JOURNAL OF NATURAL REMEDIES
DOI: 10.18311/jnr/2015/504
Toxicity of Millettia ferruginea (Hochst)
Baker against the Larvae and Adult Ticks of
Boophilus decoloratus a One-Host Tick in Cattle
Manash Kumar Choudhury* and Yoseph Shiferaw
Department of Chemistry, Dilla University, Dilla, P.O Box 419, Ethiopia
Abstract
Chaos is claimed in Indian traditional medical practice to be useful in the treatment of epilepsy and
The present
article reports
the in study,
vitro toxicity
of theeffect
seedofofmethanolic
Millettia ferruginea
firstepilepsy
time against the
neurological
disorders.
In the present
pretreatment
extract of (Berbera) for the on
larvae
and was
mature
ticks in
ofrodents
Boophilus
The 20%,
40%,evaluating
60%, 80%epileptic
and 100%
concentrations
of the seed oil
and
psychosis
evaluated
usingdecoloratus.
standard procedures.
Besides
and behavioral
parameters,
neurotransmitters
such
Gamma-Amino
(GABA)
epilepsy
psychosis
dopamine, noradrenaline
and
were found to kill
allas(100%
mortality)Butyric
larvaeAcid
after
16hr, in12hr,
8hr, and
6hr in
and
2hr respectively.
The 100% concentration
of
serotonin
contentsextract
in the rodent
were estimated.
The mortality
extract pretreatment
reduced
maximal
shock;
Isoniazidfemale tick
the petroleum
(oil) ofbrain
the seed
caused 100%
of adult male,
adult
femaleelectro
and fully
engorged
(INH)
(PTZ) induced
seizures
androot
also bark
significantly
inhibited
the attenuation
of brain
GABA
levels
afterand
5hr,Pentylenetetrazole
7hr and 12hr respectively.
The
root and
showed
less toxicity
while leaves
did not
show
any activity.
by INH and PTZ in mice. These results suggested that the observed beneficial effect in epilepsy may be by enhancing the
Among petroleum ether, acetone and methanol, the acetone extract showed highest acaricidal activity.
GABAergic system. The test drug also inhibited the apomorphine induced climbing and stereotyped behavior and showed
significantly reduced levels of brain dopamine, noradrenaline and serotonin which may be due to blocking of central
Keywords:
Acaricide, Boophilus
decoloratus,
Cattle,
Fabaceae,
vitro,
Millettiasystem.
ferruginea,
Tick obtained in
dopaminergic, noradrenergic
and serotonergic
pathways
or by
enhancingInthe
GABAergic
The results
present study suggest that the title plant possesses antiepileptic and antipsychotic activities in rodents.
Anticonvulsant, dopamine, GABA,
1. Introduction
The objective was to find an easy way for natural
remedy utilizing a cheap natural resource. The present
article gives an efficient solution to control the severe
infestation caused by ticks. The infestation of cattle by
tick, Boophilus decoloratus is highly prevalent (wide
spread) in Ethiopia and all African countries including
South Africa and Australia that leads to low production
of milk, meat and inferior quality of skin. The low quality
of skin becomes useless in leather industry and causes a
serious economic loss in any country.
The aim was to utilize a particular natural source,
Millettia ferruginea to solve this acute problem and make
a trial experiment with the seed oil. Fortunately the first
attempt was successful. The tree is readily available in the
country and is a cheap source of getting medicine that
occurs in nature.
The plant Millettia ferruginea (family: Fabaceae) is a
large tree that grow up to 25 meter (approximately 75 feet)
*Author for correspondence
Email: mkchoudhury2001@yahoo.co.in
high.
It is
widely distributed all over Ethiopia within the
, sinapic
acid
agro climatic zones of 1000-2500 meter (approx 3,0007,500 feet) above the sea level. It is endemic to Ethiopia
and is commonly known as ‘Berbera’ (in Amharic),
Sotallo, Kotalu, Sari, Yego (in Afan Oromo), Hengedi-cho (in Sidama), Zaghia (in Wolaita), Dhadhato (in
Gedeofa) languages1. The mature seeds in the powder
form are used for catching fish due to its toxicity is a
common practice in the country. The powder is spread
over the water surface for stunning fish2. It is planted
along the road sides and in agro forestry for decorative
and agricultural purposes. Moreover, it serves as shade
tree for coffee (Coffea arabica) farms and erosion
control in Eastern and Southern parts of Ethiopia3-5.
The tree is used for firewood, local construction of
houses, tool handles, household tools, improving soil
fertility (nitrogen fixing leguminous tree). The plant is
traditionally used to treat skin infection6 and for dressing
‘mujele’ an infection caused by an insect present in the
soil7. The seed extract of this plant has been observed to
34
Toxicity of Millettia ferruginea (Hochst) Baker against the Larvae and Adult Ticks of Boophilus decoloratus a One-Host Tick in
Cattle
be effective in controlling storage insect pests as adzuki
bean beetle, Callasobruchus chinensis8, maize weevil,
Sitophilus zeamais9. The seed powder of Berbera caused
100% mortality of the bean weevil, Zebrotes subfasciatus
Boheman10. Recent study showed that the aqueous
crude seed extract was extremely effective in reducing
the larval population of maize stalk borer Busseola fusca
(fuller) (Lepidoptera: Noctuidae)11. A laboratory study
showed that water extract of Berbera seed powder at
different concentration levels (10-40 % w/v) caused
93-100 % mortality against the different castes of adult
Macrotermes termites12.
Various parasitic diseases are transmitted by different
types of ticks present in the environment that stick to the
bodies of different animals like cattle, sheep, goat, horse,
donkey, dog, rabbit etc. The cattle and sheep are mostly
affected due to large infestation of different types of
ticks. Ethiopia suffers a serious economic loss and it has
become a burning problem of the day in many African
and Asian countries including Australia. The ticks
not only suck blood from the cattle, sheep and other
animals but also transmit various kinds of diseases to the
animals. The cattle become anaemic and ill-health due
to loss of blood resulting a low production of milk and
meat, the skin becomes useless in leather industry due
to the presence of a large number of holes in it caused
by tick-bite. The cattle undergo premature death after a
long period of suffering. Furthermore, the diseases are
also likely to be transmitted to human being through
uncooked milk and meat causing a serious health hazard
in the society.
Boophilus decoloratus, the blue tick (family: Ixodidae
or hard tick) is a one-host tick occurs throughout the
Ethiopian region, Southern Asia especially in humid
areas. It is parasitic chiefly on cattle and equines, but
also found on sheep and goats, wild ungulates and dogs.
The engorged females have a slate-blue colour and pale
yellow legs. It transmits the disease Babesia bigemina
known as Babesiosis (rise in temperature to high fever, in
appetence (loss of appetite), red urine, anaemia, jaundice,
emaciation, death13. A female lays around 2500 eggs14.
The toxicity of Neem seed oil against the larvae
of Amblyomma variegatum, a three-host tick in
cattle15, toxicity of Neem seed oil against the larvae of
Rhipicephalus sanguineus, a three-host tick in dog16,
toxicity of Neem leaf against the larvae of Boophilus
Journal of Natural Remedies | ISSN: 2320-3358
decoloratus, a one-host tick in cattle17, toxicity of Neem
seed oil against the larvae of Boophilus decoloratus, a
one-host tick in cattle18 toxicity of Khaya senegalensis
(Meliaceae) seed oil against the larvae of Boophilus
decoloratus, a one-host tick in cattle were published
earlier19.
The isolation of rotenone, deguelin and tephrosin20,
durmillone and ferrugone21 from Berbera seeds,
barbigerone from the seeds of Tephrosia barbigera22 were
previously published. Many isoflavones namely ferrugone,
durmillone from the seeds, jamaicin, ichthynone,
7-hydroxy-5,6-dimethoxy-3ʹ,4ʹ-methylenedioxy
isoflavone, flemichapparin-B from the bark, durmillone,
5-methoxy-durmillone,
5-hydroxy
durmillone,
calopogonium isoflavone-A, 4-hydroxy-loncocarpine,
calopogonium isoflavone-B, barbigerone, preferrugone,
predurmillone,
prebarbigerone,
pre-5-methoxy
durmillone were earlier reported from this plant23-25.
The present article reports the acaricidal activity of
Millettia ferruginea seed against Boophilus decoloratus
for the first time. It describes the in vitro toxicity of the
crude petroleum ether extract (oil), acetone extract (oil)
and methanol extract (oil) of the seed, leaf, root and root
bark of this plant against the larvae and mature male
and female ticks. The toxicity of the crude extract of
the seed of this plant against the larvae of Amblyomma
variegatum, a three-host tick in cattle was published
earlier by us26.
2. Material and Methods
2.1 Apparatus and Chemicals
Soxhlet apparatus was used for extraction. All the
solvents were obtained from Fluka Chemie, Sigma
Aldrich, Germany and distilled before use. Petroleum
ether used had b.p. 60-80 oC.
2.2 Collection of Plant Material
The different parts of the plant such as seeds, leaves,
root barks and roots were collected from a place close
to Dilla University, Dilla, Ethiopia. The freshly collected
plant materials were washed with water and dried under
shade at room temperature. The dried samples (root
bark and root) were reduced to small pieces, ground to
a coarse powder using metal coffee grinder. The seeds
were powdered using a grinder machine.
www.informaticsjournals.org/index.php/jnr | Vol 16 (1) | Jan 2016
Manash Kumar Choudhury. et al.
2.3 Extraction in Soxhlet
The powdered seed (5g) was extracted with petroleum
ether (bp.60-80 0C) in a 100 ml round bottom flask
for 6 hours at the rate of 6 cycles/hour. Viscous yellow
coloured oil (1.87 g, yield 37.5%) was obtained after
complete removal of the solvent in a rotavapor. The
analysis of one-way ANOVA indicated that statistically
no significant variation (p < 0.05) was observed in
the yield in the extraction after 6 hours. Similarly, the
powder leaf (5g), powder root (5g) and powder root bark
(5g) were separately extracted with petroleum ether (b.p.
60-80oC) and viscous oil was obtained in each case after
evaporation of solvent. Again powder seed (5g) was
extracted with acetone and powder seed (5g) extracted
with methanol separately.
2.4 Collection of Ticks
The larvae of one-host tick, Boophilus decoloratus, adult
male and female ticks were used in this study. The
engorged female (full of blood), mature male and female
ticks were collected from the bodies of cattle in Oromiya
regional state, Borana zone, Abaya woreda, Samaro
kebele near Dilla town, Ethiopia. The engorged female
tick was detached from the body of the cattle by hand
picking, put in a clean test tube plugged with cotton and
incubated at 300C with a relative humidity of 80-100%.
After the complete laying of eggs the tick died and the
dead female tick was discarded. The eggs laid in the tube
were transferred into a clean tube with a cotton plug. The
eggs were kept under the same incubating conditions
until they hatched into larvae and then starved in the
incubator at 300C for one week before use. The adult
ticks were collected in a similar way from the bodies of
cattle and kept in a clean glass tubes plugged with cotton
and the bioassay carried out soon after 30 minutes
of collection. All the ticks were properly identified at
Holeta Research Centre, Ministry of Agriculture and
Rural Development, National Animal Health Diagnostic
and Investigation Centre, Addis Ababa, Ethiopia.
2.5 Bioassay Analysis
The solvent was completely removed from the oil in
rotary evaporator before the experiment. Petri dishes
(9 cm diameter) with one sheet of filter paper inside of
the same size were used. Five different volumes of seed
oil were dissolved in 1 ml CHCl3 separately to prepare
Journal of Natural Remedies | ISSN: 2320-3358
35
solutions of different concentrations, i.e., 1ml (dissolved
in 1 ml CHCl3, 100%), 0.8 ml (dissolved in 1 ml CHCl3,
80%), 0.6 ml (dissolved in 1 ml CHCl3, 60%), 0.4 ml
(dissolved in 1 ml CHCl3, 40%), 0.2 ml (in 1 ml CHCl3,
20%) and 1 ml distilled water (0%, control) were evenly
soaked (spread with a spatula) in different filter papers
of each Petri dish and CHCl3 was completely removed
by exposing the plates at room temperature and blowing
with cold air. Fifteen (15) larvae were put inside in each
plate with the help of a soft brush and then monitored
at certain interval of time. The number of larvae found
dead in each plate was recorded. The experiment was
repeated four times.
3. Results and Discussion
Experiment showed that the toxic constituent(s) is/are
totally absent in the leaf. The toxicity was found highest
in the seed followed by the root and root bark. 100%
mortality was observed after 2 hours with seed oil, 2½
hours with root oil and 3 hours with root bark oil. The
results are summarized in Table 1.
100% mortality of the adult male, adult female and
fully engorged female was observed after 5, 7 and 12
hours respectively when tested with the seed oil obtained
from petroleum ether extract. This result indicated that
the acaricidal activity of the extract of the seed depends
on sex and stage of the tick species. The adult females and
engorged females took longer time to die than the adult
males; the engorged one took longest time. The female
ticks showed higher resistance towards the chemical(s)
present in the seed. The results are summarized in
Table 2.
Further experiment showed that the mortality of
larvae was concentration and time dependent. 100%
mortality was observed with 20%, 40%, 60%, 80% and
100% seed oil after 16 hours, 12 hours, 8 hours, 6 hours and
2 hours respectively. One way ANOVA analysis applied
to the data showed statistically significant difference in
mortality at the probability level of p < 0.05. With 0%
concentration (water, control) there was no mortality at
any time. The results are summarized in Table 3.
Variable toxicities were observed when different
extracts obtained from petroleum ether, acetone and
methanol tested against the larvae. Extract (oil) obtained
from acetone showed highest toxicity. There was 100%
www.informaticsjournals.org/index.php/jnr | Vol 16 (1) | Jan 2016
36
Toxicity of Millettia ferruginea (Hochst) Baker against the Larvae and Adult Ticks of Boophilus decoloratus a One-Host Tick in
Cattle
Table 1: Toxicity of petroleum ether extract (oil) of leaf, seed,
root and root bark against the larvae of Boophilus
decoloratus
Plant material
Total number of larvae died after ½ hour
interval of time with % mortality
1 ½ hr
2 hr
2 ½ hr
3 hr
Leaf (1ml)
0
0%
0
0%
0
0%
0
0%
Seed (1ml)
14.8
99%
15
100%
Root (1ml)
11.8
79%
14.8
99%
15
100%
Root bark (1ml)
11.0
73%
13.3
89%
14.8
99%
15
100%
Water (control, 0%)
0
0%
0
0%
0
0%
0
0%
15 larvae were used in each plate, the values are given as mean of 4 replicates
Table 2: Toxicity of the petroleum ether extract (oil) of the
seed against adult male, adult female and fully
engorged female of Boophilus decoloratus
Volume of PE
extract used
Nature of tick
Number of
ticks used
100% mortality
of ticks after
1 ml
Adult male
20
5 hours
1 ml
Adult female
20
7 hours
1 ml
Fully engorged
female
20
12 hours
The values are given as mean of 4 replicates
Table 3: Toxicity of petroleum ether extract (oil) against larvae of Boophilus decoloratus at different
concentrations and different interval of time
Plate No
Volume of
extract soaked
Number of dead larvae during interval of 2 hrs with % mortality
0-2 hr
2-4 hr
4-6 hr
6-8 hr
8-10 hr
10-12 hr
12-14 hr
14-13 hr
1.5
93%
1
100%
0
0%
0
0%
1
0.2ml (20%)
0.5
3%
1.5
13%
2.3
29%
2.2
43%
3
63%
3
83%
2
0.4ml (40%)
1
7%
2.5
23%
3
43%
3.5
67%
2
80%
3
100%
3
0.6ml (60%)
5
33%
4
60%
1.5
70%
4.5
100%
4
0.8ml (80%)
8.0
53%
4
80%
3
100%
5
1.0 ml (100%)
15
100%
6
H2O
1ml (0%)
0
0%
0
0%
0
0%
0
0%
0
0%
0
0%
15 larvae were used in each plate, the values are given as mean of 4 replicates
Journal of Natural Remedies | ISSN: 2320-3358
www.informaticsjournals.org/index.php/jnr | Vol 16 (1) | Jan 2016
Manash Kumar Choudhury. et al.
mortality after 1½ hour, 2 hours and 5 hours with acetone,
petroleum ether and methanol extract respectively. The
results are summarized in Table 4. Statistical analysis of
the results obtained in Table 3 is shown in Table 5.
The acetone extract of the seed showed the highest
efficacy than that of petroleum ether and methanol
extracts. The comparative studies of the present findings
revealed that the petroleum ether extract of the seed
(oil) possesses much higher acaricidal activity against
Amblyomma variegatum25. 100% concentration of
the seed oil took 1.5 hours for 100% mortality for A.
variegatum and 2 hours for B. decoloratus. In other
words Amblyomma variegatum is more sensitive than
Boophilus decoloratus towards the toxicity of seed oil of
this plant. The seed oil was found to be safe to the skin
of the cattle. When the oil (100% concentration) was
applied to the body (skin) of the cattle and kept under
close observation for more than a week, no irritation or
inflammation on the skin of the cattle was observed. No
larvae or adult male/female were found within the close
vicinity of the oil applied. This showed the toxic nature
of the oil towards the tick.
Financial constrained smallholding farmers cannot
afford to buy insecticide and application equipment.
For these reasons attention is being given to the use of
37
ecologically safe and cheaper methods such as botanicals
against various pests and insects. The isolation of crude
oil from seed is inexpensive and has been made simple
that can be easily adopted by farmers for the management
of ticks. We discovered that petroleum ether is easily
replaced by water, the cheapest solvent for isolation of
the oil. The powder seed is heated with water and on
cooling at room temperature, oil floats on the surface.
This oil is easily separated by decantation and directly
applied on the body of a cattle and get rid of ticks very
quickly. This method is very simple, fast and efficient;
it does not need any organic solvent or chemical. Here
lies the beauty of the work. The oil isolated by this
procedure and oil extracted with petroleum ether or
acetone showed identical Rf values on TLC indicating
the active compound(s) present did not undergo any
chemical change on heating and they are thermally
stable compounds. The water and petroleum extracts
both exhibited same toxicity results in laboratory
experiment. Since the seed oil of Millettia ferruginea is
inexpensive, readily available and bio-degradable, the
seed of this plant undoubtedly will be a better substitute
for any synthetic insecticides which are expensive, not
bio-degradable and suffer from serious side effects. The
isolation, characterization and bioassay experiments
with isolated compounds are in progress.
Table 4: Acaricidal activity of different extracts against larvae at different interval of time with % mortality
Extract
Total number of larvae died and % Mortality at different interval of time
½ hr
1 hr
1½ hr
2 hr
PE (1ml)
6.00
40%
10.65
71%
14.55
97%
15
100%
Acetone
(1ml)
6.45
43%
11.70
78%
15
100%
---
2.25
15%
3.45
23%
CH3OH (1ml)
2½ hr
3 hr
3½ hr
4 hr
4½ hr
5 hr
7.50
50%
8.70
58%
11.25
75%
11.55
77%
13.50
90%
15
100%
6.00
40%
15 larvae were used in each plate; the values are given as mean of 4 replicates
Table 5: Statistical analysis of the results obtained in Table 3. Results of ANOVA tests
justifying the effect of time interval on the mortality of larvae of Boophilus
decoloratus exposed to different concentrations of petroleum ether extract.
Source of
variation
SS
DF
MS
F
F-critical
P-values
Between groups
115.091
7
16.44157143
2.668490626
2.312741187
0.027070235
Within groups
197.164
32
6.161375
Total
312.255
39
SS: sum square, DF: degree of freedom, MS: mean square, F: F statistics
Journal of Natural Remedies | ISSN: 2320-3358
www.informaticsjournals.org/index.php/jnr | Vol 16 (1) | Jan 2016
38
Toxicity of Millettia ferruginea (Hochst) Baker against the Larvae and Adult Ticks of Boophilus decoloratus a One-Host Tick in
Cattle
The seed oil on further purification over column
chromatography afforded a colourless oil and few
isoflavonoid compounds. These isoflavones are expected
to be bioactive compounds and the evaluation of
bioactivity is in progress. After preliminary investigation
the colourless oil was characterized as a high molecular
weight triglyceride with saturated and unsaturated
fatty acids in the ester linkage by spectral analysis. The
complete structure elucidation is in progress. The oil
obtained (37.5% yield) from the seed has a potential
application for biodiesel, soap and glycerol (glycerine)
production. At present the whole world is looking for
bio-diesel since the petrol and diesel are very expensive.
The present trend in the world is to explore natural
resources for curing diseases of human being, plant
kingdom and live stock animals. Scientists are nowadays
encouraging and appreciating this type of simple and low
cost involved work which has an immediate application
in the society.
The results of ANOVA single factor test at P = 0.05
level of significance using Microsoft Excel are shown in
Table 5. The F-value is found to be 2.668490626 for ‘time
interval’ and the value is greater than its F-critical value
2.312741187. The ‘time interval’ factor is significant at
P = 0.05 level. The P value of 0.027070235 (P < 0.05)
indicates that the ‘time interval’ factor is statistically
highly significant (95%) on the mortality of larvae
exposed to different concentrations.
4. Acknowledgement
The authors are thankful to Mr. Asish Das, Department
of Electrical Engineering, National Institute of Technical
Teachers’ Training and Research, Block-FC, Sector-III,
Salt Lake City, Kolkata-700 106, India for his technical
assistance in statistical analysis during preparation of the
manuscript.
5. References
1. Bekele A. Useful trees and shrubs for Ethiopia:
Identification, propagation and Management for 17
agro climatic zones, Nairobi: RELMA in ICRAF project,
Nairobi, Kenya. 2007.
2. Karunamoorthi K, Bishaw D, Mulet T. Toxic effects
of traditional Ethiopian fish poisoning plant Millettia
ferruginea (Hochst) seed extract on aquatic macro
Journal of Natural Remedies | ISSN: 2320-3358
invertebrates. European Review for Medical and
Pharmacological Sciences. 2009; 13:179–85.
3. Teketay D, Tegineh A. Shade trees of coffee in Harare,
Eastern Ethiopia. The International Tree Crops Journal.
1991; 7:17–27.
4. Tadesse H, Legesse N, Olsson M. Mellittia ferruginea from
Southern Ethiopia: impacts on soil fertility and growth of
maize. Agroforestry Systems. 2000; 48:9–24.
5. Azene B. Useful trees and shrubs of Ethiopia; identification,
propagation and management for 17 agroclimatic zones.
In Technical Manual No.6. RELMA in ICRAF Project
Kenya. 2007. p. 550. ISBN: 978-92905-92129.
6. Mesfin F, Demissew S, Telehaymanot T. An ethno botanical
study of medicinal plants of Wonago, Woreda, SNNPR,
Ethiopia. J Ethno Biol and Ethno Med. 2009; 5(28):1–8.
7. Teklelehaymanot T, Giday M. Ethno Botanical study
of medicinal plants used by people in Zeige Peninsula,
North-western Ethiopia. J Ethno Biol Ethno Med. 2007;
3(12):1–12.
8. Damte T, Chichaybelu M. The efficacy of some botanicals
in controlling Adzuki bean beetle, Callosobruchus
chinensis in stored chickpea. Tropical Sci. 2002; 42:192–5.
9. Jembere B. Evaluation of the toxicity potential Millettia
ferruginea (Hochest) Baker against Sitophilus zeamais
Mots. International J Pest Management. 2002; 42(1):29–
32.
10. Jembere B, Namukobe J, Benard TK, Dagne E. Extracts
of Millettia ferruginea, Tephrosia vogellii and Tephrosia
pentaphylla against the bean weevil Zabrotes subfasciatus
(Boheman). Sinet: Ethiopian J Sci. 2007; 30:49–54.
11. Tilahun B, Azerefegne F. Efficacy of the aqueous crude
seed extract of Millettia ferruginea (Fabaceae) on the maize
stemborer Busseola fusca (Lepidoptera: Noctuidae) in the
field. International J Tropical Insect Sci. 2013; 33(4):256–63.
12. Getahun D, Jembere B. Evaluation of toxicity of crude
extracts of some botanicals on different castes of
Macrotermes termites. Paste Management Journal of
Ethiopia. 2006; 10:15–23.
13. Shaw RD, Thorburn JA, Wallace HG. Cattle Tick Control,
Prepared by Members of The Wellcome Research
Organisation: Cooper Division Wellcome Foundation Ltd,
London, England. 1970. p. 15.
14. Soulsby EJL. Helminths, Arthropods and Protozoa of
Domesticated Animals, 7th edition, English Language
Book Society (ELBS)/Bailliere Tindall, London, England.
1982. p. 468.
15. Ndumu AA, George JBD, Choudhury MK. Toxicity of
neem seed oil (Azadirachta indica) against the larvae
www.informaticsjournals.org/index.php/jnr | Vol 16 (1) | Jan 2016
Manash Kumar Choudhury. et al.
16.
17.
18.
19.
20.
of Amblyomma variegatum a three-host tick in cattle.
Phytotherapy Research. 1999; 13:532–4.
Choudhury MK. Toxicity of neem seed oil (Azadirachta
indica) against the larvae of Rhipicephalus sanguineus a
three-host tick in dog. J Parasitic Diseases. 2001; 25(11):
46–7.
Choudhury MK. Efficacy of neem leaf (Azadirachta indica,
family: Meliaceae) in the Control of larvae of Boophilus
decoloratus, a one-host tick in cattle. J Nepal Pharm
Assocn. 2003; 21(1):41–3.
Choudhury MK. Toxicity of neem seed oil against the
larvae of Boophilus decoloratus, a one-host tick in cattle.
Indian J Pharm Sci. 2009 Sep-Oct; 562–3.
Choudhury MK, Boshe FD. Toxicity of Khaya senegalensis
seed oil against the larvae of Boophilus decoloratus: a onehost tick in cattle. Proceedings of Zoological Soc. 2013;
66(2): 137–40. Published online 17 Jan. DOI: 10.1007/
s12595-012-0058-8.
Clark EP. The occurrence of rotenone and related
substances in the seeds of the Berebera tree. A procedure
Journal of Natural Remedies | ISSN: 2320-3358
21.
22.
23.
24.
25.
26.
39
for the separation of deguelin and tephrosin. J Am Chem
Soc. 1943; 65:27–9.
Highet RJ, Highet PF. The structure of two isoflavones
from the Abyssinian Berebera tree. J Org Chem. 1967;
32:1055–8.
Vilain C. Barbigerone, a new pyranoisoflavone from seeds
of Tephrosia barbigera. Phytochemistry. 1980; 19:988–9.
Dagne E, Bekele A, Waterman PG. The flavonoids of
Millettia ferruginea subsp. Ferruginea and subsp. Darassana
in Ethiopia. Phytochemistry. 1989; 28(7):1897–900.
Dagne E, Bekele A, Noguchi H, Shibuya M, Sankawa U.
O-geranylated and O- prenylated flavonoids from Millettia
ferruginea. Phytochemistry. 1990; 29(8):2671–73.
Dagne E, Bekele A. C-prenylated isoflavones from Millettia
ferruginea. Phytochemistry. 1990; 29(8):2679–82.
Choudhury MK, Shiferaw Y, Hussen A. Toxicity of
Millettia ferruginea darasana (family: Fabaceae) against
the larvae and adult ticks of Amblyomma variegatum
Fabricius, a three host tick in cattle. J. Parasitic Diseases.
2013; 39:298–302. DOI: 10.1007/s12639-013-0311-8.
www.informaticsjournals.org/index.php/jnr | Vol 16 (1) | Jan 2016