Original Research Article

Nanobiomedicine
Volume 3: 1–17
ª The Author(s) 2016
Development, in vitro and in vivo Reprints and permissions:
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evaluations of novel lipid drug delivery DOI: 10.1177/1849543516673445
nab.sagepub.com

system of Newbouldia laevis (P. Beauv.)

Chukwuebuka Umeyor1, Emmanuel Anaka1,

Franklin Kenechukwu2, Chinazom Agbo2, and Anthony Attama2

Abstract
Newbouldia laevis (P. Beauv.) is a tropical rainforest plant used in traditional folk medicine for the treatment of malaria,
cough, joint pains, stomach ache, oedema and inflammation. The main thrust of this research work was to study the
analgesic/anti-nociceptive properties of N. laevis-loaded solid lipid microdispersions. N. laevis leaves were extracted using
ethanol, and the extract was formulated into solid lipid microdispersions using lipid matrix comprising a rational blend of
Precirol1 ATO 5 and Softisan1 154. Characterization of the solid lipid microdispersions include determination of
morphology, particle size, pH, thermal property, encapsulation efficiency percentage and analgesic/anti-nociceptive
property. The results obtained showed that the particles were spherical with sizes ranging from 40 mm to 125 mm.
The solid lipid microdispersions maintained a stable pH within the acidic region of 5–6 with insignificant variations (p >
0.05) over a period of 90 days. Thermal analysis showed that N. laevis was entrapped in the lipid matrix used for the
formulations. Solid lipid microdispersions recorded a maximum encapsulation efficiency up to 88.1%. N. laevis-loaded solid
lipid microdispersions also produced good analgesic/anti-nociceptive property comparable with the standard diclofenac
potassium. N. laevis-loaded solid lipid microdispersions showed good analgesic/anti-nociceptive effect and could be used in
the treatment and management of pain.

Keywords
Solid lipid microdispersions, Precirol1 ATO 5, Softisan1 154, analgesic/anti-nociceptive, phytomedicine

Date received: 10 March 2016; accepted: 14 September 2016

Introduction
The use of herbs by man for therapeutic purposes started
right from the development of human culture. Until the 1
Nanomedicines and Drug Delivery Research Group, Department of
discovery and introduction of modern therapeutics, various Pharmaceutics and Pharmaceutical Technology, Faculty of
plant parts have been utilized by man in the form of decoc- Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka, Anambra
tions, macerations, teas, concoctions and so on, for the State, Nigeria
2
Drug Delivery and Nanomedicines Research Group, Department of
treatment, management and cure of various ailments. For Pharmaceutics, Faculty of Pharmaceutical Sciences, University of
many centuries, these crude drugs and their folkloric uses Nigeria, Nsukka, Enugu State, Nigeria
have been handed down from one generation to another,
and in the past millennium, reports abound of the screening Corresponding Author:
of extracts of these medicinal plants for possible use in the Chukwuebuka Umeyor, Nanomedicines and Drug Delivery Research
Group, Department of Pharmaceutics and Pharmaceutical Technology,
alleviation of various health challenges.1 These waves of Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka
research findings have made it imperative for formulation 422001, Anambra State, Nigeria.
scientists to embark on the formulation, characterization Email: ec.umeyor@unizik.edu.ng

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2 Nanobiomedicine

and delivery of these ‘green’ medicines for possible trans- ‘Ikhimi’ and ‘Akoko’ by the Hausa, Igbo, Bini and Yoruba
lation to clinical use, in line with the recent advocacy on communities, respectively. In Africa, N. laevis is widely
green initiatives to save our planet, as well as be at the used in folk medicine for the treatment of malaria, cough,
frontiers of providing novel and standardized medicines for stomach aches and pains, toothache, breast cancer and con-
clinical applications. stipation. In south-east and Midwest Nigeria, the plant is
Lipid-based drug delivery systems (LBDDSs) have been well known for the treatment of inflammation, oedema,
reported to modulate the physico-pharmacological proper- septic wounds, eye problems and sexually transmitted
ties of drugs due to their propensity to alter the pharmaco- infections (STIs).12–15 Phytochemical screening of the
kinetic and biodistribution profiles of drugs. This plant has revealed the abundance of alkaloids, phenylpro-
modulation leads to enhanced bioavailability of drugs panoids, flavonoids, tannins and glycosides in its leaf, root
encapsulated in the lipid matrix due to increased absorption and flowers.16,17 Aqueous and ethanolic leaf extracts of the
of the bioactive components through different mechanisms plant have been reported to increase the frequency of spon-
to modify their release. LBDDSs also affect the intestinal taneously contracting tissues and directly stimulate uterine
environment, stimulate the lymphatic transport of released contractions in albino rats.18 Similarly, ethanolic extracts
drugs and interact with enterocyte-based transport. This has of the leaves and stem of N. laevis have been reported to
made LBDDS an attractive alternative for the delivery of possess antioxidant activity in diabetic rats.19
both hydrophilic and lipophilic drugs.2,3 The use of solid Since the fundamental importance of using plant-based
lipids in LBDDS imparts very good stability profile to the medicines is their relative safety compared with synthetic
formulation and maximizes the rate and extent of drug drugs, and affordability of treatment, the objectives of this
dissolution. Also, it has the ability to control as well as study were to formulate SLMs encapsulating N. laevis
sustain the release properties of an incorporated drug by leaves extract and to study its analgesic/anti-nociceptive
lowering its mobility within the solid lipid core.4 Solid lipid property.
microdispersions (SLMs) are LBDDS prepared using a uni-
form blend of lipid matrices, surfactant and water and
encapsulating a hydrophilic or lipophilic drug for improved Materials and methods
absorption upon administration. SLMs have been shown to
protect encapsulated drug against chemical degradation, Materials
provide sustained release and ensure longer shelf life of the The following materials were obtained directly from their
active drug. Generally, they are biocompatible and biode- manufacturers and used without further purification:
gradable, physicochemically stable and could be produced Softisan1 154, Kolliphor1 188 (BASF SE, Ludwigshafen,
at a relatively cheap cost. They are widely accepted as an Germany), Precirol1 ATO 5 (Gattefossé, Saint-Priest
important and promising alternative drug delivery system Cedex, France), ethanol (Sigma-Aldrich, Germany),
because they are endowed with several advantages of dif- diclofenac potassium (Healthy Life Pharma, Mumbai,
ferent traditional drug carrier systems, while avoiding some Maharashtra, India). Distilled water was collected from
of their disadvantages.5–7 an all-glass still. All other reagents used were analytical
Most phytochemicals from plants are very water solu- grade and used as supplied.
ble, for example, glycosides, flavonoids and so on, and this
hydrophilicity limits their effectiveness because they are
poorly absorbed with low bioavailability when they are Collection and extraction of N. laevis
applied topically or administered orally.8 As a result of this Fresh leaves of N. laevis were harvested from a thick bush
drawback, encapsulating these phytoconstituents using at Agulu, Anaocha Local Government Area, Anambra
lipid-based drug carriers has been found to greatly enhance State, Nigeria, in June 2014, were authenticated by a tax-
their bioavailability with faster and improved systemic onomist in our institution and given the voucher number
absorption.9 In a study, lipid vesicles of boswellic acid N.A.U.H. no. 22, and the specimen was deposited in the
were developed using phosphatidylcholine, and anti- herbarium of the Faculty of Pharmaceutical Sciences,
inflammatory activity evaluation showed a clear increase Nnamdi Azikiwe University, Awka. The leaves were dried
in the activity of boswellic acid with a notable increase in under shade for 14 days and milled using a cutter mill (JCT
absorption.10 Recently, Usnea barbata carbon dioxide- Thakur, Hoshiarpur, Punjab, India). Powdered N. laevis
supercritical extract was formulated as a lipid emulsion leaves (400 g) were extracted by continuous Soxhlet
with improved stability and antibacterial activity profiles.11 extraction using ethanol, with the extractor connected to
These reports support the need to develop dosage forms of a cooling chamber. The residue was discarded and the
natural products using lipid-based carrier systems. extract was concentrated using a rotary evaporator (Stuart,
Newbouldia laevis (P. Beauv.) belongs to the Bignonia- Barloworld Scientific, Essex, UK) under reduced pressure
ceae family and is commonly known as smooth Newboul- to yield a dark, green mass labelled as N. laevis extract
dia or boundary tree. In Nigeria, it is known with different (NLE), which was stored in an airtight plastic container
names among different communities: ‘Aduruku’, ‘Ogirisi’, until required.
Umeyor et al. 3

Screening for secondary metabolites Table 1. Composition of Newbouldia laevis-loaded SLMs.a

Phytochemical assay of the crude ethanolic extract of N. Softisan1 Precirol1 Kolliphor1

laevis leaves was carried out in order to ascertain the pres- 154 ATO 5 188 Distilled NLE
ence or absence of secondary metabolites in the extract Batch (%w/w) (%w/w) (%w/w) water (qs) (%w/w)
using standard conventional protocols.20,21 X1 2.5 2.5 2.0 100 1.0
X2 2.5 2.5 2.0 100 3.0
X3 2.5 2.5 2.0 100 5.0
Acute toxicity study X4 2.5 2.5 2.0 100 –*
White albino rats were maintained at standard housing con- Y1 1.7 3.3 2.0 100 1.0
ditions, and the animals were fed with standard pellets Y2 1.7 3.3 2.0 100 3.0
Y3 1.7 3.3 2.0 100 5.0
(Vital Feeds, Nigeria) and water ad libitum during the
Y4 1.7 3.3 2.0 100 –*
experiment. Ethical clearance for this study was obtained Z1 3.3 1.7 2.0 100 1.0
from the Animal Ethics Committee of the Faculty of Phar- Z2 3.3 1.7 2.0 100 3.0
maceutical Sciences, Nnamdi Azikiwe University, Awka, Z3 3.3 1.7 2.0 100 5.0
following animal experimental protocols in accordance Z4 3.3 1.7 2.0 100 –*
with the European Community Guidelines (European Eco-
SLMs: solid lipid microdispersions; NLE: Newbouldia laevis extract.
nomic Community (EEC) Directive of 2010; 2010/63/ a
For each batch, X1–X3, Y1–Y3 and Z1–Z3 contain Newbouldia laevis,
EEC). A total of 25 rats grouped into I–V and comprising while X4, Y4 and Z3 contain no Newbouldia laevis.
five rats per group were used for this study. Rats were *Unloaded.
fasted for 24 h prior to administration of NLE. Groups I,
II, III and IV were administered 250, 500, 1000 and 2000 (Yorco, York Scientific, Ghaziabad, Uttar Pradesh, India)
mg kg1 body weight single dose of NLE, respectively, by and the lyophilized samples were stored in airtight plastic
oral gavage, while the control group V received normal containers until future use.
saline (10 mL kg1). Feeding of rats resumed immediately
after NLE administration, and the lethal dose (LD50) of the
plant extract was calculated following standard methods.22 Determination of percentage recovery
Furthermore, time-dependent cage-side clinical observa-
The lyophilized SLMs were weighed to get the yield of
tions for toxicity signs such as paw licking, motor activity,
SLMs formulated per batch. The percentage (%) yield was
tremors, convulsions, spasticity, salivation, diarrhoea, wri-
calculated using the formula
thing and change in skin colour were made 2 weeks post-
administration. Body weights were also measured shortly W1
Percentage ð%Þ recovery ¼  100 (1)
before the administration of NLE and weekly thereafter. W2 þ W3
where W1 is the weight of the SLMs formulated (g), W2 is
Preparation of lipid matrix the weight of the drug added (g) and W3 is the weight of the
The lipid matrices consisting ratios of 1:1, 1:2 and 2:1 lipids used and surfactant (g).
mixtures, respectively, of Precirol1 ATO 5 and Softisan1
154 were prepared by fusion.20 Briefly, the lipids were Morphology and time-resolved particle size analysis
weighed out using an electronic balance (Pyrex, Germany),
and melted together at 70 C in a thermoregulated water A weighed amount of SLMs (50 mg) from each batch was
bath and stirred until solidification to get lipid matrices, dispersed in distilled water and smeared on a microscopic
labelled as ‘PreciSoft’ lipid matrix. slide using a glass rod. The mixture was covered with a
cover slip and viewed with a Hund1 binocular photomicro-
scope (Weltzlar, Germany) attached with a digital camera
Formulation of solid lipid microdispersions (Moticam, China) at a magnification of 100. Several
The hot-melt homogenization method23 was adapted in microparticles were counted and their sizes recorded (n ¼
the formulation of NLE-loaded SLMs according to the 100). A mean of particles sizes was calculated as the rep-
quantities of ingredients shown in Table 1. In each case, resentative size for a batch. This was done 24 h and 60 days
the PreciSoft matrix was melted at 70 C, and the surfac- post-formulation.
tant aqueous phase containing Kolliphor1 188 at the same
temperature was added to the molten lipid matrix with
gentle stirring with a homogenizer (Stuart) at 2000 r
Time-dependent pH study
min1 for 20 min to produce hot primary emulsions, A weighed amount of SLMs (50 mg) from each formula-
which were collected in beakers. Unloaded SLMs that tion was dissolved in 50 mL of distilled water in a 250-mL
served as negative control were also formulated. Each beaker. This dispersion was used to determine the pH of
batch of SLMs was lyophilized using a freeze dryer each sample in triplicates using a pH meter (Jenway 3505,
4 Nanobiomedicine

East Norwalk, Connecticut, USA). This was done at differ- Table 2. Phytochemical constituents of Newbouldia laevis extract.
ent time intervals of 24 h, 30 days and 90 days.
Secondary Metabolite Presence
Alkaloids þ
Thermal analysis Saponins 
Melting transitions and changes in heat capacity of Tannins þþ
Flavonoids þþ
Precirol1 ATO 5, Softisan1 154, PreciSoft lipid matrices,
Steroids 
and loaded SLMs were determined using a differential Terpenoids 
scanning calorimeter (Netzsch DSC 204 F1, Germany). Cardiac glycosides þþ
One milligram of each batch was weighed into aluminium Proteins 
pan, hermetically sealed and the thermal behaviour was
þ: moderately present (about 50%); þþ: abundantly present (70% and
determined in the range 50 C–350 C under a 20 mL min1 above); : absent.
nitrogen flux at a heating rate of 10 C min1. The thermal
property of the NLE was also determined in the range 50 C–
350 C. The baselines were determined using an empty pan, Determination of analgesic/anti-nociceptive property
and all the thermograms were baseline corrected. Analgesic activity was tested in mice using the hot plate
method.24 Mature mice of either sex (150–200 g) were
Determination of encapsulation efficiency divided into 10 groups of five mice per group. NLE-
loaded SLMs equivalent to 250 mg were administered
About 10 mg of each batch of the SLMs was dispersed in100 orally to test groups. The control groups received normal
mL of distilled water in a 100-mL volumetric flask. The dis- saline 10 mL kg1, while the reference group received 10
persion was allowed to equilibrate for 48 h at room temperature mg kg1 of diclofenac potassium. Mice were placed on
(25 C + 2 C), centrifuged at 4000 rpm and filtered. The hotplate maintained at 55 C + 1  C and the reaction
filtrate was adequately analysed for crude drug extract content latency (seconds) for licking of hind paw or jumping was
spectrophotometrically (Jenway UV/Vis 6505) at 250 nm. This recorded. Recordings were taken before treatment with the
was done in triplicates for each batch. The amount of drug different drugs and at 30, 60, 90 and 120 min post-
encapsulated in the microparticles was calculated with refer- treatment. Results were expressed as the difference
ence to a standard Beer’s plot for NLE. Encapsulation effi- between the baseline reaction latency and the reaction
ciency (EE %) was calculated using the following formula latency at each time interval.
Actual drug content
Encapsulation efficiencyð%Þ ¼  100
Theoretical drug content Statistical analysis
(2) Statistical analysis was carried out using SPSS version 14.0
(SPSS Inc., Chicago, Illinois, USA). All experiments were
performed in replicates (n ¼ 3) for validity of statistical
Determination of drug loading capacity analysis. Results were expressed as mean + standard
Drug loading capacity (DLC) estimates the ratio between deviation. Analysis of variance and Student’s t-tests were
the entrapped Active Pharmaceutical Ingredient (API) and performed on the data sets and differences were considered
total weight of the lipids. DLC was determined using the significant for p < 0.05.
following relationship
Wa
DLC ð%Þ ¼  100 (3) Results
Wl
where Wl is the weight of lipid used in the formulation and Secondary metabolites screening
Wa is the actual amount of NLE encapsulated in the SLMs. The result of the phytochemicals screening is presented in
Table 2. The result indicates the absence of saponins, ster-
Pharmacodynamic studies oids, terpenoids and proteins. Alkaloids were moderately
present (about 50%), while tannins, flavonoids and cardiac
Mature mice were maintained at standard housing condi- glycosides were abundantly present (70% and above).
tions, and the animals were fed with standard pellets (Vital
Feeds) and water ad libitum during the experiment. All
animal experimental protocols were carried out in accor-
Acute toxicity test
dance with guidelines of the Animal Ethics Committee of Acute toxicity study is used to determine LD50 value,
the Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe which is a useful indicator of the safety margin and dose
University, Awka, Nigeria, and European Union (EU) range at which a drug can be used such that there is no
Directive 2010/63/EU for animal experiments. harmful or lethal effect on the animal. The result of the test
Umeyor et al. 5

Table 3. Effect of NLE on body weights of rats.a

Time Group I (g) Group II (g) Group III (g) Group IV (g) Group V(g)
Day 1 195 + 0.25 205 + 1.50 198 + 1.08 185 + 1.83 178 + 1.20
Day 7 197 + 1.12 203 + 1.10 196 + 1.23 183 + 0.23 174 + 2.05
Day 14 199 + 0.52 204 + 0.67 199 + 2.45 184 + 0.94 177 + 1.67
NLE: Newbouldia laevis extract.
a
All values are expressed as mean + standard deviation.

Table 4. Percentage recovery of Newbouldia laevis–loaded SLMs. 7.0 mm to 66.5 + 5.2 mm after 60 days. Similarly, drug-
loaded ‘Z’ batches gave sizes ranging from 59.2 + 9.3 mm
Batch Amount recovered (g) Percentage yield to 89.0 + 4.4 mm after 60 days. After 60 days, unloaded
X1 6.33 86.3 SLMs recorded size ranges of 52.0 + 4.4–125.1 + 3.2 mm
X2 7.29 88.0 across all batches.
X3 8.25 89.2
X4 6.44 92.0
Y1 6.61 88.0 Time-dependent pH studies
Y2 8.27 90.0 The result of the pH of SLMs studied over time is presented
Y3 8.41 90.8
Y4 6.41 92.8
in Table 6. From the table, ‘X’ batches of SLMs formulated
Z1 7.07 88.4 with 1:1 lipid matrix had pH range of 6.03 + 0.1–6.79 +
Z2 9.41 91.7 0.2 after 90 days. ‘Y’ batches formulated with 1:2 lipid
Z3 11.00 94.1 matrix had pH ranging from 5.03 + 0.4 to 6.77 + 0.4,
Z4 6.10 85.7 whereas ‘Z’ batches formulated with 2:1 lipid matrix had
SLMs: solid lipid microdispersions.
their pH ranging from 5.43 + 0.2 to 6.87 + 0.1.

showed that the plant extract was generally safe even at a Thermal analysis
high dose of 2.5 g kg1, and no mortalities were recorded in The result of the thermal analysis is presented in Figures 7
the study animals. Furthermore, there was no significant to 16. The differential scanning calorimeter (DSC) thermo-
(p > 0.05) change in body weights (Table 3) and clinical gram of Softisan1 154 showed melting peak of 59 C with
behaviour of rats throughout the period of the study. enthalpy of 8.873 mW mg1, while the thermogram of
Precirol1 ATO 5 showed melting peak of 69.2 C with
Percentage recovery of SLMs enthalpy of 24.31 mW mg1. SLMs formulated from the
lipid matrices produced using these lipids gave DSC ther-
The percentage recovery of SLMs post-formulation is mograms with lower enthalpies, indicating low crystallinity
shown in Table 4. For the ‘X’ batches, X1, X2 and X3 gave and high drug entrapment.
percentage yields of 86.3%, 88.0% and 89.2%, respec-
tively. In the ‘Y’ batches, Y1, Y2 and Y3 produced per-
centage yields of 88.0%, 90.0% and 90.8%, respectively, EE (%) and DLC
while within the ‘Z’ batches, Z1, Z2 and Z3 gave percent- Results of the EE % and DLC of the SLMs are shown in
age yields of 88.4%, 91.7% and 94.1%, respectively. How- Table 7. From the table, EE % decreased with the increase
ever, X4, Y4 and Z4 yielded 92.0%, 92.8% and 85.7%, in drug loading with maximum EE % recorded for ‘X’ (1:1)
respectively. batches, ‘Y’ (1:2) batches and ‘Z’ (2:1) batches at 77.2%,
81.0% and 88.1%, respectively, at 1% w/w of NLE. Con-
versely, DLC increased with the increase in drug loading
Morphology and time-resolved particle size
with maximum DLC per 100 g of lipid recorded for ‘X’,
Morphology of the SLMs is presented as photomicrographs ‘Y’ and ‘Z’ batches at 20.0, 28.0 and 34.0 g, respectively, at
in Figures 1 to 6, while time-resolved particle sizes are 5% w/w of NLE.
presented in Table 5. The photomicrographs of representa-
tive SLMs showed stable and spherical microparticles
evenly distributed throughout the microscopic images of
Analgesic/anti-nociceptive property
the NLE-loaded SLMs, while the photomicrographs of The result of analgesic/anti-nociceptive study is presented
unloaded SLMs showed irregularly shaped particles. After in Table 8. From the result, analgesic/anti-nociceptive
60 days, drug-loaded ‘X’ batches recorded sizes ranging activity of the SLMs was comparable with the basal values
from 74.2 + 5.9 mm to 75.1 + 9.0 mm. Also, drug- after 30 min but were enhanced 60 min and above when the
loaded ‘Y’ batches recorded sizes ranging from 53.6 + pain reaction time was greatly increased. The delayed
6 Nanobiomedicine

Figure 1. Photomicrograph of batch X1 showing spherical Newbouldia laevis-loaded SLMs (bar: 100). SLMs: solid lipid
microdispersions.

Figure 2. Photomicrograph of batch X4 showing spherical, unloaded SLMs (bar: 100). SLMs: solid lipid microdispersions.
Umeyor et al. 7

Figure 3. Photomicrograph of batch Y1 showing spherical Newbouldia laevis-loaded SLMs (bar: 100). SLMs: solid lipid
microdispersions.

Figure 4. Photomicrograph of batch Y4 showing spherical, unloaded SLMs (bar: 100). SLMs: solid lipid microdispersions.
8 Nanobiomedicine

Figure 5. Photomicrograph of batch Z1 showing spherical Newbouldia laevis-loaded SLMs (bar: 100). SLMs: solid lipid
microdispersions.

Figure 6. Photomicrograph of batch Z4 showing spherical, unloaded SLMs (bar: 100). SLMs: solid lipid microdispersions.
Umeyor et al. 9

Table 5. Time-resolved particle size analysis. remained non-toxic to the rats. It was also observed that the
animals remained healthy as there were no unusual clinical
Particle size (mm + SD)* Particle size (mm + SD)*
Batch after 24 h after 60 days
changes in locomotor activity or behaviour, and no ataxia
and no signs of intoxication were observed. This indicates
X1 43.0 + 2.5 74.2 + 5.9 that all the animals tolerated the extract. Furthermore, there
X2 51.4 + 6.4 74.5 + 3.6 was no significant (p > 0.05) difference in the rate of food
X3 61.4 + 3.7 75.1 + 9.0
and water consumption between the study and control
X4 40.0 + 7.5 125.1 + 3.2
Y1 47.1 + 7.0 53.6 + 7.0 groups of animals, and this might be responsible for the
Y2 57.1 + 5.5 54.1 + 6.8 insignificant (p > 0.05) changes in the body weight of the
Y3 73.0 + 4.0 66.5 + 5.2 rats. The implication of these indicators is that the in vivo
Y4 41.4 + 8.5 70.0 + 2.4 administration of any formulation of N. laevis may not be
Z1 54.3 + 9.5 59.2 + 9.3 harmful to the biological system.
Z2 65.7 + 5.5 66.6 + 1.3 From the table of percentage recovery of SLMs, it could
Z3 77.5 + 6.0 89.0 + 4.4
be seen that very good amounts of SLMs were recovered
Z4 51.4 + 7.7 52.0 + 4.4
for all the batches. This indicates that hot melt homogeni-
SD ¼ standard deviation. zation method is reliable in the formulation of SLMs.21,26
*n ¼ 3. Within the loaded batches, there is an increase in SLMs
recovery with the increase in NLE loading, for example,
Table 6. pH analysis of Newbouldia laevis-loaded SLMs. SLMs recovered increased from 86.3% (X1) to 88.0% (X2)
and then to 89.2% (X3). This means that the amorphous
Batch pH (after 24 h)a pH (after 30 days)a pH (after 90 days)a lipid matrices created imperfections on melting leading to
X1 6.45 + 0.1 6.45 + 0.2 6.44 + 0.5 increased drug entrapment. Across batches, it could be
X2 6.12 + 0.1 6.14 + 0.9 6.41 + 0.3 observed that SLMs recovery was higher for the ‘Y’
X3 6.03 + 0.1 6.04 + 0.5 6.05 + 0.3 batches formulated with 1:2 ratio of the lipid matrix. This
X4 6.73 + 1.3 6.79 + 0.1 6.79 + 0.2 means that at the ratio of 1:2 lipid matrix, there were exten-
Y1 6.35 + 2.3 6.37 + 0.3 6.36 + 0.1 sive rearrangements in the crystalline structure of the lipids
Y2 6.05 + 0.3 6.06 + 0.3 6.04 + 0.9 leading to high solubilization of NLE. The high recovery
Y3 5.92 + 0.7 5.92 + 0.1 5.03 + 0.4
Y4 6.77 + 0.4 6.73 + 0.6 6.73 + 0.2
obtained for the unloaded SLMs batches (X4, Y4 and Z4)
Z1 6.63 + 0.1 6.63 + 0.4 6.61 + 1.2 might be due to poor drying of the formulations, as the
Z2 5.61 + 0.2 5.63 + 0.1 5.70 + 0.9 recovered particles were somewhat sticky and viscous in
Z3 5.43 + 0.5 5.43 + 0.2 5.64 + 0.5 appearance. However, none of the batches recovered was
Z4 6.82 + 0.1 6.87 + 0.1 6.86 + 0.3 up to 100%. This could be due to losses from weighing,
a
Values are expressed as mean + standard deviation; n ¼ 5
transference, filtration and drying.
When particles of SLMs are viewed edge-on using a
photomicroscope, the particles as shown on the photomi-
sensation to pain observed was reflective of that produced crographs appear to be two-dimensional, and the spherical
by the standard drug (positive control), diclofenac potas- shape of the SLMs may not be fully appreciated. However,
sium, and the effect was sustained throughout the study all the NLE-loaded SLMs appeared spherical, smooth and
period. stable. After lyophilization, some batches of the SLMs
were free-flowing, while batches that retained certain level
of moisture were viscous with retarded flow. After 24 h
Discussion post-formulation, varying sizes of particles were obtained,
Findings from the screening for secondary metabolites and these variations could be due to factors such as con-
were in agreement with earlier reports.16,17 The presence centrations of lipid matrices and surfactant used. It was
or abundance of secondary metabolites has been attri- observed that the particle size increased with the increase
buted to the nature of soil where the plant grows and the in drug loading. Therefore, drug loading produced signifi-
prevailing microclimate conditions.25 This result indi- cant (p < 0.05) increase in average particle sizes of the
cates that the medicinal values of the plant could be due SLMs, and batches with the highest drug loading produced
to the presence of one or more of these metabolites. This the largest particle sizes due to increased drug concentra-
could be verified from fractionation, isolation and iden- tion entrapped in the lipid matrix, for example, X1 loaded
tification or characterization of compounds present in the with 1% w/w NLE produced average particle size of 42.86
crude extract, formulating using an appropriate drug + 2.5 mm, while X3 loaded with 5% w/w NLE produced
delivery system and studying its pharmacotherapeutic particles with average size of 61.43 + 3.7 mm. Also, Z1
potentials. loaded with 1% w/w NLE produced average particle size of
The result of the acute toxicity study indicates that the 54.29 + 9.5 mm, while Z3 loaded with 5% w/w NLE
extract has a very high safety margin on administration and produced an average particle size of 77.5 + 6.0 mm. The
10 Nanobiomedicine

Figure 7. DSC thermogram of Precirol1 ATO 5. DSC: differential scanning calorimeter.

Figure 8. DSC thermogram of Softisan1 154. DSC: differential scanning calorimeter.

same scenario was observed when the SLMs were viewed information will enable a formulator to adapt reliable and
after 60 days. However, a comparison of the particle sizes acceptable protocols in the formulation of a drug.21 From
across batches depicted slight (p > 0.05) increases in par- the result, the pH values of all the SLMs ranged between
ticle sizes after storage. This could be due to particulate 5.03 + 0.4 and 6.87 + 0.1, which implies that the formu-
aggregation in the formulation due to Ostwald ripening lation is slightly acidic, hence will be most likely absorbed
leading to greater particle size. It could also be due to the in acidic environment of the gastrointestinal tract. The
effect of temperature variations (25 + 2 C–27 + 2 C) acidic pH recorded by the SLMs could be due to the pres-
during storage as SLMs are better stored in cool place. ence of free fatty acids in the lipid matrices. There was no
A very good knowledge of the pH of maximum stability significant (p > 0.05) change in pH throughout the duration
of a drug or its stability profile is important, as the of study. This shows that the formulations were stable and
Umeyor et al. 11

Figure 9. Representative DSC thermogram of lipid matrix (Softisan1 154 and Precirol1 ATO 5 1:1). DSC: differential scanning
calorimeter.

Figure 10. DSC thermogram of Newbouldia laevis extract. DSC: differential scanning calorimeter.

showed no signs of degradation. Furthermore, it was decline in pH as confirmed by their steady-state pH

observed that pH decreased with the increase in NLE load- throughout the study period.
ing; this implies that NLE could be acidic or caused the The DSC thermogram of Softisan1 154 showed an
release of free fatty acid in the NLE-loaded SLMs, hence endothermic melting peak of 59 C with enthalpy 8.873
the observed reduction in pH of the SLMs. This is because mW mg1, while the DSC thermogram of Precirol1 ATO 5
the unloaded SLMs (X4, Y4 and Z4) did not exhibit such showed an endothermic melting peak of 69.2 C with
12 Nanobiomedicine

Figure 11. DSC thermogram of SLMs loaded with 1% w/w of Newbouldia laevis extract based on 1:1 lipid matrix. DSC: differential
scanning calorimeter; SLMs: solid lipid microdispersions.

Figure 12. DSC thermogram of SLMs loaded with 5% w/w of Newbouldia laevis extract based on 1:1 lipid matrix. DSC: differential
scanning calorimeter; SLMs: solid lipid microdispersions.

enthalpy 24.31 mW mg1. These values correspond to the 43.42 mW mg1, 66.7 C and 61.08 mW mg1 and 64.7 C
literature values of the melting points of Precirol1 ATO 5 and and 39.87 mW mg1 (at 1:1, 1:2 and 2:1 Softisan1 154 and
Softisan1 154 with a slight variation, which could be due to Precirol1 ATO 5), respectively. The single endothermic melt-
sensitivity of the DSC instrument. When these two lipids were ing peaks and low enthalpies produced indicate reduced crys-
blended, the resulting PreciSoft lipid matrix produced the fol- tallinity and rearrangement for drug localization due to carbon
lowing endothermic melting peaks and enthalpies: 67 C and chain adjustments from the interaction of saturated even-
Umeyor et al. 13

Figure 13. DSC thermogram of SLMs loaded with 1% w/w Newbouldia laevis based on 1:2 lipid matrix. DSC: differential scanning
calorimeter; SLMs: solid lipid microdispersions.

Figure 14. DSC thermogram of SLMs loaded with 5% w/w Newbouldia laevis based on 1:2 lipid matrix. DSC: differential scanning
calorimeter; SLMs: solid lipid microdispersions.

numbered, unbranched and natural fatty acids of Softisan1 NLE produced three endothermic peaks at 81.3 C, 218.5 C
154 and mixture of mono-, di- and triglycerides of palmitic and 138.8 C with corresponding enthalpies of 8.046, 5.277
acid and stearic acid in Precirol1 ATO 5. This presents an and 7.33 mW mg1. It also showed an exothermic melting
important alternative to lipid modification using chemical peak at 347.8 C with enthalpy of 2.795 mW mg1. When
methods because the latter lead to the production of lipids with NLE was loaded into the lipid matrices to formulate SLMs
decreased in vivo compatibility. DSC thermogram of crude (Figures 11 to 16), it produced microparticles with low melting
14 Nanobiomedicine

Figure 15. DSC thermogram of SLMs loaded with 1% w/w Newbouldia laevis based on 2:1 lipid matrix. DSC: differential scanning
calorimeter; SLMs: solid lipid microdispersions.

Figure 16. DSC thermogram of SLMs loaded with 5% w/w Newbouldia laevis based on 2:1 lipid matrix. DSC: differential scanning
calorimeter; SLMs: solid lipid microdispersions.

peaks and enthalpies, for example, X1, Y1 and Z1 from 1:1, 1:2 concentration of NLE made bioavailable in the biological sys-
and 2:1 lipid matrices produced the following melting peaks: tem following administration, and this will be useful in order to
65, 64.6 and 64 C with the corresponding enthalpies: 20.44, initiate any meaningful pharmacotherapeutic event. However,
19.61 and 24.17 mW mg1. This signifies high drug load- an in vivo release study would clearly prove this.
ing, despite the crude character of NLE. The implication of this EE is an essential characteristic of SLMs, which indi-
finding is that there might be the possibility of a high cates their propensity to entrap and accommodate active
Umeyor et al. 15

Table 7. Analyses of encapsulation efficiency and drug loading capacity.

Batch Theoretical drug content (g) Actual drug content (g) Encapsulation efficiency (%) Drug loading capacity (g API/100 g lipid)
X1 1.0 0.8 77.2 16.0
X2 3.0 0.9 29.6 18.0
X3 5.0 1.0 20.7 20.0
Y1 1.0 0.8 81.0 16.0
Y2 3.0 1.3 43.8 26.0
Y3 5.0 1.4 27.5 28.0
Z1 1.0 0.9 88.1 18.0
Z2 3.0 1.5 48.5 30.0
Z3 5.0 1.7 34.6 34.0
API: Active Pharmaceutical Ingredient.

Table 8. Analgesic/anti-nociceptive effect.

Group Dose (mg) Basal values (min) 30 min 60 min 90 min 120 min
X1 250 4.0 + 0.5 5.0 + 0.2 5.1 + 0.2 6.0 + 0.3 6.1 + 1.2
X2 250 4.0 + 0.9 4.3 + 0.6 10.0 + 1.2 9.6 + 0.1 10.1 + 0.6
X3 250 4.0 + 1.2 5.2 + 0.1 10.1 + 0.5 8.5 + 1.0 10.0 + 0.2
Y1 250 5.1 + 1.1 5.2 + 1.3 6.0 + 0.1 7.0 + 0.3 8.2 + 1.9
Y2 250 4.1 + 0.3 4.6 + 1.0 5.3 + 0.9 6.0 + 1.0 10.3 + 0.5
Y3 250 3.0 + 0.1 4.1 + 0.3 5.1 + 0.5 5.7 + 0.7 6.1 + 0.3
Z1 250 2.9 + 0.2 3.2 + 0.1 5.7 + 0.5 5.7 + 0.1 5.7 + 0.1
Z2 250 4.0 + 0.4 4.1 + 0.3 4.9 + 0.2 5.2 + 0.8 7.0 + 1.2
Z3 250 3.0 + 0.1 4.0 + 0.2 5.0 + 1.1 4.0 + 0.8 5.0 + 1.4
Diclofenac potassium 10 mg kg1 2.0 + 0.7 4.9 + 0.2 5.7 + 1.0 12.0 + 0.1 11.0 + 0.3
Normal saline 10 mL kg1 3.1 + 0.1 3.2 + 0.4 2.8 + 1.0 3.4 + 0.7 3.1 + 0.2

drugs. From the table, the actual drug content increases for all the batches, giving the maximum DLCs as 20, 28 and
with the increase in drug loading for all the batches. The 34 g of NLE per 100 g of lipid for 1:1, 1:2 and 2:1 lipid
order of increase across the batches is X < Y < Z. Again, matrices. This result shows that the ‘Z’ batches produced
across batches, the EE % decreased with the increase in the highest DLC, and this is a confirmation of the highest
drug loading giving maximum EE % for lipid matrices 1:1, EE % obtained with the ‘Z’ batches.
1:2 and 2:1 as 77.2%, 81.0% and 88.1%, respectively. This From the result of the analgesic/anti-nociceptive study
can be explained from the fact that each of the lipid matrix of the SLMs, it could be seen that all the batches of SLMs
used for the formulation of the SLMs has reached its max- have good anti-nociceptive effect comparable with the
imum level of drug solubilization and entrapment, for standard drug, diclofenac potassium. The relative values
example, in batches X1, X2 and X3 formulated using 1:1 of the anti-nociceptive action of the formulations when
lipid matrix, increasing drug loading from 1% w/w to 3% compared to the basal value indicate an increased delayed
w/w led to a decrease in EE % from 77.2% to 29.6%. This sensation to pain of heat due to an increase in duration of
was also observed in SLMs formulated using lipid matrices response of the mice to stimulus (heat). However, this rela-
1:2 and 2:1. Furthermore, batches Z1, Z2 and Z3 formu- tivity lasted for 30 min; but from 60 min, there was
lated with lipid matrix 2:1 produced the highest EE % enhanced delay in sensation to pain of heat, which lasted
compared with the ‘X’ and ‘Y’ batches formulated with for another 60 min. This may be due to increased absorp-
1:1 and 1:2 lipid matrices. The high encapsulation effi- tion of the N. laevis in vivo in the presence of the lipid
ciency in ‘Z’ batches is a clear indication that they pro- carrier. This is because earlier reports have shown that
duced the highest imperfections and disorderliness, which drugs incorporated in lipid matrices have better absorption
entrapped the highest amounts of NLE in comparison to than unencapsulated drugs. 18 The pain reaction time
batches X and Y.27 This implies that N. laevis would be increased with time as a result of delay in reaching pain
readily available systemically for the control of inflamma- threshold in the test animals. The anti-nociceptive action of
tion upon administration, and this would be higher for the formulations was higher for the ‘X’ batches followed by
batch ‘Z’ formulations than batches ‘X’ and ‘Y’. On the the ‘Y’ batches, while the ‘Z’ batches produced the least
other hand, DLC shows the amount of encapsulated drug pain reaction time. The high anti-nociceptive effect pro-
relative to the amount of lipids used in the formulation. duced by the ‘X’ batches could mean that NLE was
From Table 6, DLC increased with the increase in drug released in substantial amount from the 1:1 lipid matrix
loading. The highest DLC was obtained at 5% w/w of NLE into the study mice producing a high pain threshold. The
16 Nanobiomedicine

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