 |
 |
| Figure 1 (a): Map of South Africa showing Limpopo Province in red (Wikipedia 2011) |
Figure 1 (b): Map of Limpopo showing Blouberg in red (Wikipedia 2011) |
| Livestock Research for Rural Development 28 (12) 2016 | Guide for preparation of papers | LRRD Newsletter | Citation of this paper |
The aim of the study was to determine the anthelmintic activity of Cassia abbreviata, Schotia brachypetala, Senna italica, Pappea capensis and Peltophorum africanum against egg and third larval stages of H. contortus. The bark of C. abbreviata, P. capensis and P. africanum, the root bark of S. italica and the leaves of S. brachypetala were harvested, dried at room temperature in the laboratory and thereafter extracted in hot and cold distilled water. Concentrations of 2.5, 5.0 and 7.5 mg/mL of these extracts were tested for inhibitory activity against H. contortus egg hatching and larval development.
Hot and cold water extracts of P. africanum inhibited the highest percentage of eggs (25 and 22%, respectively) from hatching at the lowest concentration of 2.5 mg/mL. These extracts did also inhibit 86 and 80% of larval development, respectively. The highest rates of larval mortality were recorded for P. africanum and S. italica hot water extracts (68% and 65% within two hours at the lowest concentration and 63% and 64 % for cold water extracts. The percentage of larval mortality increased as the concentrations increased. However, the experimental results were not solely dependent on extract concentration because all the third stage H. contortus larvae died within 72 hours irrespective of the level of concentration. This work therefore supports the use of the five experimental plant species used in the present study for the control of gastrointestinal nematodes by the Bapedi people of the Blouberg Municipality in Limpopo Province of South Africa and recommend that further studies like toxicity testing and in vivo trials be made to validate the use of these plants as an alternative to anthelmintic drugs.
Keywords: aqueous extracts, ethnoveterinary medicine, Haemonchus contortus
Internal parasite infestations constitute a major constraint to sheep production in South Africa (Perry & Randolph 1999), with Haemonchus contortus considered the most important of all the gastrointestinal nematodes that restrain the survival and productivity of sheep and goats owned by the rural poor in the developing world (Perry et al 2002). The control programmes for H. contortus usually result in the control of other internal worms (Kandu-Lelo 2010).
In Africa, commercial anthelmintics are expensive and sometimes unavailable leading to the use of poor quality or altered products (Luseba & Tshisikhawe 2012). The misuse of these synthetic medicines has led to the development of anthelmintic resistance (Lans & Brown 1998). According to Kaplan (2004), H. contortus features prominently amongst the reports of anthelmintic resistance that has emerged in all countries of the world that produce small ruminants. In Africa, anthelmintic resistance has been reported in both the commercial and resource poor farming sectors (Tsotetsi et al 2013). This justifies an urgent need to find alternatives to synthetic drugs (Shen et al 2010) such as ethnoveterinary remedies. South Africa’s indigenous people have the knowledge of plant species with anthelmintic activity. Furthermore, because of limited availability of drugs, high cost, development of resistance, chemical residue in milk and meat, the majority of world population depends on traditional remedies (Jeyathilakan et al 2011).
Farmers claim that medicinal plants are more effective than pharmaceuticals for chronic pathologies (Luseba et al 2007). Luseba and Van der Merwe (2006) reported the use of ethnoveterinary medicines by Setswana-speaking people in the Madikwe area of the North West province and the Tsonga speaking people of the Limpopo Province, respectively. However, ethnoveterinary medicine has proven to be ethnical and locality-specific (Luseba & Van der Merwe 2006). Thus, research findings in other areas might not be applicable to the present study area. Therefore, the aim of this study was to investigate the anthelmintic activity of plant species used to treat parasite infections of livestock by Pedi-speaking communities in the Blouberg Municipality of the Limpopo Province, South Africa for the preservation of local knowledge by way of documenting it.
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| Figure 1 (a): Map of South Africa showing Limpopo Province in red (Wikipedia 2011) |
Figure 1 (b): Map of Limpopo showing Blouberg in red (Wikipedia 2011) |
Interviews and plant collections were conducted in the Blouberg Municipality (Figures 1 (a) and 1(b) which falls under the Capricorn District Municipality and comprises the arid sweet bushveld (Acocks 1988) in the Limpopo Province. Rainfall is 400 mm per annum and the rainy season usually extends from November to February but rainfall distribution is irregular and unpredictable. Average minimum and maximum temperatures are 12° C and 25° C, respectively (Mara research station, South Africa). A permit (Permit no. RB 102/13) for collection of plant materials was obtained from the Limpopo Department of Environmental affairs in Polokwane before plant specimens of each species were collected, labelled and pressed according to the methods of Fish (1999).
Purposeful sampling of homogenous groups where participants are most likely to give good insight of the phenomenon of interest was conducted (Patton 1990). A total of 60 livestock farmers with profound knowledge on plant species used for the treatment of internal parasites were interviewed. Many rural small stock farmers in Blouberg Municipality fall outside the periphery of formal livestock markets. However, statistics show that small stock rearing remains an integral part of their everyday life and it is an important source of income (Grwambi et al 2006). Semi-structured interviews were conducted using an open-ended questionnaire between May and August 2013 which aimed at collecting data relating to tree and shrub species that are used against helminthosis by livestock farmers in the Blouberg Municipality of the Limpopo Province. Areas of discussion were guided by descriptions such as farmer’s age, gender, socio-economic profile, animal husbandry and local knowledge in animal healthcare (Luseba & Van Der Merwe 2006). Comprehensive information of plants used by the local people such as local name, indications, method of preparation, administration and dosage to formally record the ethno-botany of the area was recorded.
Plants cited more than two times as having anthelmintic properties by interviewed farmers were selected for the current study (Kansonia and Ansay 1997). They were collected and thereafter authenticated by a botanist (Dr. B Egan) at the University of Limpopo. In order to have an appropriate quantity of samples for laboratory analyses, approximately 1-2 kg of fresh plant materials were collected for each plant species and from two or more shrubs or trees around Blouberg Municipality of the Limpopo province (33°47′50″S 18°27′43″E). The plant materials (leaves, bark and root-bark) were collected from October to December 2013, dried at room temperature in the laboratory and weighed frequently on a daily basis to assess the moisture content until constant weight was obtained after subsequent weighing. They were then ground to fine powder using a Macsalab mill (Model 200Lab, Eriez®, Bramley, RSA). Five grams of each powdered material was extracted in hot and cold water at 10 mL/g respectively overnight. The extracts were filtered using Whatman® No.1 filter paper (Whatman, United Kingdom) and filtrates were frozen at -80oC before drying using a Lyoquest 50 freeze dryer (Labotec, South Africa). The extracts were reconstituted in distilled water for their respective stock solutions. Then, the stock solutions were diluted to the required concentrations of 2.5, 5.0, and 7.5 mg/mL for the bioassay analyses.
According to Kandu-Lelo (2010) the control programmes for H. contortus usually result in the control of other internal worms, hence results derived from experiments using H. contortus as a model can be applied to other nematode species. Faecal samples were collected directly from recta of adult ewes experimentally infected with H. contortus field strain. The sheep belonged to Dr. P.C Van Schalkwyk of Biozetica Agri-Source (Pty) LTD and were kept on plot A 64 Buffelshoek, Mooinooi near Rustenburg in the North West Province of South Africa (25°79′08″ S 27°55′15″ E). Collected samples were immediately transported to the Helminthology laboratory of the Agricultural Research Council, Onderstepoort Veterinary Institute in a cooler box. Faecal samples were analysed using the McMaster technique (Soulsby 1982) to confirm the presence of nematode eggs and faecal cultures prepared (Reinecke 1973) to identify/confirm the nematode genera using Van Wyk et al (2004).
Four assays were conducted. In order to determine the anthelmintic activity of the prepared plant extracts, Haemonchus eggs were recovered from faeces through an egg recovery assay (Maphosa et al 2010) with some modifications.
The egg hatch assay was conducted as published by McGaw et al (2007) with some minor modifications. The larval development assay was conducted as described by Bizimenyera et al (2006).
Larval mortality assay was conducted according to the method described by McGaw et al (2007) with some modifications. All live and motile L3s in each well were counted and mortality was expressed as a percentage. All tests were replicated three times.
The highest frequencies of trees and shrubs were cited by males compared to females as shown in Table 1. Older people aged over 40 years, of which 20 (33.3%) were females and 40 (66.6%) males, reported to know more plant species used to treat internal parasites in livestock than younger people. All in all, the number of females aged under 40 years were 5 which is 8% of the total number of participants and 25% of the female participants whereas on the other hand the number of male participants under the age of 40 were 15 which is equal to 25% of the total number of people who took part in the study and 37.5% of the male participants.
|
Table 1. Frequencies of trees and shrubs cited by both male and females of different age groups in Blouberg Municipality |
||||||||||||
|
Plant |
Females |
Males |
||||||||||
|
Freq: |
(%) |
Freq: Above |
(%) |
Total |
(%) |
Freq: |
(%) |
Freq: Above |
(%) |
Total |
(%) |
|
|
Peltophorum africanum |
4 |
44.44 |
7 |
29.1 |
11 |
33.33 |
6 |
40 |
25 |
27.77 |
31 |
29.5 |
|
Senna italica |
2 |
22.22 |
4 |
16.66 |
6 |
18.18 |
1 |
6.67 |
19 |
21.1 |
20 |
19.05 |
|
Cassia abbreviata |
1 |
11.11 |
3 |
12.5 |
4 |
12.12 |
1 |
6.67 |
12 |
13.33 |
13 |
12.38 |
|
Schotia brachypetala |
1 |
11.11 |
4 |
16.66 |
5 |
15.15 |
1 |
6.67 |
9 |
10 |
10 |
9.52 |
|
Pappea capensis |
1 |
11.11 |
2 |
8.33 |
3 |
9.09 |
2 |
13.33 |
8 |
8.88 |
10 |
9.52 |
|
Ochna pulchra |
0 |
0 |
1 |
4.16 |
1 |
3.03 |
1 |
6.67 |
4 |
4.44 |
5 |
4.76 |
|
Capparis sepiaria |
0 |
0 |
1 |
4.16 |
1 |
3.03 |
1 |
6.67 |
3 |
3.33 |
4 |
3.81 |
|
Mormodica balsamina |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
6.67 |
2 |
2.22 |
3 |
2.86 |
|
Jethropha zeyheri |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
3 |
3.33 |
3 |
2.86 |
|
Dicerocarym senecioides |
0 |
0 |
1 |
4.16 |
1 |
3.03 |
0 |
0 |
2 |
2.22 |
2 |
1.9 |
|
Gymnosporia senegalensis |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
2 |
2.22 |
2 |
1.9 |
|
Punicum granatum |
0 |
0 |
1 |
4.16 |
1 |
3.03 |
1 |
6.67 |
1 |
1.11 |
2 |
1.9 |
|
Where Freq. = Frequency; % = percentage of frequency |
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Table 2 shows the thirteen plant species belonging to seven families that were identified of which the plant family containing more species was the Fabaceae. The five plants that were used for biological assays were selected because according to Kansonia & Ansay 1997, there is consistency when one plant is cited for the same use by more than two respondents.
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Table 2. Plants and plant parts administered for the treatment of internal parasites in animals in the Blouberg Municipality |
||||||
|
Botanical and family names |
Voucher no |
Common names |
Habitat |
Part used |
Preparation |
Indication |
|
Capparis sepiaria
L. var.subglara (Oliv.) DeWolf
|
MM0001 |
Moopatladi: Sep; Capper bush: Eng. |
Shrub |
R |
Crushed and immersed in water |
Immune booster, internal parasites and blood purifier |
|
Ochna pulchra
Hook.f
|
MM0002 |
Monamane:Sep; Lekker breek: Afr. |
Tree |
B |
Crushed and immersed in water |
Heartwater, internal parasites and diarrhoea |
|
Cassia abbreviata
Oliv. Subsp. bearana
|
MM0003 |
Monepenepe:Sep; Sjambok pod: Eng. |
Tree |
B |
Crushed and immersed in water |
Internal parasites, blackwater fever, headache, toothache, stomach-ache and abortion agent |
|
Punica granatum
L.
|
MM0004 |
Garenata: Sep; Pomegranate: Eng |
Shrub |
F |
Sliced, dried and immersed in water |
Internal parasites, stomach-ache and diarrhoea |
|
Peltophorum africanum
Sond. |
MM0005 |
Mosehla: Sep; African wattle:Eng; Huilboom: Afr |
Tree |
B |
Crushed and immersed in water |
Diarrhoea, worms in sheep and human |
|
Gymnosporia senegalensis
(Lam.) Loes.
|
MM0006 |
Sephato: Sep; Rooi pendoring: Afr |
Shrub |
L |
Dried, milled to powder and mixed with water |
Heartwater, internal parasites, diarrhoea and immune booster |
|
Dicerocarym senecioides
(Klotzch) Abels.
|
MM0007 |
Mosehlo: Sep; Devil thorn: Eng. |
Forb |
W |
Dried, ground and mixed with water |
abortion agent, retained placenta and internal parasites |
|
Pappea capensis
Eckl. Zeyh
|
MM0008 |
Morobadiepe: Sep; Jacket plum: Eng. |
Tree |
B |
Crushed and immersed in water |
Internal parasites, purgative and a cure for ringworm |
|
Clerodendrum glaborum
E. Mey. Var. glabrum
|
MM0009 |
Motlhokotlhoko: Sep; Smooth tinderwood: Eng. |
Shrub |
L |
Dried, ground to powder and mixed with water |
Purgative, worms and head- ache |
|
Schotia brachypetala
Sond.
|
MM0010 |
Molope: Sep; African walnut: Eng |
Tree |
L |
Dried, milled to powder and mixed with water |
Diarrhoea, worms in sheep and human |
|
Mormodica balsamina
L.
|
MM0011 |
Segwere sa thaba;Sep |
Vine |
B |
Sliced and immersed in water |
Internal parasites, heartwater and blood purifier. |
|
Senna italica
Mill.
|
MM0012 |
Morotoditshosi: Sep |
Legume |
RB |
Crushed and immersed in water |
Worms, heartwater, gallsickness, intestinal diseases, and chicken pox in humans |
|
Jatropha zeyheri
Sond.
|
MM0013 |
Sefapabadia: Sep; Verfbol: Afr. |
Tree |
BLB |
Sliced and immersed in water |
Worms, purgative, blood purifier |
|
Sep=Sepedi; Eng=English; Afr=Afrikaans; B=Bark; BLB=Bulb; F=Fruit; L=Leaves; RB=Rootbark; W=Whole plant |
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The highest inhibition of 22 and 25%, at the lowest concentration of 2.5 mg/mL, were recorded for P. africanum cold and hot water extracts, respectively whereas P. capensis showed the lowest inhibition percentages of all the tested plants. Generally, the egg hatch assay indicated that hot water extracts significantly inhibited (p = 0.05) the hatching of a higher number of eggs compared to the cold water extracts except for P. capensis 2.5 mg/mL and 5.0 mg/mL where there was no significant difference (p=0.05) between cold and hot water plant extracts (Table 3).
|
Table 3. Mean inhibition percentages for the egg hatch assay for Haemonchus contortus, using different concentrations (conc.) of crude cold and hot water extracts of five plants |
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|
Plant |
Cold water |
Hot water |
Controls |
|||||||
|
Conc. (Mg/ml) |
2.5 |
5.0 |
7.5 |
2.5 |
5.0 |
7.5 |
+ |
- |
||
|
C. abbreviata |
13.00±0.00s |
19.00±0.00op |
37.00±1.00g |
15.33±1.52qr |
21.00±0.00no |
40.00±1.00f |
100 |
0 |
||
|
S.brachypetala |
14.66±2.08rs |
23.33±1.52lm |
39.00±2.00fg |
17.00±2.00pq |
25.66±1.52k |
42.66±1.52e |
100 |
0 |
||
|
S. italica |
17.66±1.52p |
28.66±0.57j |
47.66±0.57d |
20.66±0.57no |
32.00±1.00i |
53.00±0.00c |
100 |
0 |
||
|
P. capensis |
5.33±1.52u |
11.00±2.00t |
13.66±0.57rs |
7.33±1.52u |
13.00±1.00st |
17.66±1.52p |
100 |
0 |
||
|
P. africanum |
22.00±2.00mn |
31.00±2.00i |
55.33±0.57b |
25.00±0.00kl |
34.33±1.52h |
58.00±1.00a |
100 |
0 |
||
|
Means with the same letter (a-u) are not significantly different (p =0.05). + = Positive; - = Negative |
||||||||||
The results for larval development and viability are reported in Table 4. Similar to the egg hatch assay, the larval development assay indicated a linear dose related inhibitory response. The highest inhibition of larval development at the lowest concentration was observed for P. africanum with 80.00 and 86.33% for cold and hot water plant extracts respectively whereas the lowest inhibition percentage at the lowest concentration was observed for P. capensis with 66.00 and 69.00% for cold and hot water plant extracts respectively. There was a significant difference between P. africanum cold and hot water extracts while the same was observed for S. italica (p = 0.05). The positive control thiabendazole induced a 100% larval development inhibition whereas the negative control inhibited 0% of pre-infective L1 and L2 larvae from developing to the parasitic infective L3 larvae stage. The plant extracts proved to be much more effective against the infective stage larvae than with respect to the pre-infective stage larvae.
|
Table 4. Mean inhibition percentages for the larval development assay for Haemonchus contortus, using different concentrations (conc.) of crude cold and hot water extracts of five plants |
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|
Plant |
Cold water |
Hot water |
Controls |
||||||
|
Conc. (Mg/ml) |
2.5 |
5.0 |
7.5 |
2.5 |
5.0 |
7.5 |
+ |
- |
|
|
C. abbreviata |
69.00±0.00k |
75.00±2.00ij |
87.66±1.52ef |
74.33±0.57ij |
83.00±2.00g |
91.66±2.51c |
100 |
0 |
|
|
S.brachypetala |
70.33±1.52k |
80.66±1.52h |
90.33±1.52cd |
76.00±1.00ij |
86.00±0.00f |
95.66±0.57b |
100 |
0 |
|
|
S. italica |
76.33±0.57i |
86.66±0.57f |
100.00±0.00a |
80.00±0.00h |
89.00±2.00ed |
100.00±0.00a |
100 |
0 |
|
|
P. capensis |
66.00±2.00l |
70.33±1.52k |
76.00±0.00ij |
69.00±2.00k |
74.00±0.00j |
80.00±2.00h |
100 |
0 |
|
|
P. africanum |
80.00±1.00h |
89.00±0.00de |
100.00±0.00a |
86.33±1.52f |
90.33±3.05cd |
100.00±0.00a |
100 |
0 |
|
|
Means with the same letter (a-l) are not significantly different (p =0.05). + = Positive; - = Negative |
|||||||||
The results for the larval mortality assay with hot and cold water extracts. The larval mortality rate was dose dependent in both hot water and cold water extracts. The highest rates of mortality were recorded for P. africanum and S. italica cold water extracts, with 63% mortality and 64% mortality rates within two hours at the lowest concentration, and 68% and 65% for hot water extracts respectively. The positive control Thiabandazole® killed all the L3 larvae within 2 hours of exposure even at the lowest concentration of 2.5 mg/mL, while as expected there was no larval mortality in the negative control, which was distilled water. All the hot water plant extracts recorded the highest inhibition percentages compared to all the cold water plant extracts at all concentrations.
About 72 hours were needed to result in a total larval mortality irrespective of the concentration. The percentage of mortality increased as the concentrations increased; however, the experimental results were not concentration dependent because all the third stage H. contortus larvae died within 72 hours at 2.5, 5.0 and 7.5 mg/mL, respectively (Table 5). Even at the lowest concentration of 2.5 mg/mL in both cold and hot water there was no extract from all five of the tested plants that induced larval mortality of less than 50 percent. No significant difference was found to exist in all the plant extracts, and in all the three different concentrations, at the observation time of 72 hours (p = 0.05).
|
Table 5. Mean mortality percentages for the larval mortality assay for Haemonchus contortus, using different concentrations of crude cold and hot water extracts of C. abbreviata, S. brachypetala, S. italica, P. c apensis and P. africanum |
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|
Plant |
Time |
Cold water extracts |
Hot water extracts |
Controls |
|||||
|
2.5 mg/mL |
5.0 mg/mL |
7.5 mg/mL |
2.5 mg/mL |
5.0 mg/mL |
7.5 mg/mL |
+ |
- |
||
|
CA |
02 |
57.66±1.15r |
60.66±1.52q |
77.66±0.57hi |
57.00±2.00r |
58.66±0.57r |
58.33±0.57r |
100 |
0 |
|
24 |
80.33±1.52g |
81.00±1.00g |
94.00±2.00c |
65.00±2.00o |
75.00±2.00j |
85.00±2.00f |
100 |
0 |
|
|
48 |
91.66±1.52d |
95.00±1.00c |
97.00±1.00b |
87.66±1.52f |
92.00±2.00d |
96.33±0.57b |
100 |
0 |
|
|
72 |
100.0±0.00a |
100.0±0.00a |
100.0±0.00a |
100.0±0.00a |
100.0±0.00a |
100.0±0.00a |
100 |
0 |
|
|
SB |
02 |
59.00±0.00q |
64.33±0.57o |
70.00±1.00l |
55.00±2.00s |
58.00±0.00r |
60.00±1.00q |
100 |
0 |
|
24 |
70.33±1.52l |
72.33±1.52k |
80.00±1.00g |
63.00±0.00p |
65.66±1.52o |
80.33±1.52g |
100 |
0 |
|
|
48 |
73.00±2.00k |
81.00±2.00g |
86.00±1.00f |
74.00±0.57j |
89.00±2.00e |
96.33±0.57b |
100 |
0 |
|
|
72 |
100.0±0.00a |
100.0±0.00a |
100.0±0.00a |
100.0±0.00a |
100.00±2.00a |
100.0±0.00a |
100 |
0 |
|
|
SI |
02 |
64.33±1.52o |
67.00±0.00n |
72.00±0.00k |
65.33±1.52o |
66.33±0.57n |
74.00±0.00j |
100 |
0 |
|
24 |
70.00±1.00l |
71.66±0.57l |
77.00±1.00hi |
75.33±0.57j |
77.66±2.51hi |
80.00±1.00g |
100 |
0 |
|
|
48 |
78.33±1.52h |
80.00±1.00g |
86.00±1.00f |
81.66±1.52g |
89.00±4.58e |
88.00±1.00e |
100 |
0 |
|
|
72 |
100.0±0.00a |
100.0±0.00a |
100.0±0.00a |
100.0±0.00a |
100.0±0.00a |
100.0±0.00a |
100 |
0 |
|
|
PC |
02 |
50.00±2.00t |
58.00±2.00r |
60.00±1.52q |
51.66±1.52t |
60.00±1.00q |
74.00±1.00j |
100 |
0 |
|
24 |
56.00±6.08r |
62.66±1.52p |
69.66±0.57m |
64.00±0.00o |
84.33±0.57f |
86.00±0.00f |
100 |
0 |
|
|
48 |
77.00±2.00hi |
79.33±0.57h |
89.00±1.00e |
75.66±1.52j |
90.33±0.57d |
98.33±0.57b |
100 |
0 |
|
|
72 |
100.0±0.00a |
100.0±0.00a |
100.0±0.00a |
100.0±0.00a |
100.0±0.00a |
100.0±0.00a |
100 |
0 |
|
|
PA |
02 |
63.66±1.15p |
64.00±1.00o |
68.66±0.57m |
68.00±0.00m |
68.00±1.00m |
69.00±2.00m |
100 |
0 |
|
24 |
74.00±1.00j |
85.00±2.00f |
88.00±3.00e |
78.66±0.57hi |
90.66±1.52d |
90.00±1.00d |
100 |
0 |
|
|
48 |
80.00±1.00g |
91.00±1.00d |
96.00±1.00b |
83.66±1.52g |
96.33±0.57b |
97.00±2.00b |
100 |
0 |
|
|
72 |
100.0±0.00a |
100.0±0.00a |
100.0±0.00a |
100.0±0.00a |
100.0±0.00a |
100.0±0.00a |
100 |
0 |
|
|
Where CA = Cassia abbreviata; SB = Schotia brachypetala; SI = Senna italica; PC = Pappea capensis; PA = Peltophorum africanum.+=Positive; - Negative. Means with the same letter (a-t) are not significantly different (p =0.05) |
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The frequencies of plants mentioned during the survey coincide with the anthelmintic activities of the plants against H. contortus. For instance P. africanum was mentioned more often than any other plant for the treatment of worm infestations in animals, while the biological assays revealed that P. africanum was the most potent plant; on the other hand, P. capensis which was the least popular plant was not as effective as P. africanum against the H. contortus at three different stages of development. There was also no significant difference (p=0.05) on the low inhibition percentage of both cold and hot water P. capensis plant extract for egg hatch assay.
The results from the egg hatch assay signify that, although egg hatch inhibition was observed, not all eggs were inhibited from doing so. Egg hatch inhibition percentage for P. africanum cold water extract which proved to be the most potent plant extract which recorded 22 % at the lowest concentration and 55 % at the highest concentration whereas hot water plant extract of the same plant inhibited 25% at the lowest concentration and 58% at the highest concentration Although there was some inhibition, most eggs managed to hatch and according to Molefe el al (2012) this might be because the egg is, at this stage, disseminated into the environment and protected with a thick wall, causing it to be resistant to various environmental conditions.
This results of the present study suggests that P. africanum and S. italica are still effective in preventing the pre-infective larvae from developing to the infective L3 larvae stage even at low concentrations. As expected, there was a significant difference between 2.5, 5.0 and 7.5 mg/mL concentrations in both hot and cold water plant extracts for egg hatch and larval development assays.
The most outstanding feature of the larval mortality assay results was that there was no significant difference (p=0.05) between the cold and hot water plant extracts in all the concentrations at 72 hours which could mean that the longer the worms remain in contact with the plant extracts, the lower their chances of survival. Although the results from the present study supports the theory that P. africanum has an ovicidal effect on H. contortus eggs, no significant difference was found between P. africanum cold water extract and S. italica hot water extract; furthermore, no significant difference was established between S. brachypetala cold water extract and C. abbreviata hot water extract irrespective of the concentration (p = 0.05).
There was no significant difference between P. africanum 2.5 mg/mL cold water extract and S. italica at the concentration of 2.5 mg/mL hot water extract for the larval development assay as well. Similarly, there was no significant difference for egg hatch inhibition (p = 0.05) between P. africanum 2.5 mg/mL hot water extract and S. brachypetala 5.0 mg/mL hot water extract, nor between S.italica 2.5mg/mL hot water extract, P. africanum 2.5 mg/mL cold water extract and Cassia abbreviata 5.0 mg/mL hot water extract. In other words, P. africanum and S. italica were effective even at low concentrations.
Even though P. africanum proved to be the most potent of the five plant extracts, all these (both hot and cold water extracts) managed to inhibit further development of the free living pre-infective L1 and L2 larvae into the infective L3 larvae, and the result was 100% mortality within 72 hours. It was also evident that hot water extracts were responsible for higher rates of larval mortality in all the treatments. The efficacy of any plant extract, at the lowest concentrations, against the gastrointestinal nematodes proves the anthelmintic activity of that plant (Maphosa et al 2010). It is therefore concluded that C. abbreviata, S. brachypetala, S. Italica, P. cappensis P. africanum exhibit such an activity. It is also important to note that the survey revealed water as a common solvent used in the preparation of concoctions to be given to the animals for the treatment of internal parasites, which concurs with what Bizeminyera et al (2006) reported. The high polarity of the bio-active compounds in the plants also means that these compounds may be extractable by the polar solvents available to rural users. Molefe (2013) also reported the high effectiveness of water extracts compared to acetone extracts for the egg hatching and larval mortality assay.
On one extreme, P. africanum proved to be the most potent plant while on the other, P. capensis was the least potent, as determined by the highest egg hatch and larval development inhibition percentages and the highest larval mortality percentage at the lowest extract concentration of 2.5 mg/mL with minimal contact time of 2 hours between the larvae and the plant extract. On the intermediate level were S. italica, S. brachypetala and C. abbreviata, respectively.
It is clear that P. africanum bark and S. italica root-bark recorded high anthelmintic activity and are therefore good candidates for treatment of gastrointestinal infections. However, the mechanisms of their effectiveness still remain to be tested in vivo. Furthermore, safety and toxicity studies must be conducted in vivo to determine the minimum non-lethal concentrations needed for the treatment of nematode infections.
Overall, the study revealed that the hot water extracts of all tested plants were more effective than cold water extracts with respect to the egg hatch inhibition, larval development and larval mortality assays. Therefore, hot water plant extracts of P. africanum bark and S.italica root-bark could be considered as a replacement for synthetic drugs. However, S. brachypetala, C. abbreviata and P. capensis exhibit moderate anthelmintic activity; as a result, they cannot be used as a sole replacement for synthetic drugs but rather as an integrated approach to achieve sustainable parasite control in ruminant production systems (Githiori et al 2006).
Adamu et al (2013) stated in their report on work done on the efficacy and toxicity of thirteen plant leaf acetone extracts used in ethnoveterinary medicine in South Africa, as regards egg hatching and larval development of H. contortus, that to their surprise the aqueous extract of Markhamia obtusifolia displayed double the activity of the acetone extract. According to these authors these results indicate that the anthelminthic activity of aqueous extracts of plants which had already been investigated using organic solvents should be determined. The results of the current work are crucial in advancing towards finding long term solutions in alternative treatment for since control programmes for Haemonchus contortus usually result in the control of internal worms (Kandu-Lelo 2010).
Hot and cold water extracts of P. africanum inhibited the highest percentage of eggs from hatching at the lowest concentration. These extracts also exhibited high inhibition percentages of larval development. The highest rates of larval mortality were recorded for both P. africanum and S. italica hot and cold water extracts at the lowest concentration. This work therefore supports the use of these plant species in the control of internal parasites by the Bapedi people of the Blouberg Municipality in Limpopo Province. This is particularly the case because any validation of the use of plants in traditional medicine which is based on the results obtained using organic solvents might be considered irrelevant since, traditionally, water is used instead as a solvent for most of the preparations.
The authors are grateful for the nematode eggs obtained from Dr P C Van Schalkwyk of Biozetica Agri-source Pty (LTD). The co-operation, patience and guidance of Dr B Egan and Prof P Masoko (University of Limpopo, RSA) for plant identification and grinding respectively, Agricultural Extension and Veterinary staff of Blouberg Municipality, Ms L Morey of ARC central office, Pretoria Hatfield for statistical analysis and Limpopo Department of Agriculture for financial support.
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Received 24 September 2016; Accepted 11 November 2016; Published 1 December 2016