleum ether extract of Gongronema latifolium leaves. Anthroquinones, polyphenols and phlobatannins were not detected while flavonoids occurred in trace amount. These are antioxidants with great antibacterial potency (Trease and Evans, 1989).  The effects of the aqueous extract of the plant leaves on the tussis infected Hubbard broilers over a 6-week period are shown in Figures 1 and 2. The antitussive potential of the plant has been established (Fig.1). The leaf extract significantly reduced the mortality rate of the broilers by 25% within 3 weeks (at 10 weeks old) of treatment and by 40% in 6 weeks, when the broilers were 13 weeks of age. These coincide with the reduction in the number of bacteria in the respiratory system of the broilers. The number of aerobic bacteria in the trachea was reduced from 36 x 102 cfu/ml of viscera suspension to 8 x 102 cfu/ml within 3 weeks of treatment. The number of pleuro-pneumonia -like organism was also reduced from 12 x 102 cfu/ml to 4 x x 102 cfu/ml within 2 weeks of treatment, and totally eliminated within 3 weeks of administering the extract.   The pleuro-pneumonia like organism (PPLO) particularly the Mycoplosma species and the aerobic bacteria including klebsiella and Pseudomonas species isolated from the bird’s trachea have been implicated in bird’s respiratory diseases (Jawetz et al., 1984). Other aerobic bacteria isolated, Staphylococcus aureus, Streptococcus and Corynebacterium species are known etiological agents for human tussis (Jawetz et al., 1984), which may also contribute to fowl cough (Jasper, 1990). The most affected of all the isolates in vivo were Mycoplasma, Corynebacterium, Klebsiella and Pseudomonas species (Table 2). Some of which were easily eliminated from the bird’s respiratory system. This is a pointer to the fact that G. latifolium may be effective in the treatment of cough caused by the listed pathogens, although its efficacy duration may be longer than the conventional antibiotics used for the treatment of fowl cough. The remarkable antibacterial potency of the plant leaves may be ascribed to the synergistic actions of antioxidants (saponins, alkaloids, tannins, flavonoids, cardiac glycosides, and stero/triterpenes) in the plant leaf extract.  The antimicrobial properties of flavonoids, saponins and alkaloids have been established (Trease and Evans, 1989). However, the apparent slow action of the plant extract may be attributed to the presence of constituents (proximate components) other than antioxidants which might have retarded the potency of the extract. Staphyloccocus aureus was noticeably the most stubborn and prevalent of the bacteria species isolated from the birds trachea. The susceptibility of Klebsiella, Corynebacterium and Mycoplasma species may be ascribed partly to the weak competitive parasitism of the bacteria and to the age-dependent increase in the resistance of the broilers. All the survivors (broilers) exhibited variable loss in weight (Fig.2). The untreated birds recorded a significant loss in weight while birds treated with the plant extract regained loss weight within 5 weeks of treatment. The gain in weight may be as result of a reduction in the physiological effect of the disease on affected birds and increase in the metabolic activities of the birds.  The ability of Gongronema latifolium leaves extract to check the proliferation of pathogenic bacteria and the elimination of Mycoplasma species in the trachea of the sick birds have justify its use by local Antioxidant and Antitussive Properties of Gongronema latifolium * Corresponding author:  Essien, J. P. 49farmers in controlling fowl cough. This has also confirmed its antitussive and antidiarrhoeal efficacy in humans. However, the present knowledge of the chemical properties of the leaf extracts makes it difficult to identify with certainty the actual antitussive component of the plant leaves. Further research is therefore necessary to isolate and identify the active components and their antibacterial specificity for higher efficacy and safety in rural poultry management.  Table 1: Phytochemisty of Gongronema latifolium leaves  Property Observation (precipitate, ppt) Remark Concentration (mg/100g) dry seeds Saponins Persistent foam ++ 0.69mg/100g  Brown ppt. ++  Tannins Green ppt ++ 0.0435mg/100g Poly phenols No ppt -  Alkaloids Green ppt ++ 0.046mg/100g  Red ppt ++  Phlobatannins No ppt -  Flavonoids Faint-yellow ppt +   Faint-yellow ppt + 0.016mg/100g Anthroquinones Yellow ppt -  Sterol/triterpenes Pink ppt ++  Cardiac glycosides Reddish brown ppt ++      + Present in trace amount   ++ Remarkably present   - Absent       10090807060504030201008 9  10 11 12 13Treated birdsUntreated birdsMortality rate (%)Fig. 1: Mortali ty rate of tussis infected broilers treated with .  leaf extract. G latifoliumDuration of treatments (weeks)   Table 2: Bacteriological properties of the sick Hubbard broiler’s trachea  Properties   Age of birds (weeks) Untreated birds (control)  Treated birds  7 8 9 10 11 12 13 7 8 9 10 11 12 13 Aerobic count (cfu 102/ml) 36 36 39 40 38 42 52 36 24 15 8 11 10 11 Count of PPLO (cfu x 102/ml) 12 12 15 17 16 19 18 12 11 4 0 0 0 0 Isolates               Corynebacterium sp + + + + + - - + + + + - - - Klebsiella sp + + + + + - - + - - - - - - Mycoplasma sp + + + + + + + + + + - - - - Pseudomonas sp + + + + + + + + + + + + + - Streptpcoccus sp + + + + + + + + + + + + + - Staphylococcus aureus + + + + + + + + + + + + + +  + = Present;   -  =  Absent or present in an undetectable number     Antioxidant and Antitussive Properties of Gongronema latifolium * Corresponding author:  Essien, J. P. 503217 8 Treated birdsUntreated b irds9 11 12 13 1410Fig. 2:  Changes in weight of  sick broilers treated   leaf extractG. latifoliumWeight of broilers (1.5kg)Duration of  treatments (weeks)  REFERENCES Anaso, H. U; Onochie, C. C. (1999). A comparative study of nutrients in Gongronema latifolium, Piper Guidneense and Piper nigrum, to testify high acceptability in local dishes. Journal of Science Engineering and Technology 6 (2): 1321 – 1832.  AOAC (1975) Official Methods of Analysis, 12th Edn. Washington, D. C. Association of Official Analytical Chemists.  Brain, K. R., Fazil, F., Hardman, R; Wood, A B (1968). The rapid quantitative determination of C25 epimeric steroidal sapogenins in plant. Phytochemistry 7: 1815 – 1823.  Cowan, S T (1985). Cowan and Steel’s Manual for Identification of Medical Bacteria 2nd Edition. Cambridge University Press. London pp. 46-79.  FDA, (1984). Bacteriological Analytical Manual (6th Edn). AOAC Publication, Arlington, VA, USA.  Harbone, J B (1973) Textbook of Phytochemical Methods. Champam and Hall Ltd. London pp.45-51.  Iwu, M. M. (1993). Handbook of African Medicinal Plants. C. R. C. Press Incorporated. Tokyo. pp.47-240.  Jasper, A. W. (1990). Oral vaccine for control of Newcastle disease. In Poultry International (Terry Evans ed) Jaarbeurs Utrecht, Holland pp.22-23.  Jawetz, E., Melnick, J. L., Adelberg, E. A. (1984). Review of Medical Microbiology (16th Edition) Longe Medical Publication Los Altas, California. Pp.361-367.  Paul, M. O., Aderibighe, D. A., Sule, C., Lamikanra, A (1983). Antimicrobial sensitivity patterns in hospital and non-hospital strains of Staphylococcus aureus isolated from nasal carriers. Journal of Hygeine. 89, 253-259.  Sofowora, A. (1982). Medicinal Plants and Traditional Medicine in Africa (2nd edu.) Spectrum Books Publishers Ltd. Ibadan. Pp.140-145.  Sokal, R. R., Rolf, F. J. (1969). Biometry. W. W. Freeman and Company. San Francisco pp.340.  Thomas, C. G. A. (1979). Medical Microbiology. (4th Edition) Baihiere Tindall London pp.183-186.  Trease, G. E. Evans, W. C. (1989) Textbook of Pharmacognosy. Balhiere Tindall London pp.544-636.   

Antioxidant and Antitussive Properties of Gongronema Latifolium Leaves Used Locally for the Treatment of Fowl Cough in Nigeria

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