Download on murine uterine contractility. : 0 Vol. 22, No. 1 , 2010 by Alawa

EFFECTS OF NATRON (KANWA) VARIETIES ON MURINE VIRGIN UTERINE
CONTRACTILITY
Alawa N. Judith1,, , Kwanashie O. Helen2, Singh S. Prasad1 and Alawa B. I. Clement3
Departments of Human Anatomy1, Pharmacology and Clinical Pharmacy2and National Animal Production
Research Institute3, Ahmadu Bello University, Zaria, Nigeria
Abstract
Natron is traditionally used as an aid in childbirth, postnatal care and employed in the
treatment of menstrual disorders. The folkloric use of natron has been well documented
but few scientific studies addressing its efficacy or side effects exist. This study reports
the pharmacological response of isolated strips of murine uterine tissue to oxytocin as
standard and six varieties of natron which are commonly used in ethnomedicinal
preparations. Results of uterine contractility showed that only three varieties namely Far
kanwa (4.0 – 16.0 mg/ml), kanwa Ungruni (4.0 – 16.0 mg/ml) and Ja kanwa (1.6 – 16.0
mg/ml) stimulated contractions while neither kanwa Balma, Manda nor Shanu did. Ja
kanwa exhibited greatest potency with a rate of 1:300 of that of oxytocin. It elicited a
dose-dependent transcient contractile response followed by relaxation of the isolated rat
uterus. These results confirm the efficacy of Ja kanwa in childbirth and further supports
the rationale of its folkloric use as an abortifacient.
Key words: Natron, Uterine, Murine, Contractility
1
INTRODUTION
Natron, a sesquicarbonate or hydrated carbonate of sodium is generally referred to as
Kanwa in the northern part of Nigeria and a product of the salt industry in many parts of
northern Africa. It occurs as a mixture of different substances with sodium constituting
about 30% and other minerals such as potassium, iron, zinc etc in varying proportions.
There are different varieties and its composition varies with respect to their locations
(Ekanem and Harrison, 1997; Ikwuegbu, 1985). Natron has several uses: it is used in
cooking as a food tenderizer especially in pulses (Ekanem and Harrison 1992; Edijala,
1980), to curdle milk, in the tanning industry and in the preparation and enhancement of
flavor of local beverages and snuff (Buchanan and Pugh, 1969). It is used extensively in
ethnoveterinary practices for the treatment of skin diseases and digestive problems. It
also serves as a salt lick and mineral supplement in ruminants (Alawa et al, 2000) and
used in decoctions for the treatment of reproductive ailments such as retained placenta
and difficulty in urination. Its uncontrolled use in livestock may lead to abortions in
pregnant animals (Gefu et al, 1998). Ethnomedicinal potency has been documented for
ailments such as stomach ache, constipation and toothache (Buchanan and Pugh,
1969). The historical use of natron as an aid in childbirth has been well documented. In
the kanuri tribe of Borno, it is used to increase uterine contractility and motility for safer
childbirth (Fage, 1982). In the Hausa-Fulani tribe of northern Nigeria, it is used in
postnatal care of the puerperium as nursing mothers consume large quantities of natron
(about 40 g equivalent to 450M of Na) daily in a pap of guinea corn as part of the fortyday postpartum practice in the belief that it increases the quantity and quality of breast
milk (Sanderson et al 1979). Consequently this has been implicated in the incidence of
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peripartum cardiac failure (PPCF) among nursing mothers in this region (Davidson et al
1974). Traditionally it is used to treat various ailments related to endocrine and
reproductive systems such as treatment of painful uterus, inducing uterine contractions
or abortions, management of retained placenta and post partum bleeding, infections,
infertility, colic pains and treatment of irregular and painful menstruation. One of its
folkloric claims is its use as an abortifacient which has not been substantiated
scientifically. This study aimed to determine the effect of six varieties of natron
commonly used in the northern part of Nigeria on the isolated uterine contractility in
virgin Wistar rats demonstrated that some varieties stimulated uterine contractions in
the rats hence providing some pharmacological justification for the ethnic use of natron
as a herbal oxytocic or abortifacient.
MATERIALS AND METHOD
Collection and Identification of Samples
Six samples of natron in common use were bought from the local market in Samaru,
Kaduna State. They were identified and authenticated by the Chief technologist, at the
Mineral Stone laboratory in the Department of Geology, Ahmadu Bello University, Zaria.
Numbers I-VI were arbitrarily assigned to identify each sample for this study only. The
corresponding Hausa names are Far kanwa (sample I), kanwa Ungruni (sample II), Ja
kanwa (sample III), kanwa Balma (sample IV), kanwa Manda (sample V) and kanwa
Shanu (sample VI).
3
Preparation of Samples
Each sample was visually observed to establish its physical properties. This is shown in
Table 1. The samples were then carefully ground with mortar and pestle and sieved
through a 9 µ-pore size sieve. Sample specimens were weighed on a Mettler balance,
mixed thoroughly and stored in a stoppered bottle under room temperature until used.
Sample Analysis
The six samples were analyzed both quantitatively and qualitatively for chemical
composition using analytical reagent grade chemicals and distilled water.
Quantitative analysis
This is used to determine the cations or metallic elements present in each sample. The
sample solutions were coned and quartered to obtain the sample for the quantitative
analysis. Each solution was made up to the 1 litre mark in the conical flask with distilled
water. 0.004M-lanthanum chloride was introduced into the sample solutions of natron
for the detection of sodium and potassium. This is to suppress the ionization
interference observed during their determination. The analysis of sodium and potassium
was done by the flame photometer. The analyses for magnesium, manganese, zinc,
iron,
calcium
and
lead
were
performed
in
the
Unicam
atomic
absorption
spectrophotometer (Model 969). The instrument was adjusted for optimum flow of flame
gas and test solutions. The capillary tube was aspirated with distilled water initially then
with air to clear the liquid passage. The calibrations involved the aspiration of the blank
solution and setting it to zero reading after which the concentrated standards were
4
aspirated. The readings were taken and these were used in the construction of the
calibration curves.
Qualitative analysis
This is used to determine the anions present in the samples. Standard methods of
qualitative analysis were used and this involved titration against standards using colour
indicators such as methyl orange. The results were checked against those obtained by
pH titration for other samples.
Experimental Animals
Female adult albino rats inbred in the Animal House of the Department of
Pharmacology and Clinical Pharmacy, A.B.U. Zaria. They were allowed to acclimatize
for a week in the animal unit of the Department of Human Anatomy before the study. All
rats were fed with commercial pellets (Pfizer Nigeria Plc., Ikeja, Nigeria) and watered ad
libitum throughout the duration of the study. This research was carried out in
accordance to the rules in Nigeria governing the use and care of laboratory animals as
accepted internationally.
Rat Uterus Preparation
Non-pregnant young Wistar rats weighing approximately 150 g were used in each
experiment to determine the effect of the six different samples of natron on the isolated
uterus. The choice of rat was informed by the fact that, for isolated uterine preparation
work, of various animal species, the rat is the best model that could be extrapolated to
man. Each rat was treated with stilbesterol intraperitoneally inorder to induce estrous.
This was done to prime the uterus, to make the uterine horns more distinguishable and
5
enhance sensitivity to drug effect. A blow on the head and cervical dislocation sacrificed
the rats, the uterine horns were rapidly excised and cleaned of surrounding tissues and
transferred into an oxygenated petri dish containing the physiological solution. They
were divided into about 4 sections to reduce the length. The horns were opened along
the mesenteric border and the myometrial tissue cut into longitudinal strips of 2 cm
which were mounted by means of a thread loop to an internal hook located at the base
of a removable tissue holder. A second loop was tied at the other end of the tissue and
attached to the isometric transducer (Ugo Basile). Each tissue was mounted in a
conventional organ bath (25 ml) containing De–Jalon’s solution composed of [5 litres:
Nacl (45 g), KCl 10% (21 ml), D. glucose (2.5 g), NaHCO3 (2.5 g), CaCl2 (M) dissolved
and made upto 5 litres with de-ionised water] at a constant temperature of 37oC and pH
of 7.4. It was continuously aerated with 95% oxygen/ 5% carbondioxide mixture and
connected in parallel to an Ugo Basile microdynanometer (7050) o which contractions
were recorded. Uterine contractions were ecorded on a paper by Universal Harvard
Osillograph with chart speed of 6 mm/min and sensitivity of x3. Three different
concentrations were prepared 100 mg/ml, 250 mg/ml and 500 mg/ml by dissolving the
samples in distilled water. Oxytocin (10 µg/ml and 40 µg/ml) was used as the standard
drug.
The mounted tissue was allowed to equilibrate for about 30 minutes before
challenging with the drug or natron sample. A contact period of 2 minutes was allowed
between drug or sample and the tissue. Adequate rest periods (usually 20 minutes but
sometimes longer) after washing thrice was given until tracing returned to the baseline
before challenging again with the drug or sample. Maintenance of buoyancy and
prevention of sagging was achieved by supplying the bathing fluid as fast as possible
6
after each drain as this could produce irregular results. Each sample like oxytocin was
tested on at least three uterine preparations from different rats.
RESULTS
All the samples were crystalline in texture and the sample colours varied. Most were
light coloured from white and light pink to grey while others were dark coloured including
brown and black. This is shown in Table 1 below.
Table 1: Physical Properties of Natron Samples
NATRON SAMPLE
APPEARANCE
COLOUR
I) Farar Kanwa
Mixed range of large to
micro-crystals
White grey with
blackish grey stains
II) Ja Kanwa
Crystal aggregates
Light pink with white
patches
III) Ungruni
Transparent needle
shaped aggregates
Blackish grey
IV) Balma
Crystal aggregates
Dirty white – grey
V) Manda
Mixed crystalline
and earthy mass
White mixed with
brown zones
Crystalline particles
White –grey
VI) Kanwa Shanu
of mixed grain sizes
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Result of Chemical Analysis
This gives the amount of cations and anions present in each sample. Sodium levels
were high in all the samples with an approximate value of 30% and highest occurrence
was in Ja Kanwa (sample III) where it was 40%.
Potassium levels were low as
expected (less than 10%). Highest occurrence was in Kanwa Shanu (sample VI). Iron
was present in all the samples but was of low levels (less than 10%). Highest
occurrence was in Manda (sample V). Calcium occurred in all the samples in
appreciable amounts and significantly high in Ungruni (sample II) and Ja Kanwa
(sample III) where it was approximately 30%. Magnesium, manganese and zinc
occurred in very little quantities of less than or approximately 1% in all the samples.
Carbonates (CO3)2- were detected in all the samples and it occurred with bicarbonates
(HCO3-) except in sample II (Ungruni) in which bicarbonate occurred in trace amounts.
All the samples contained sulphates in appreciable amounts with sample IV (Balma)
containing the highest amount of over 50%. Chloride was also detected in all the
samples but in very little quantity usually less than 5%. Values determined for individual
mineral elements are presented
8
Table 2a: Yields of cations in six natron samples following quantitative analysis
Samples
Elements (% Cations)
Na
K
Fe
Mg
Mn
Zn
Ca
Farar kanwa
28.0
2.8
4.9
0.9
0.3
0.8
10.1
Ungruni
25.9
3.7
7.0
0.1
0.1
0.8
27.0
Ja kanwa
39.5
3.6
6.5
0.5
0.1
1.0
30.5
Balma
31.4
3.6
7.1
0.1
0.1
1.0
8.5
Manda
37.6
3.3
8.0
0.2
0.2
1.0
16.1
Shanu
26.3
5.4
6.3
0.4
0.3
0.9
11.8
Table 2b: Yields of Anions in six natron samples following quantitative analysis
Samples
Elements (% Anions)
Carbonates
Farar kanwa
9.0
Bicarbonates
Sulphates
Chloride
4.9
32.1
2.3
Ungruni
19.9
Trace
27.7
1.1
Ja kanwa
13.9
1.7
33.1
0.7
Balma
3.0
1.2
53.6
1.2
Manda
4.1
0.8
41.7
1.1
Shanu
3.8
0.8
27.1
1.6
9
Effects of natron samples and Oxytocin on uterine contractions in rats
Different doses of the six natron samples ranging from (1.6 – 16 mg/ml organ bath
concentration) were tested on the rat’s uterus. Three natron samples Farar kanwa,
Ungruni and Ja kanwa stimulated contractions of the rat uterus (Table 3) while three
samples, kanwa Balma, Manda and Shanu did not contract the uterus despite being
used in the same concentrations as the others. Ja Kanwa elicited highest contraction,
which was seen to increase with concentration, hence it could be said to be
concentration dependent. Ungruni followed this while Farar Kanwa elicited the least
(figure 1a, b, c). Oxytocin, which was the standard drug used, (40–1600 ng/ml organ
bath concentration) contracted the rat uterus in a dose –dependent manner (figure 1d).
The rat uterus preparation was highly responsive to it, as a low concentration of 40
ng/ml produced appreciable contraction. Futhermore, oxytocin response was immediate
as there was no time lag between contact of the drug with the tissue and response
observed while response to the natron samples not immediate as there was a time lag
between introduction of the drug and tissue contraction.
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Table 3: Contractile Effect of Natron (Kanwa) on Virgin Rat Uteri
OBC‫٭‬
Log OBC
Mean Responses of samples
(height in mm)
Farar Kanwa
(µg/ml)
Ungruni
Ja Kanwa
1600
3.20
2.00±0.58
2.67±2.18
1.67±0.88
3200
3.51
2.67±1.33
3.33±1.86
2.33±1.20
4000
3.60
2.33±1.20
4.67±3.71
2.50±1.44
8000
3.90
4.57±2.90
5.67±3.67
3.83±1.74
16000
4.20
5.67±3.13
6.73±0.33
110.50±2.04
‫ ٭‬OBC—Organ Bath Concentration
Table 4: Contractile Effect of Oxytocin on virgin Rat Uteri
Organ Bath Conc.
Log. Org. Bath
Height of Contraction
(ng/ml)
Conc.
(mm)
40
1.60
23
80
1.90
39
160
2.20
42
320
2.51
50
400
2.60
54
800
2.90
57
1600
3.20
62
11
Fig 1a: Contractile effects of Farar Kanwa on virgin rat uterus
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Fig 1b: Contractile effects of Kanwa Ungruni on virgin rat uterus
13
Fig 1c: Contractile effects of Ja Kanwa on virgin rat uterus
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Fig 2:Comparison of log concentration/response curves of natron samples on isolated uterine strips
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Fig 1d: Concentration-dependent contractile effects of oxytocin on virgin rat uterus
16
Fig 1e: Non-contractile effects of Manda, Balma and Shanu samples on virgin rat uterus
17
DISCUSSION
Results of the chemical analysis showed that sodium was the predominant metal in all
the samples which is in line with the results obtained from various studies involving the
chemical composition of natron (Alawa et al, 2000; Ikwuegbu et al, 1985; Ankrah and
Dovlo, 1978 and Ekanem, 1977). Sodium constituted more than 30% of the total
composition in three of the six natron samples. Potassium levels were low (ranging from
2.8 to 5.4%, which is as expected, as the name “potash” is actually a misnomer. These
elements are known as the macro-elements and are mainly utilized in the body for
structural purposes (calcium) or in maintenance of acid to base balance (sodium,
potassium and chlorine). Results demonstrated that samples which contracted the
uterus, had relatively high amount of sodium when compared to those which did not
contract the uterus. It may be reasonable to assume that uterine contractions may be
mediated via sodium influx into the cell with a subsequent increase in intracellular
calcium. This could be compared to the mode of action of prostaglandin E 2 and F2α,
which stimulate uterine contraction and sensitize the uterus to oxytocin. This is
mediated via sodium influx and increase in intracellular calcium (Bygdeman, 1992).
These two elements are essential in muscle contraction; sodium is used to increase the
force of contraction (Rogart, 1981) while calcium is essential for continual contractility
and it is well established that excitation contraction coupling in smooth muscle is
initiated by a rise in cystolic calcium (Suzuki et al 1990). This indicates that the chemical
composition of natron is positively correlated with its contractile activity. This is one of
the findings of this research work.
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Also results showed that the contractions elicited by the natron samples were far less
(300folds) and not immediate as compared to oxytocin the standard drug despite the
higher concentrations used, difference being mg/ml as against ng/ml. This suggests that
the samples are interacting with contractile mechanisms in smooth muscles but inorder
to bring an effect similar to that of oxytocin, it must be used in very large quantities or
should be used with a potentiator, which will increase its effect on the uterus. The
response was also not spontaneous as there was a time lag between introduction of the
drug and tissue contraction. This may suggest that the samples were not acting directly
on the smooth muscles via L- type channels which allow the influx of calcium (kuriyama
et al, 1995) but rather via receptor operated channels or voltage operated channels
(Aziba, 2008). However among the samples, which produced contraction, Ja kanwa
elicited the highest contraction as shown on the log concentration/response curves
(figure 2). This may be the scientific basis for which it is used as an abortifacient
substance particularly as the pregnant uterus is normally more sensitive to drugs than
the non-pregnant uterus. Thus, it may bring about abortion in the early stages of
pregnancy if used in high enough concentration. This action may be compared to that of
oxytocin, which is, a hormone produced by the hypothalamus and stored in the
neurohypohysis (posterior lobe of the pituitary gland). Oxytocin causes contraction of
the myometrium of the uterus hence it could be used in the induction of labour, uterine
inertia and abortion. Its use in labour is because it has a selective stimulating action on
the uterine muscle and its degree of activity is related to the physiological state of the
uterus, which is greatest during the last stages of pregnancy (Hopkins, 1993).
19
Ja kanwa has also been implicated in the expulsion of retained placenta in
ethnoveterinary practices (Abdu et al, 2000) but this action can be related more to
ergometrine than to oxytocin. Ergometrine is an alkaloid of the fungus ergot that
develops in the ear of rye and some other cereals (Hopkins, 1993). It is used to induce
the expulsion of retained placenta when not normally expelled. This is because it brings
about sustained contractions of the uterus, which is required to push out the placenta.
This property may not be exhibited by oxytocin as it has a more rapid but shorter action
and could lead to the rupture of the uterus. Thus the contractile effect of ja kanwa was
can be compared to this as it not only contract the uterus but had a sustained effect on
the uterine contraction. This may be directly or via another means which is yet to be
determined.
Since the folkloric uses elicited by Ja kanwa are similar to those produced by both
oxytocin and ergometrine, it can then be compared to syntometrine, which is a product,
formulated to produce results likened to a combination of oxytocin and ergometrine that
is (i.e.) prompt and sustained action. Syntometrine contains synthetic oxytocin and
ergometrine (Hopkins, 1993). Further work on the effect of natron on receptors ad other
channels is needed to elucidate its specific mode of action.
ACKOWLEDGEMENT
The author wishes to thank Mr John kono and Mallam Ibrahim Adamu for their technical
assistance in the laboratory.
20
REFERENCES
1. Abdu, P.A.; Gefu J.O; Jagun, A.G.; Mohammed, A.K. and Alawa, C.B.I. (2000). A Survey
of Ethnoveterinary Practices of Agropastoralists in Nigeria. In: Gefu, J.O.; Abdu, P.A.
and Alawa, C.B. (Eds.) Ethnoveterinary Practices Research and Development.
Proceedings of the International Workshop on Ethnoveterinary Practices, 25 – 27.
2. Alawa, C.B.I.; Adamu, A.M.; Ehoche, O.W.; Lamidi, O.S. and Oni O.O. (2000).
Performance of Bunaji Bulls fed maize stover supplemented with urea and local mineral
lick (Kanwa). Journal of Agriculture and Environment, 1(2): 35 – 42
3. Ankrah, E.K. and Dovlo, F.E. (1978). Properties of Trona and its effect on cooking time
of cowpeas. Journal of Science, Food and Agriculture, 50: 345 – 349
4. Aziba P.I. (2008).Effects of methanolic root extract of Ledebouria ovaltifolia
(Hyacithaneae) on isolated smooth muscle preparation in rats. African Journal of
Biomedical Research, 11: 323 – 327
5. Buchanan, K.M. and Pugh, J.C. (1969). Land and People in Nigeria. 7th Edition.
University of London Press, 120 to 125.
6. Bygdeman, M. (1992). Prostaglandin analogues and their uses. Baillieres Chemical
Obstetrics and Gynaecology, 104: 275 – 277.
7. Davidson, N.McD, Trevitt, L. and Parry E.H.O. (1974). Peripartum Cardiac Failure.
Bulletin of World Health Organization, 51: 203 – 208.
8. Edijala, J.K. (1980). Effects of Processing on Thiamin, Riboflavin and Protein contents of
Cowpeas, Alkali (Potash) treatment. Journal of Food Technology, 15: 445 – 453.
9. Ekanem, E.J. (1977). Preliminary Analysis of Samples of “Kanwa” for sodium, potassium
and other materials. Unpublished M.Sc. Thesis. Department of Chemistry, Ahmadu
Bello University Zaria
10. Ekanem, E.J. and Harrison G.F.S. (1992). The raw material potential of lake salt
(Kanwa). A Poser for Nigerian Industries. Paper presented at 3rd annual conference of
Science Association of Nigeria, Makurdi.
11. Ekanem, E.J. and Harrison G.F.S. (1997). Assessment of Lake salt for sourcing dietary
minerals. Nigerian Journal of Chemical Research, 2: 33 - 38.
12. Fage J.D. (1982). The Cambridge history of Africa: From the earliest times to 500BC.
Cambridge University Press. Volume 1: 222 – 225
13. Gefu, J.O.; Abdu, P.A. and Alawa C.B.I (1998). Ethnoveterinary Practices of livestock
producers in Nigeria, 80 – 84.
21
14. Hopkins, S.J. (1993). Drugs and Pharmacology for Nurses. 11th Edition.
Publishers Limited, Singapore, 385 – 387.
Longman
15. Ikwuegbu, O.A.; Gbodi, T.A. and Ogbonna, G.A. (1985). Effects of Local Salt (Kanwa)
and Proprietary mineral salt licks in the ration of milk cows. Nigerian Veterinary Journal,
14(1): 66 – 69.
16. Kuriyama K. Yanada S., Nozaki T and Kamijoh T (1995). Radiation effects on Li-vacancy
ordering in β-LiAl. Physiological Reviews B, 52:3020 – 3022
17. Rogart, R. (1981). Sodium Channels in Nerve and Muscles Membrane. Annual Review
of Physiology, 43: 711
18. Sanderson, J.E.; Adesanya, C.O.; Anjorin, F.I. and Parry, E.H.O. (1979). Post–partum
Cardiac Failure – Heart Failure due to Cardiac Overload. American Heart Journal, 97:
613 – 621.
19. Suzuki H. and Chen G. (1990). Endothelium derived hyperpolarizing factor (EDHF) on
endogenous potassium channel opener. News in Physiological Science, 5: 212 – 215
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