Formulation of a Diuretic Oral Suspension of Basella alba

L. (Basellaceae)
Noreen Mae E. Chavez, Bianca Mae G. Limjoco, Melchi Esrom L. Opelario, Edmin Christian R. Tunod, Ma. Beatrice M.
Vega

Faculty of Pharmacy, University of Santo Tomas, Metro Manila, Philippines

ABSTRACT
The ethanolic leaf extract of Basella alba L. has been shown to possess diuretic activity owing to the presence of
saponins. The study focused on the formulation of a diuretic oral suspension, as potential alternative to oral solid diuretic
medications, using the ethanolic leaf extract of Basella alba L. as active ingredient. The ethanolic leaf extract of Basella
alba L. is an aromatic, non-greasy, dark green, viscous mass. The test for saponins produced a positive result in the froth
test, but a negative result in the hemolysis test. Differential scanning calorimetry (DSC) analysis showed potential inter-
actions with sodium chloride, carboxymethylcellulose, sodium benzoate, citric acid, and saccharin. An oral suspension
containing 50 mg/mL of Basella alba leaf extract was formulated, and viscosity, pH, and redispersibility were assessed.
Using the Lipschitz method, diuretic activity was evaluated using four groups of Sprague Dawley rats: Group 1 was
treated with base suspension, Group 2 received 10 mg/kg Furosemide, and Groups 3 and 4 were treated with 250 mg/kg
and 500 mg/kg of the oral suspension formulation, respectively. The urine volume and electrolyte content of the urine
were used as indicators of diuretic activity. The formulated oral suspension with the dose of 500 mg/kg showed a compa-
rable diuretic effect to furosemide having a diuretic index of 1, while the formulated oral suspension with a dose of 250
mg/kg produced a mild diuretic effect. There was a significant increase in urine Na+ and K+ output after administration of
both test concentrations of Basella alba oral suspension (p<0.0001). The study findings suggest that the formulated oral
suspension containing Basella alba leaf extract exhibits dose-dependent diuretic activity and maybe a suitable alternative
to oral solid diuretic medications.

Keywords: Basella alba Linn., diuretic, furosemide, oral suspension, saponins

1. Introduction sitivity, ototoxicity and electrolyte imbalance due to di-

uresis (Se & Sang, 2015).
Diuretics refer to drugs which enhances the rate Basella alba L., from the Basellaceae family, is
of urine flow. Water and electrolytes such as sodium, locally known in the Philippines as “Alugbati”. It is a fast
potassium, chloride and bicarbonate are also regulated in growing perennial climbers that is commonly known as
the presence of diuretics. These are the body's response to Ceylon spinach or Malabar Spinach (Kumar, et al., 2013).
maintain a state of homeostasis. There are different types Phytochemical Studies shows that the leaves of
of diuretics according to their mechanism of action. Basella alba L. have been found to contain vitamin K
Furosemide, the prototype drug from a type of diuretic (Adegoke and Ojo, 2017). It also contains basella
called Loop diuretics inhibits Sodium Potassium Chloride saponins A, B, C, and D, which are responsible for the
transporter in the thick ascending loop of Henle. It is also diuretic activity. An alternative theory is that the potassi-
known for increasing renal blood flow and reducing the um content of the plant also contribute to the diuretic ac-
pulmonary congestion and left ventricular filling pres- tivity (Hostettmann and Marston, 2015). Sridevi, K.. ,
sures in the heart (Katzung & Trevor, 2017), thus making Ravishankar, K. & Kiranmayi, G.V.N. (2014) stated that
it the drug of choice for heart failure. Adverse effects as- the ethanolic leaf extract of Basella alba L. can increase
sociated with taking this drug are hypovolemia, hypersen- urine output as well as in the sodium, potassium, chloride
and bicarbonate excretion.
 Suspensions are a class of dispersed system in Sprague-Dawley rats weighing approximately
which a finely divided solid is dispersed uniformly in a 100-200g were utilized as test animals and were obtained
liquid dispersion medium (Nutan, M. & Reddy, I., 2010). from Joselito Plegaria Biological and Zoological Supply.
In formulating a suspension, excipients are employed to a The rats were acclimatized in metal cages in a room tem-
certain extent to allow the production of a stable and un- perature of 25 ± 2°C in the animal housing facility of Re-
compromised preparation. The essential components of a search Center for the Natural Sciences and Applied Sci-
pharmaceutical suspension that are commonly used are ences (RCNAS), University of Santo Tomas for 7 days.
the active ingredient, wetting agent/surfactant, flocculat- The animals were kept at constant 12 hours light/dark
ing agent, suspending agent, buffers, preservatives, sol- cycle and were given free access to food and water for the
vent, and flavorant. entire duration except 17-24 hours prior to the experi-
Physicochemical tests according to the ASEAN ment.. This study protocol was approved by the Institu-
Guideline on Stability of Drug Products Oral Solutions tional Animal Care & Use Committee of University of
and Suspensions (2013) should be evaluated for appear- Santo Tomas (UST-IACUC).
ance (including formation of precipitate, clarity for solu-
tions), color, odour, assay, degradation products, pH, vis- 2.1.1 Extraction Procedure
cosity, preservative content and microbial limits. Addi-
tionally for suspensions, redispersibility, rheological Fifty grams of leaves were cleared from dirt by
properties, mean size and distribution of particles should washing the leaves with water. After washing, the leaves
be considered. After storage, sample of suspensions were left to dry in a shade inside a room for a span of two
should be prepared for assay according to the recom- weeks. After thorough drying, the dried leaves were
mended labeling (e.g. shake well before using). coarsely powdered using a mechanical grinder. The pul-
Lipschitz Method is a standard method and a very verized leaves were subjected to Soxhlet method of ex-
useful tool for screening of potential diuretics. The traction. 200 mL of ethanol was added to the round bot-
method is based on water and sodium excretion in test tom flask, attached to the Soxhlet extractor and condenser
animals (Nayak, et al.,2012). Analysis of the urine in- on an isomantle. The ground leaves were loaded into the
cluded estimation of sodium and potassium ions (Sridevi, thimble. Using the isomantle, the solvent was heated and
K., et al. 2014) and Ion selective electrodes or ISEs is was made to evaporate, moving through the apparatus to
used due to its ability to provide a wide range of ap- the condenser. The process ran for a total of 16 hours
plications in determining ions in water and other medi- (Redfern, et al., 2014). Once the process has finished, a
ums where such determinations are not subject to inter- rotary evaporator at 50°C under reduced pressure was
ferences (Department of Chemistry and Biochemistry, used to evaporate the ethanol solvent, leaving a small
New Mexico State University, n.d.). yield of extracted plant material (about 2 to 3 mL) in the
glass bottom flask. The extract was stored until the time
This study centers on the development of an oral of the formulation.
suspension to aid in the administration of the diuretic
formulation to geriatrics. Since there are no commercially 2.1.2 Test for Saponins
available oral diuretic suspensions, the study aimed to
determine if the ethanolic leaf extract which possesses 2.1.2.1 Froth Test
diuretic activity will elicit the same activity if it is formu-
lated into an oral suspension. One gram of the extract was dissolved in 10 mL
of distilled water in a test tube and vigorously shaken for
2. Materials and Methods 1-2 minutes.
2.1 Study Design
Formation of honeycomb froth of 1 cm in height
The leaves of Basella alba Linn. plant were pur- that lasted for a minimum of 30 minutes indicated the
chased from Dizon Farms. Analytical grade ethanol used presence of saponins (Gunjal, et al., 2015).
for Soxhlet extraction was purchased from Belman Labo-
2.1.2.2 Hemolysis Test
ratories. All excipients used for the formulation of the
suspension such as carboxymethylcellulose, citric acid,
A blood agar plate was used with four equidistant
sodium benzoate, saccharin, and sodium chloride were
wells with depths of 4 mm cut using a sterile well cutter.
purchased from Alysons' Chemical Enterprises. The ref-
120 µl each of the ethanolic extract, ethanol, aqueous
erence drug whis is Furosemide (Lasix) was purchased
Gugo solution (positive control) , and sterile water (nega-
from Mercury Drugstore.
tive control) were dispensed into the wells and kept cov-
ered for 24 hours at room temperature (Almutairi & Ali,
2015).
 A presence of a zone of clearance or “hemolytic 

halo” is an indication of the presence of saponins. 2.1.5 Physicochemical Tests for Oral Suspensions

2.1.3 Differential Scanning Calorimetry 2.1.5.1 Organoleptic Evaluation

The materials tested for compatibility with the Organoleptic properties of formulated suspension
active ingredient are found in Table 1. Binary mixtures were described based on color, odor, and consistency.
were obtained by manual mixing of 1:1 ratios of the Samples were placed at 300 C and 500 C temperature con-
ethanolic leaf extract and excipients using a mortar and ditions for one week and reassessed using the same para-
pestle. Thermal behavior was assessed by weighing 4–6 meters.
mg of the sample, enough to uniformly cover the alu-
minum crucible. A heating rate of 20°C min−1 was used 2.1.5.2 pH Determination
during this study. The thermograms for each excipient
and ethanolic extract was compared to the thermogram of The pH of the formulated suspension was mea-
the binary mixture. If there are no insignificant changes in sured using a calibrated pH meter. Three independent
the peaks of the thermogram of the extract and the binary measurements were made. The pH of samples stored at
mixture, the excipient and the ethanolic leaf extract are room temperature and oven conditions for one week were
considered compatible. Glycerin was also tested for ther- compared to the pH of the suspension at time 0.
mal stability using the oven by subjecting it to 50 ℃ for a
duration of 4 weeks. 2.1.5.3 Viscosity Determination

Table 1. Excipients used for compatibility test Viscosity of the different samples were measured
using a Brookefield viscometer, spindle 4 at 250 rpm.
Components of an Oral
Excipients 2.1.5.4 Redispersibility Evaluation
Suspension

Flocculating agent Sodium chloride Cylinder was used to determine the redispersibili-
ty of the suspension based on the method reported by
Viscosity/ thickening Mittal and co-workers (2014). The suspension was al-
Carboxymethycellulose lowed to settle in a measuring cylinder. The mouth of the
agent/ suspending agent
cylinder was closed and inverted to 180º and the number
Buffer Citric acid of inversions necessary to restore a homogeneous suspen-
sion was determined. If the homogeneity of the suspen-
Preservative Sodium benzoate sion was attained in one inversion, then the suspension is
Sweetener Saccharin considered 100% easily redispersible. Every additional
inversion decreases the percentage of ease of redis-
persibility by 5%.

2.1.6 Test for Diuretic Activity

2.1.4 Formulation of the Oral Suspension
2.1.6.1 Lipschitz Method
To prepare a 50 mL oral suspension, 1.4 g of car-
boxymethylcellulose was first triturated in a mortar and The test was performed following the Lipschitz
pestle. Followed by the addition of the ethanolic leaf ex- method with minor modifications. 12 male Sprague Daw-
tract of Basella alba L. mixed with 10 mL glycerin. 10 ley rats each housed individually in a metabolic cage.
mL volume of water was added prior to the addition of Food was withdrawn 17 hours before the experiment but
the following: 0.01 g of citric acid, 0.5 g of saccharin, 0.1 water was given ad libitum. On the day of the experiment,
g of sodium chloride and 0.1g of sodium benzoate. Final- gentle pressure was applied to the urinary bladders of the
ly, water was added in portions to bring up the final vol- rats to have a uniform content of urine. Followed by that
ume of 50 mL with continuous trituration to ensure a uni- was the administration of 15 mL saline solution as a
form product. priming dose to all of the test animals regardless of body
weight (Ganegamage, Abeytunga, and Ratnasooriya,
2014). An oral gavage was used to introduce the base
suspension, furosemide (Lasix) dispersed in the base sus-
pension, and the formulated suspension with the dosage
strength of 50 mg/mL. The first group which is the nega-
tive control group was treated with the base suspension checked using Anderson-Darling test. Duncan Multiple
and the second group which will serve as the positive Range test was used to check what levels of the treatment
control group, was treated with 10 mg/kg furosemide are significantly different from each other with respect to
(Lasix) dispersed in the base suspension. After the admin- the dependent variable. All tests were performed at 0.05
istration of the two suspensions a washout period was level of significance.
done for 1 week. After the washout period, the first and
second group acted as the third and fourth groups, respec- 3. Results
tively. The third group and fourth group received varying 3.1 Ethanolic Leaf Extract of Basella alba L.
250 mg/kg and 500 mg/kg doses of the formulated oral
suspension respectively. The ethanolic leaf extract of Basella alba L. is a
non-greasy, dark green mass, with a viscous consistency
For the collection of the urine sample, the tip of the tails and an aromatic odour.
of the rats were tugged and the urine volume for analysis
of Na+ and K+ was recorded 5 hours after drug adminis- 3.2 Test for Saponins
tration (Sridevi, K., et al. 2014). From the performed pro-
cedure, the diuretic index of the urine output was calcu- The ethanolic leaf extract produced a honeycomb
lated. Diuretic index is the ratio between the volume of froth with the height of 1 cm which lasted for a few min-
excretion of the test groups and the standard group, utes. However, it did not produce a zone of hemolysis or
(Asif, Jabeen, Atif, Majid, & Qamar-Uz-Zaman, 2015) “hemolytic halo”.

Diuretic Index (I.D.) = VT / VS 3.3 Differential Scanning Calorimetry Analysis

Where The thermogram of the ethanolic extract of Basel-

la alba L. showed that a thermal process occurred be-
VT - Total volume of urine excreted by the test groups tween 106.81°C to 135.80 °C, producing and endothermic
peak at 116.50 °C, and an enthalpy of 243.9739 J/g. This
VS- Total volume of urine excreted by the standard is probably related to the loss of volatile constituents of
the sample, such as ethanol. Moreover, thermographs
2.1.6.2. Urinary Electrolyte Excretion presenting the various excipients utilized and its binary
mixtures caused the disappearance of the peak of the ex-
Urine samples were submitted to Majime Animal
tract but retained the peaks of the excipient.
Diagnostics Laboratory in Antipolo for analysis of sodi-
Aside from the aforementioned solid excipients,
um and potassium ions. The diagnostic laboratory utilized
glycerin and the binary mixture of the extract with glyc-
Hitachi Ion Selective Electrodes for both sodium and
erin stored at 50℃ for one week were found to be physi-
potassium content determination. Urine samples were
cally stable.
incubated for 24 hours. The urine was allowed to equili-
brate at room temperature, then mixed properly to ensure
3.4 Formulation of the Oral Suspension
homogeneity. A volume of 250 µl from a urine sample
was aspirated and added to 250 µl of the urine diluent
Three prototypes were made until the final com-
which is ISE diluent Gen 2. Inversion of the mixed urine
position of the formulation was achieved. The final com-
sample and diluent was repeated for 5-10 times. The urine
position is stated in table 2. The first and second proto-
samples were then loaded into the instrument. All calcula-
type produced a gummy consistency so the suspending
tions were performed by the system using a machine-
agent was reduced. In addition to that, the first two proto-
stored calibration curve.
types exceed the suggested amount of saccharin in an oral
suspension and have pH values which were below the
2.2 Statistical Analysis
specified range of 4-6, therefore saccharin and citric acid
Two separate Analysis of Covariance (ANACO- were reduced to render a more appropriate formulation.
VA) tests were performed. One using sodium as the de-
pendent variable and one using potassium as the depen-
dent variable. The treatments were the categorical vari-
able and urine amount was the continuous variable that
may affect sodium or potassium. The assumptions of
ANACOVA are normality of error terms and constancy of
variance. Since there is a duality nature between the two
assumptions, only the normality of error terms was
 Table 2. Quantitative Compositions of the Prototype stand for 1 week at a 40º C oven has the largest differ-
Formulations ence, which may be caused by heat which can influence
the viscosity of a suspension. The result is that liquids
Prototype
Prototype show a reduction in viscosity with increasing tempera-
Excipient Prototype1 3
2 tures.
(FINAL)
CMC 5.0 g 3.5 g 2.5 g 3.5.4 Redispersibility

Glycerin Upon standing of the suspension at baseline, sus-

10 mL 5.0 mL 10 mL
pension allowed to stand for 1 week at room temperature,
Extract and suspension allowed to stand for 1 week at 40ºC oven
2.5 g 2.5 g 2.5 g
in a measuring cylinder, the suspensions all required one
Citric acid inversion to render them homogenous. The results indi-
0.1 g 0.05 g 0.01 g cate that the three formulated suspensions are all 100%
Saccharin easily redispersible regardless of the environment and
5.0 g 2.0 g 0.5 g temperature condition.
NaCl 1.0 g 0.5 g 0.1 g 3.6 Test for Diuretic Activity
Sodium
3.6.1 Lipschitz Method
0.1 g 0.1 g 0.1 g
benzoate
The diuretic activity of the sample formulations
Water 50 mL qs 50 mL qs were determined as the diuretic index of the different
50 mL qs ad formulations. The result for the base suspension showed a
ad ad
diuretic index of .60 which is not included in any range
specified thus attributed to no diuretic activity. The 250
mg/kg suspension resulted to a diuretic index of 0.82
which falls in the range for mild diuretic effect, lastly the
3.5 Physicochemical Tests for Oral Suspensions
500 mg/kg suspension produced the greatest diuretic in-
3.5.1 Organoleptic Evaluation
dex among the three, the diuretic index is 1.00 which is in
the range of moderate diuretic effect.
The suspension at baseline, the suspension al-
lowed to stand for 1 week at room temperature, and the
Table 3. Diuretic Indices of the Formulated Suspen-
suspension allowed to stand for 1 week at 40º C oven re-
sions
tained the same color, odour, and taste. However, the sus-
pension allowed to stand for 1 week at 40º C oven dif- Treatment Diuretic Index
fered in consistency because it produced a less viscous
property when compared to the first two suspensions. Base suspension 0.60

Formulated oral
3.5.2 pH 0.82
suspension (250 mg/kg)
The pH of the suspension at baseline, suspension Formulated oral
allowed to stand for 1 week at room temperature and the 1.00
suspension (500 mg/kg)
suspension allowed to stand for 1 week at 40ºC oven were
4.102, 4.586, and 4.574 respectively. Therefore, the pH of
each suspension conforms with the specifications indicat-
ed for suitability for use. 3.6.2. Urinary Electrolyte Excretion

There is enough evidence that the ANACOVA

3.5.3 Viscosity
will give reliable results at 0.05 level of significance.

 However, to confirm if there is an overall statistically sig-
The suspension at baseline and the suspension nificant difference among the treatment levels, a post hoc
allowed to stand for 1 week at room temperature didn’t test was conducted.
largely differ, this may be attributed to the constant tem-
perature environment in which they were formulated and For the Duncan test of Na+ ion, the formulated
located. On the other hand, the suspension allowed to oral suspension has a significantly higher mean than the
base suspension and a significantly lower mean than the the excipients. On the other hand, glycerin is compatible
standard (Furosemide) suspension. with the excipient based from its physical attributes. The
results of the formulations showed that increased amounts
Table 6. Duncan Grouping for Sodium Ion of the suspending agent will render a suspension with a
gummy consistency. Saccharin, in amounts higher than
Duncan
Mean Treatment the specified range, will render a suspension too sweet-
Grouping
tasting and will be harmful to patients. Lastly, citric acid
A 16.500 Furosemide suspension will extremely lower pH if added in excess. The three
formulated oral suspensions showed uniformity on their
Formulated oral suspension organoleptic properties. However, a Slight decrease in
B 12.667
(500 mg/kg) viscosity for sample stored at 400 was observed and may
be considered a natural behavior of systems at elevated
B temperature All pH determinations were within the ac-
ceptable range of 4-6. The use of citric acid as a buffer
Formulated oral suspension
B 11.000 efficiently resisted pH changes and stabilized the suspen-
(250 mg/kg)
sions for a given period and temperature condition.
C 7.833 Base suspension Therefore, the pH of each suspension conforms with the
specifications indicated for suitability for use (Patil, P.D.,
Means with the same letter are not significantly different
Desai, S.R., & Disouza, J., I. 2016). The formulated sus-
pensions subjected in differing environments are all 100%
For the Duncan test of K+ ion, the formulated oral suspen-
sion has a mean not significantly higher standard easily redispersible. The formulated oral suspension with
(Furosemide) suspension and significantly higher mean the dose of 500 mg/kg showed a comparable diuretic ef-
than the base suspension. fect to furosemide. As reported by Sridevi, Ravishankar,
and Kiranmayi (2014), the 500 mg/kg dose of formulated
Table 7. Duncan Grouping for Potassium Ion suspension showed an enhanced diuretic effect compared
to the dose of 250 mg/kg. Both doses of the formulated
Duncan
Mean Treatment oral suspensions gave significantly higher Na+ and K+
Grouping
output than the base suspension (p<0.05). One important
A 21.000 Furosemide suspension factor which determines the variability of the response to
diuretics is the level of endogenous urine electrolyte se-
A cretion, since high levels of these in the urine manifest
diuresis. (Campus, n.d.).

Formulated oral suspension

B A 19.167
(500 mg/kg) 5. Conclusion
B The study shows that the formulated oral suspen-
Formulated oral suspension sion of Basella alba L. is effective in inducing diuresis
B 18.000 and may be used as an alternative medication to solid di-
(250 mg/kg)
uretic dosage forms.
C 11.667 Base Suspension
6. Recommendations
Means with the same letter are not significantly different
For future establishment and investigation, the
4. Discussion researchers recommend the following:
1. Confirm excipient compatibility using an alternative
The ethanolic leaf extract produced a positive method like Fourier-transform infrared spectroscopy
result in the froth test but a negative result in the hemoly- (FTIR).
sis test. This may be due to the low concentration and 2. Conduct stability testing of formulated suspension.
incomplete diffusion of the ethanolic extract on the blood 3. Develop stability indicating assay for formulated sus-
agar plate. Thermograms of the ethanolic extract, in com- pension.
parison with the corresponding excipients and its corre- 4. Investigate mechanism of action of the diuretic activity
sponding binary mixture are found to be incompatible on of Basella alba L. extract.
the basis of disappearance of the characteristic peak
found in the ethanolic extract alone. This may be an indi-
cation of an interaction between the active ingredient and
Conflicts of Interest
Bajaj, S., Singla, D., & Sakhuja, N. (2012). Stability
No conflict of interest to declare. Testing of Pharmaceutical Products. Journal of
Applied Pharmaceutical Science. 02 (03): 129-138.
Acknowledgements
Bamidele O, Akinnuga AM, Olorunfemi JO, Odetola OA,
The authors would like to express their deepest Oparaji CK, Ezeigbo N (2010). Effects of Aqueous Ex-
gratitude to the Research Center of Natural and Applied tract of Basella alba leaves on Haematological and Bio-
Sciences (RCNAS) of the University of Santo Tomas chemical Parameters in Albino rats. African Journal of
(UST), the UST Herbarium and Asst. Prof. Gina C. Cas- Biotechnology. 9(41):6952-6955.
tro from the Faculty of Pharmacy of the University of
Santo Tomas for providing invaluable guidance and Bernal1, N. et al. (2014). Development, Physical-Chemi-
knowledge throughout the research. cal Stability, and Release Studies of Four Alcohol-Free
Spironolactone Suspensions for Use in Pediatrics. De-
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