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Journal of Chemical and Pharmaceutical Research, 2015, 7(1):870-878
Research Article
ISSN : 0975-7384
CODEN(USA) : JCPRC5
Assessment of the suspending properties of naturally occurring
Entandrophragma angolense gum in oral sulphamethoxazole suspension
Oladapo A. Adetunji and Mosunmola O. Odole
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, University of Ibadan,
Ibadan, Nigeria
_____________________________________________________________________________________________
ABSTRACT
The need to reduce costs of pharmaceutical suspensions and consequently enhance affordability has culminated in
the search for a pool of naturally occurring excipients that are biodegradable, economically feasible and can also
compete with established agents. A study was conducted on the suitability of Entandophragma angolense gum as a
suspending agent in oral pharmaceutical suspensions in comparison with official gelatin BP and Acacia gum. The
gum was characterized and used in formulating sulphamethoxazole suspensions containing different concentrations
(1.0 - 4.0 % w/v) of the gum; comparison was made with similar formulations containing gelatin BP and Acacia
gum. Rheological properties, particle size analysis, suspending properties at varying pH values and stability at
different temperatures were the assessment criteria for comparing the suspending ability of the polymers.
Entandrophragma angolense gum is devoid of alkaloids, but contains carbohydrates, reducing sugars and saponins,
which are pointers to its emulsifying properties. At all concentrations employed, the suspending ability of the
polymers was found to be in the order: Entandophragma angolense > Acacia gum > Gelatin. The gum, thus, has
potential as a suspending agent that can be exploited for use in the pharmaceutical industry.
Keywords: Entandophragma angolense, Natural polymers, suspending agents, sulphamethoxazole, particle size.
_____________________________________________________________________________________________
INTRODUCTION
Many people have difficulty in swallowing solid dosage forms and therefore require the drug to be dispersed in a
liquid. If the drug is insoluble or poorly soluble in a suitable solvent, then formulation as a suspension is usually
required [1]. A pharmaceutical suspension is a coarse dispersion system in which insoluble solid particles (dispersed
phase) are dispersed in a liquid phase (dispersion medium), and like other disperse systems, it is thermodynamically
unstable, thus, making it necessary to include in the dosage form, a stabilizer or suspending agent which reduces the
rate of settling and permits easy redispersion of any settled particulate matter both by protective colloidal action and
by increasing the consistency of the suspending medium [2].
Suspending agents are (i) inorganic materials, (ii) synthetic compounds, or (iii) polysaccharides. Natural gums like
Acacia, Tragacanth, Khaya, Karaya and Entandrophragma angolense belong to the latter group.
Gum is a widely available, naturally occurring substance obtained from the trunk or branches of specific plants,
which has been in use since ancient times. Gums have been described as materials that can be dissolved or
dispersed in water to form more or less viscous colloidal dispersion [3]. Plant gums have evoked tremendous interest
due to their diverse pharmaceutical applications such as diluents, binders, disintegrants in tablets, thickeners in oral
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Oladapo A. Adetunji and Mosunmola O. Odole
J. Chem. Pharm. Res., 2015, 7(1):870-878
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liquids and as protective colloids in suspensions [4, 5]. The use of natural gums has increased in recent times; also
due to the advocacy of “green” materials from “green” chemistry and technologies. Natural polymers are biogenic
and their biological properties such as cell recognition and interactions, enzymatic degradability, semblance to the
extracellular matrix and their chemical flexibility make them materials of choice for drug delivery [6].
Gums are biocompatible, cheap and easily available, thus, making them attractive substitutes for costly semisynthetic and synthetic excipients [7]. The use of natural gums as adjuvants has been reported by several workers
[4,8-10]. The increase in demand for natural gums has made it necessary to explore the newer sources of gums to
meet the industrial demands [11]. Moreover, most of the natural gums or polymers are generally regarded as safe for
oral consumption, thus emphasizing their current use in pharmaceutical dosage forms designed for oral
administration [12]. The present work characterized Entandrophragma angolense (Welw) gum (Meliacea) and
investigated the suitability of the gum as a suspending agent in oral sulphamethoxazole suspensions.
Entandrophragma angolense gum was compared with similar formulations containing gelatin BP and Acacia gum.
The suspending potentials of Entandrophragma angolense gum were evaluated using different assessment criteria
such as rheological properties, particle size and size distribution, suspending properties at varying pH values and
stability at different temperatures. Sulphamethoxasole (BCS Class II drug) was chosen as the model drug because of
its low solubility and thus, requires a good suspending agent for easy redispersion when formulated as an oral
suspension.
EXPERIMENTAL SECTION
Materials
The materials used include Gelatin powder BP, Acacia gum powder, and Sulphamethoxazole powder BP (BH
Chemicals Ltd., Poole ,UK) supplied by Bond Pharmaceuticals Ltd., Aawe, Nigeria. Analytical grade chloroform
water (D/S), acetone and ultra-pure deionized water were obtained from the Research Laboratories of the
department of Pharmaceutics and Industrial Pharmacy, University of Ibadan, Ibadan, Nigeria. Entandrophragma
angolense gum (family: Meliaceae) was obtained from the early morning exudates of the trunk of the tree crop
available in the Botanical Gardens of the University of Ibadan, Ibadan, Nigeria, and authenticated at the Forest
Herbarium, Ibadan (FHI No: 108883) located in the Forestry Research Institute of Nigeria, Jericho, Ibadan, Nigeria.
Collection and purification of gum
Entandrophragma angolense gum was collected from the early morning exudates of the excised bark of the gum
that was previously sprayed with ethilon (a pentacyclic triterpene) to prevent microbial growth [13]. It was washed
with chloroform water double strength and dried in an oven at 50°C for two days. The dry gum was then weighed,
blended and soaked with chloroform water double strength for 2 days. A calico cloth was used to squeeze out the
mucilage gel. The mucilage was then washed with ethanol followed by diethyl ether, dried in the oven at 50ºC for 2
days, blended into powder and sieved with 120-µm mesh sieve to obtain the powdered gum [14]. The percentage
yield of the gum was then calculated.
Phytochemical Examination
Preliminary tests were performed to confirm the nature of mucilage obtained. The chemical tests that were
conducted were to determine the presence of carbohydrates (Molisch’s test), reducing sugars (Fehling’s test),
ketones (Selivanoff’s test), alkaloids (Wagner’s and Dragendorff’s tests), saponins, anthraquinones, tannins,
glycosides, mucilages and flavonoids.
Evaluation of Toxicity
Toxicity studies were carried out according to the method reported by Kumar and other workers [14]. Male albino
rats weighing 180-200g were divided into two groups comprising of six animals each. The control group received a
daily dose of 250mg/kg of Entandrophragma angolense gum suspension in normal saline orally. The other group
received a daily dose of 4000mg/kg of Entandrophragma angolense gum suspension in normal saline orally. The
animals were observed continuously for behavioral changes for the first 4 hours and then observed for mortality if
any for 48 hours. The body weights were recorded for both groups at an interval of 10 days for 30 days. At the end
of the 30-day period, hematological parameters were studied in both groups.
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Oladapo A. Adetunji and Mosunmola O. Odole
J. Chem. Pharm. Res., 2015, 7(1):870-878
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Preparation of Sulphamethoxazole Suspension
Entandrophragma angolense gum powder (500 mg) was triturated with 5.0 g of sulphamethoxazole with a small
quantity of ultra-pure deionized water to obtain a 1.0 % w/v suspension. The resulting mixture was transferred into a
50 mL cylinder and made up to 50 mL with ultra-pure deionized water. The procedure was repeated using 1000 mg
(2.0 %w/v suspension), 1,500 mg (3.0 %w/v suspension) and 2000 mg (4.0 %w/v suspension) of gum. The
procedure was repeated using Acacia and Gelatin powders [15].
EVALUATION OF SUSPENSION
Sedimentation Volume
Each suspension (50 mL) was stored in a 50 mL measuring cylinder for 7 days at 25 0C. Observations were made
every hour for 5 hours and then every 24 hours for 7 days [2]. The sedimentation volume, F (%) was then calculated
using the following equation.
F = 100Vu/Vo
(1)
Where Vu is the ultimate volume of sediment and Vo is the original volume of the suspension
Flow rate
The time required for each suspension sample to flow through a 10 mL pipette was determined and the apparent
viscosity, in mL was calculated using the equation
Flow rate = Volume of pipette mL
Flow time (in secs)
(2)
Ultra-pure deionized water was used as the control.
Viscosity
The viscosity (in poise) of the samples was determined at 25 0C, 37 0C, 45 0C, 55 0C and 70 0C using the Brookfield
Viscometer, Dr-2x-Pro [14].
Particle Size Analysis
After shaking, 5 mL of each sample (which was stored for 24 hours at 25 ºC) was separately transferred into 100 mL
cylinder. Ultra-pure deionized water (75 mL) was then added and mixed, 5 mL aliquot was removed at a distance of
5cm below the surface of the mixture at 1, 5, 10, 20 and 30 minutes. This was transferred into an evaporating dish
and evaporated to dryness in an oven at 105 ºC and the residue weighed. The particle diameter, d, was then
calculated using the Stokes’ equation [14].
d = 18ηh
(PS-P0) g t
(3)
where, h is the distance of fall of the particle in the particle (cm), t is the time (s),η is the viscosity of the dispersion
medium (poise), (PS-P0) is the density gradient between the dispersed particles and the liquid, g is the gravitational
constant in cm-2.
Redispersion
Fixed volume of each suspension (25 mL) was kept in similar calibrated tubes, stoppered and stored at 25Cº. At 5day intervals, one tube of each sample was removed and shaken vigorously to redistribute the sediment and the
presence of deposits if any was recorded.
Determinations of pH
At the end of each 5-day interval, the pH of the sample was measured after the observation of the ease of dispersion
has been made.
872
Oladapo A. Adetunji and Mosunmola O. Odole
J. Chem. Pharm. Res., 2015, 7(1):870-878
______________________________________________________________________________
RESULTS AND DISCUSSION
The percentage yield of the dry gum obtained from exudates of the excised trunks of Entandrophragma angolense
tree was 41.46 %w/w. The high value of the Entandrophragma angolense gum obtained after the purification
exercise is as a result of the time of collection of the gummy exudates, which was done early in the morning. This
was also observed by Bellal and other workers when they documented the difference in yield of Acacia gum due to
the time of collection [16] This high yield further establishes the potential for the use of the gum in the
pharmaceutical industry. One of the properties of a good suspending agent is to ensure homogeneity, for at least the
period between shaking the container and removing the required dose [1, 14]; this property was observed in the
results of the physicochemical (Table 1) and phytochemical (Table 2) analysis of the gum. The results presented
revealed Entandrophragma angolense gum to be slightly acidic with a bulk density of 0.61g/mL. Enhanced
solubility and swelling capacity in water were obtained at elevated temperatures while the gum is devoid of reducing
sugars, ketones, alkaloids, starch, saponins, tannins and flavonoids A violet ring was formed at the junction of the
liquids on reaction with Molisch’s reagent as a confirmation of the presence of carbohydrates. Treatment of the
solution of the gum with ruthenium red showed a red colour confirming the gum has mucilaginous properties.
Entandrophragma angolense gum also has the ability to swell in water. The safety profile of the gum was confirmed
from results of the toxicity tests as there were no behavioural changes observed for the first 4 hours and no mortality
was observed after 48 hours of feeding the rats with the mucilage. There were no significant differences in the
weights of the rats in both groups after observation for a 30-day period. The effect of the mucilage on
haematological parameters (Table 3) also indicated that were no significant differences in the parameters between
the rats treated with the mucilage and the control. In traditional medicine, Entandrophragma angolense gum has
been used by pulping with the seeds of melegueta pepper (Aframomum melegueta) as a febrifuge drink, thus further
emphasizing the safety of the gum when consumed orally [17]. The sedimentation volume gives a qualitative
account of flocculation (F). As the value of F, which ranges from nearly zero to greater than one, increases, the
volume of suspension that appears to be occupied by sediment increases. Thus, if F can be made to approach unity,
the product becomes more acceptable since the volume of supernatant in the suspension is being progressively
reduced, and consequently, caking is eradicated [18] The values of the sedimentation volume of sulphamethoxazole
suspensions containing different concentrations of the gums as suspending agents for day 1 and the subsequent days
(days 2 to 7) for suspensions containing 1% w/v gum are presented in Table 4 and Figure 1 respectively. The profile
shows an increase in concentration of the suspending agents led to an increase in sedimentation volume with
Entandrophragma angolense gum manifesting superiority over gelatin and Acacia. At a concentration of 2%w/v,
Entandrophragma angolense gum was able to achieve similar sedimentation profile when compared with official
Gelatin at a concentration of 4%w/v. Only Entandrophragma angolense gum behaved as an ideal suspension on day
1 where F approached unity after 3 hours at 2.0% w/v concentration, after 5 hours at 3.0%w/v concentration and
after 7 hours at 4.0%w/v concentration. Generally, sedimentation volume reduced with time of storage over a period
of 7 days for all suspending agents used. It was observed that Entandrophragma angolense had the least
sedimentation rate while Acacia gum had the highest sedimentation rate and settled most rapidly, hence the
suspendability of the agents as evaluated by the sedimentation volume were in the order of Entandrophragma
angolense > Gelatin > Acacia. The velocity of a medium decreases as the medium comes closer to the boundary
wall of the vessel through which it is flowing. However, as a result of the “cohesive force between the wall and the
flowing layers” and inter-molecular cohesive forces (viscosity), there is a stationary layer attached to the wall, hence
viscosity is the opposing force to flow. Ideally, increase in viscosity of the medium should lead to a decrease in flow
rate [2]. An increase in the concentration of the experimental formulations resulted in a decrease in flow rate and a
subsequent increase in viscosity. The results revealed that suspensions containing acacia gum flowed fastest and
were the least viscous, while Entandrophragma angolense -containing suspensions exhibited the highest viscosity
profile. At a concentration greater than 2.0%w/v, the flow rate and viscosity for the formulations containing
Entandrophragma angolense were experimentally indeterminable because of the thickness of the formulations. It
was also observed that there was an inverse relationship between temperature and viscosity of the polymers (4%w/v)
as shown in Figure 2, with Entandrophragma angolense gum showing the highest viscosity at all the temperatures at
which observations were made. Particle size analysis was observed to be in the rank order of Acacia >
Entandrophragma angolense > Gelatin.
873
Oladapo A. Adetunji and Mosunmola O. Odole
J. Chem. Pharm. Res., 2015, 7(1):870-878
______________________________________________________________________________
Table 1: Physicochemical Composition of Entandrophragma angolense Gum (Mean± SD, n = 4)
Parameters
Swelling capacity in water at 270C (%)
Swelling capacity in water at 800C (%)
Solubility in water at 270C (%)
Solubility in water at 800C (%)
Loss on drying (%w/w)
Total ash (%w/w)
Acid insoluble ash (%w/w)
pH of 1% solution at 320C
Bulk density (gm/mL)
Tapped density (gm/mL)
Hausner’s quotient
Compressibility index (%)
Angle of repose
Moisture content
Entandrophragma angolense
51.3±0.01
82.7±0.02
38±0.01
53±0.01
0.1±0.01
1.26±0.04
0.1±0.02
4.27±1.08
0.61±0.03
0.72±0.04
1.18±0.03
15.28±0.04
12.53±2.02
9.2±0.01
Table 2: Phytochemical screening of Entandrophragma angolense gum (+: Present, -: Absent)
Tests
Test for Carbohydrates (Molisch’s test)
Test for reducing sugars (Fehling’s test)
Test for ketones (Selivanoff’s test)
Test for alkaloids (Wagner’s and Dragendorff’s
tests)
Test for saponins
Test for anthraquinones
Test for Tannins (Ferric Chloride test)
Test for glycosides (Keller-Killiani test)
Test for mucilage (Ruthenium red test)
Test for flavonoids (Shinoda test)
Mounting in 95% alcohol
Mounting in iodine
Observation
+
+
Transparent angular masses seen under the microscope
Colour of iodine retained (no blue coloured particles seen (starch is
absent)
Table 3: Hematological values of male albino rats receiving the mucilage of Entandrophragma angolense for 30 days (Mean± SD, n = 4)
Parameters
RBC (x 106 cells/mm3)
WBC (x 106 cells/mm3)
Hemoglobin (g/dl)
Platelet ( x 103 cells/mm3)
Neutrophil (%)
Eosinophil (%)
Lymphocytes (%)
Monocyte (%)
Basophil (%)
Hematocrit (%)
MCV (µm3/red cell)
MCH (pg/red cell )
MCHC (g/dl/red cell)
Mucilage treated
9.67±0.18
5.03±0.39
17.11±0.04
961±0.16
19.04±0.11
1.77±0.25
62.31±0.41
11.68±0.05
4.59±0.32
51.22±0.41
59.64±0.27
21.05±0.04
31.86±0.08
874
Control
9.61±0.06
5.01±0.16
17.05±0.22
957±0.03
18.96±0.45
1.73±0.15
62.27±0.22
11.36±0.04
4.47±0.14
51.19±0.07
58.96±0.05
21.03±0.06
31.77±0.11
Oladapo A. Adetunji and Mosunmola O. Odole
J. Chem. Pharm. Res., 2015, 7(1):870-878
______________________________________________________________________________
Figure 1: Sedimentation profile of suspensions containing 1%w/v gum
ENTA: Entandrophragma angolense gum
Table 4: Values of sedimentation volume (%) of sulphamethoxazole suspensions containing different concentrations of suspending
agents obtained on day 1
Suspending
Agents
ENTA
Acacia
Gelatin
Sedimentation volume (%)
Time (hours)
0
1
2
3
4
5
0
100
36
34
33
33
33
1.0
100 100 100
98
98
98
2.0
100 100 100 100
98
98
3.0
100 100 100 100 100 100
4.0
100 100 100 100 100 100
1.0
100
51
50
40
40
36
2.0
100
61
51
46
44
38
3.0
100
66
62
50
48
46
4.0
100
68
64
58
52
46
1.0
100
81
56
49
38
30
2.0
100
91
81
70
61
51
3.0
100
95
81
70
61
58
4.0
100
99
82
71
66
64
ENTA: Entandrophragma angolense gum
Concentration
(%w/v)
875
6
33
96
98
98
100
31
32
40
44
28
47
49
62
7
33
96
96
98
100
31
32
37
44
28
46
48
58
Oladapo A. Adetunji and Mosunmola O. Odole
J. Chem. Pharm. Res., 2015, 7(1):870-878
______________________________________________________________________________
Table 5: Effects of the type and concentration of suspending agents on the flow rate and viscosity of sulphamethoxazole suspensions
determined at 250C
Suspending agent
ENTA
Acacia
Gelatin
Concentration (%w/v)
-1
Flow rate (mLs )
1.0
1.68
2.0
1.57
3.0
Too viscous
4.0
Too viscous
1.0
1.76
2.0
1.71
3.0
1.52
4.0
0.92
1.0
1.97
2.0
1.82
3.0
1.58
4.0
1.43
ENTA: Entandrophragma angolense gum
Viscosity (Poise)
1.94
2.36
Indeterminable
Indeterminable
0.15
0.17
0.18
0.25
0.11
0.14
0.15
0.19
Figure 2: Plot of Viscosity (Poise) against temperature for suspensions containing 4%w/v gums
876
Oladapo A. Adetunji and Mosunmola O. Odole
J. Chem. Pharm. Res., 2015, 7(1):870-878
______________________________________________________________________________
Values of the particle diameter obtained from the particle size measurements using are presented in Table 6.
According to Stoke’s equation, sedimentation velocity (v) is directly proportional to the square of the diameter of
the particle [2]. The rate of fall of suspending particles in a vehicle of given density is greater for larger particles
than it is for smaller particles [18, 19]. Particle size diameter (d) is inversely proportional to the time of storage;
hence, increase in storage time of the suspensions should result in a decrease in particle size/diameter. From the
results obtained, there was a direct relationship between increasing the concentration of the gums and particle size
[20]. On the other hand, increase in storage time resulted in a gradual decrease in particle diameter as determined
from the particle size analysis carried out. Hence, particle diameter of a suspension is directly proportional to the
concentration of the suspension while time of storage of suspension is inversely proportional to particle diameter. It
was observed that suspensions containing gelatin gum had the largest particle diameter while Acacia-containing
formulations had the least particle diameter.
Table 6: Values of Particle diameter obtained from the particle size measurements
Suspending agent
ENTA
Acacia
Gelatin
Concentration (%w/v)
Particle diameter (µm)
Time (minutes)
1
5
10
20
1.0
1.171 1.144 1.211 0.725
2.0
1.226 1.182 0.902 0.553
3.0
1.234 1.224 1.142 0.724
4.0
1.251 1.234 1.211 1.161
1.0
1.179 1.178 1.178 1.175
2.0
1.211 1.211 1.208 1.201
3.0
1.322 1.312 1.296 1.291
4.0
1.393 1.391 1.390 1.383
1.0
1.602 1.588 1.455 1.301
2.0
1.808 1.762 1.618 1.711
3.0
1.986 1.901 1.798 1.661
4.0
1.991 1.888 1.801 1.601
ENTA: Entandrophragma angolense gum
30
0.525
0.321
0.420
1.122
1.175
1.198
1.282
1.381
0.889
0.911
1.433
1.223
CONCLUSION
Entandrophragma angolense gum is devoid of toxic properties and has the potential of a suspending agent at low
concentrations and can thus be used as a pharmaceutical adjuvant. The gum obtained from Entandrophragma
angolense can be a cheaper alternative to Acacia and gelatin gum because of its availability all year round, and
because of the economic advantage of affordability in terms of cost.
Acknowledgements
This research received support from the Academic Staff Union of Nigeria Universities (ASUU) Research grant. The
authors are grateful to Bond Chemicals Limited, Aawe, Nigeria for providing Gelatin BP, Acacia gum and
Sulphamethoxazole powders, and to Dr Oluwasanmi O. Aina of the Faculty of Veterinary Medicine, University of
Ibadan, Nigeria for technical support.
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