Powder Technology 328 (2018) 140–147

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Powder Technology

journal homepage: www.elsevier.com/locate/powtec

Cellulose nanoparticles encapsulated cow urine for effective inhibition

of pathogens
Koh Hann Suk a, Subash C.B. Gopinath a,b,⁎, Periasamy Anbu c, Thangavel Lakshmipriya b
a
School of Bioprocess Engineering, Universiti Malaysia Perlis, 02600 Arau, Perlis, Malaysia
b
Institute of Nano Electronic Engineering, Universiti Malaysia Perlis, 01000 Kangar, Perlis, Malaysia
c
Department of Biological Engineering, College of Engineering, Inha University, Incheon 402-751, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: Cow urine has been shown to have useful applications in medicine and therapeutics. Indeed, many studies
Received 19 June 2017 have shown that cow urine has the potential to cure various conditions, such as kidney and skin diseases.
Received in revised form 15 November 2017 The current study demonstrated the effectiveness and cost efficiency of cow urine enhanced by encapsulat-
Accepted 5 January 2018
ing in cellulose nanoparticles (CNPs). The encapsulation process was aided by Tween-20 and olive
Available online 09 January 2018
oil through the microemulsion procedure. Scanning electron, transmission electron and atomic force
Keywords:
microscopy revealed that the CNPs encapsulated cow urine were with the size range of 40 to 100 nm and
Cow urine agglomerated. Furthermore, X-ray photoelectron spectroscopy (XPS), X-ray powder diffraction (XRD)
Encapsulation and Fourier transform infrared spectroscopy (FTIR) studies revealed that various components known to
Cellulose nanoparticle exist in cow urine were also in the CNPs encapsulated cow urine. XPS analysis displays the presence of a
Bacillus subtilis single carbon between the atoms composed of two electrons in the CNPs. XRD result shows the most intense
Escherichia coli peak at 2θ = 32.14° and crystallinity. With FTIR on the encapsulated cow urine, a broad peak was observed
Aspergillus niger in the range between 655 and 662.5 cm−1. The encapsulated cow urine exerted a good inhibitory activity
toward Bacillus subtilis (11 ± 1.5 mm/50 μL) and Aspergillus niger compared to the cow urine alone,
as well as a notable inhibition of Escherichia coli (17 ± 2.2 mm/50 μL). Overall, the results of this study
demonstrated that the antimicrobial properties of cow urine can be enhanced through microencapsulation,
which may enable its efficient delivery.
© 2018 Elsevier B.V. All rights reserved.

1. Introduction Cellulose nanoparticles (CNPs) are also known as nanospheres,

Cow urine has been used as a traditional medicine for hundreds of
years and long been known to have medical usefulness in Ayurveda
medicine [1]. The use of cow urine has been accepted universally
among medical researchers, microbiologists, and pharmacologists [2].
One of the ingredients in cow urine, ‘Panchagawya,’ has the capability
to treat kidney diseases, edema, and skin diseases. Traditional medicines
such as herbs are usually paired with cow urine to treat sicknesses such
as fever and anemia. Cow urine has also been found to be potentially
useful for treating high blood pressure, heart failure, and arterial
blockages [3,4]. The relationship between cow urine and antimicrobial
activity might be due to the presence of volatile and non-volatile
components. In the medical field, cow urine is now receiving a great
deal of attention because its benefits have been confirmed scientifically.
Additionally, cow urine has useful applications in agriculture and
sericulture [1,2,4].
⁎ Corresponding author at: School of Bioprocess Engineering, Universiti Malaysia Perlis,
02600 Arau, Perlis, Malaysia.
E-mail address: subash@unimap.edu.my (S.C.B. Gopinath).
nanowhiskers, whiskers, and nanopowders depending on the method
of preparation [5]. The processes utilized to prepare CNPs can be differ-
entiated into debilitation and pre-treatment of extracted CNPs from mi-
crofibrils and biosynthesis of microfibrils from cellulose-based materials
[6]. The advantages of CNPs are their low cost, widespread availability,
non-toxic nature, higher specific strength and low density, which en-
able their use in a variety of applications, including nanocomposites
and filtration media. Accordingly, CNPs have been applied broadly
in pharmaceutical and food industries [5]. CNPs can also serve as
multi-functional agents such as engineering composites, paper, aerogels
and biomedical materials [7]. The properties of CNP-reinforced compos-
ites are influenced by the CNPs themselves, a scattering of the micro-
structure and production conditions. Moreover, CNPs exhibit greater
strength and higher percolation ability in polymer matrices. In addition,
the mechanical properties of composites have greater impaction
because the CNPs are dispersed in the polymer matrix [8]. During the
encapsulation there are some limitations, such as it need a proper
optimization, otherwise will attain the fragile condition and also cause
the variations with the size. Another limitation is due to the soluble
nature, solubility varies based on the solvents in the reaction media or
cellular milieu. Further, preparation of CNPs having a lesser diameter

https://doi.org/10.1016/j.powtec.2018.01.010
0032-5910/© 2018 Elsevier B.V. All rights reserved.
 K.H. Suk et al. / Powder Technology 328 (2018) 140–147
(b100 nm) is challenging, because it tends to be agglomerated during
the processing.
Encapsulation is accomplished by trapping one substance within
another substance, such as an active agent within a wall material [9].
Particle encapsulation has received wide attention because it can be
used to stabilize guest components or increase energy-transfer process-
es in enclosed areas [10]. Nevertheless, entrapment of components can
be challenging because of the high-energy activity associated with the
configuration of nanoparticles [11]. According to Li et al. [12], a positive-
ly charged nanoparticle has the benefit of being able to selectively
convey drugs or genes to the surface of the target tissue.
In this study, cow urine was tested for its antimicrobial activity
based on its inhibition of the growth of pathogenic bacteria. To accom-
plish this, different amounts of cow urine were evaluated to determine
the minimum inhibitory concentration (MIC). The cow urine was then
encapsulated by CNPs and analyzed for its effectiveness against patho-
gens. The cow urine encapsulated in CNPs was also characterized by
scanning electron, transmission electron, and atomic force microscopy,
as well as by X-ray photoelectron spectroscopy, X-ray powder diffrac-
tion, and Fourier transform infrared spectroscopy.
2. Materials and methods
2.1. Materials
Cow urine used for this study was collected from a local disease-
free cow at Penang Cow Farm in Malaysia. During the collection
of cow urine, we preferred the healthy cow based on the medical
records in the farm. The collected cow urine was stored in a clean
empty bottle that was tightly sealed and then placed into a polysty-
rene container and kept at 4 °C. Cow urine was filter sterilized (0.45
μm) before use. Cellulose was purchased from a local market in
Malaysia. Reagents such as sodium hydroxide (NaOH), ethanol
(Merck, USA), Tween-80 (Sigma-Aldrich, USA), olive oil (from
the local market) and urea (Sigma-Aldrich, USA) were used for the
preparation of encapsulated CNPs via the microemulsion method.
In addition, ethanol was used as a washing reagent for CNPs following
centrifugation. Antimicrobial activity was assessed using Escherichia
coli, Bacillus subtilis and Aspergillus niger as models. Yeast extract
peptone dextrose (YPD), nutrient agar (NA) and nutrient broth (Merck)
were used for cultures. All microorganisms were obtained from the
culture collection of the School of Bioprocess Engineering, Universiti
Malaysia Perlis.
Nutrient agar and YPD agar were prepared to determine the mini-
mum inhibitory concentration and for validation of bacterial inhibition.
Briefly, 14 g of NA powder were dissolved in 500 mL of distilled water or
25 g of YPD agar were dissolved in 500 mL of distilled water in two
different Erlenmeyer flasks. Both flasks were then autoclaved at 121 °C
for 20 min. The broths were allowed to cool before being poured into
Petri dishes in a laminar flow hood.
2.2. Minimum inhibitory concentration (MIC) of cow urine
The MIC was measured to determine the minimum amount of cow
urine needed to inhibit the visible growth of microorganisms. To accom-
plish this, 1, 10, 20, 40 and 50 μL aliquots of cow urine were diluted to
final volume 50 μL with sterilized distilled water. Alternatively, place a
sentence above stating that all microbial analyses were done aseptically.
Microorganisms were then spread on their respective agar plates, after
which sterilized filter disc papers were placed on the agar. Next, 50 μL
of the different dilutions of cow urine were placed on the filters. E. coli
and B. subtilis were subsequently incubated overnight, whereas
A. niger was incubated for 2–3 days, after which the inhibition zones
were measured.
141
2.3. Encapsulation of cow urine in cellulose nanoparticles (CNPs)
A microemulsion solution was prepared in a beaker using 100 μL
of Tween-20, 120 μL of olive oil and 1 mL of cow urine. Next, 10 mL of
absolute ethanol was added into the same beaker containing the solu-
tion, after which the beaker was covered with parafilm and stirred at
900 rpm for 1 h at room temperature. Another solution was prepared
separately in a beaker containing 8% NaOH, 10% urea and 1% of cellulose
with a total working volume of 50 mL. Next, 500 μL of this solution was
pipetted into the microemulsion solution drop-by-drop while stirring.
Approximately 1.7 mL of the microemulsion solution was then trans-
ferred into six different Eppendorf tubes, which were subsequently cen-
trifuged at 4500 ×g for 20 min. The encapsulated cow urine was then
washed with absolute ethanol, centrifuged at 4500 ×g for an additional
20 min, then washed with absolute ethanol prior to the final centrifuga-
tion process at 10,000 rpm for 5 min.
2.4. Atomic force microscopy (AFM)
The measurement of the sample was conducted using an atomic
force microscope (Digital Instruments Nanoscope) with a 2 μm scan
size and a scan rate of 0.8 Hz. The sample was dried overnight at
ambient temperature before analysis.
2.5. Scanning electron microscopy (SEM)
A scanning electron microscope (Hitachi, S-4300 SE) was used to
determine the structure and size of particles. Ultrasonic treatment was
conducted for approximately 5 min to disperse the aggregated CNPs.
This dispersion was then applied to a silicon substrate and placed
under the microscope to obtain the visual electric images of the particles
at 15 kV and 1000× magnification. We made the metal conducting
coating on the substrate before performing the SEM experiments, as it
is necessary when analyzing the non-metallic particles.
2.6. Transmission electron microscopic (TEM) analysis
The distribution and size of nanoparticles were also visualized using
a transmission electron microscope (JEM-2100F). Images were taken at
an accelerating voltage of 200 kV after the sample had been dried at
ambient temperature on a copper grid overnight.
2.7. X-ray photoelectron spectroscopy (XPS) analysis
Samples were diluted in of about 2 mL absolute ethanol, then centri-
fuged at 10,000 ×g for 30 min. The supernatant was removed, after
which the precipitated sample was collected and dried at ambient
temperature overnight. Next, the sample was analyzed using an X-ray
photoelectron spectroscopy (Thermo Scientific, K-Alpha) equipped
with a Mg X-ray source operating at 300 W.
2.8. X-ray diffraction (XRD) analysis
Samples were analyzed by XRD (DMAX-2500, Rigaku) using
CuKα as a radiation source. The operation was conducted at 40 kV and
100 mA in the range of 10 ≤ 2θ ≤ 90.
2.9. Fourier transform infrared spectroscopy (FTIR)
A Fourier transform infrared (FTIR) spectroscopy (Spectrum 65,
Perkin Elmer) was utilized to measure the FTIR spectra of nanoparticles,
which was reported in absorbance with a resolution of 4 cm−1 and
a range of 4000–400 cm1. Encapsulated cow urine, cow urine and
unencapsulated CNP samples were each scanned 40 times and the
readings were recorded and averaged.
142 K.H. Suk et al. / Powder Technology 328 (2018) 140–147
Fig. 1. Schematic representation of CNP encapsulated cow urine for the pathogenic inhibition. The effects of CNP encapsulated cow urine and non-encapsulated CNPs are shown.
2.10. Validation of bacterial inhibition by encapsulated cow urine
2.10.1. Disc diffusion method
Regular sized (6 mm) filter paper discs were made using a hole-
puncher. The discs were then placed on agar plates and swabbed with
bacteria or pinpoint inoculated with A. niger. The nanoparticles were
subsequently pipetted onto the discs and their inhibitory effects were
compared to each other and those of discs without CNP. In addition,
results were compared to those obtained using ampicillin as a standard
control and cow urine as an intermediate control. Samples were
incubated at 37 °C overnight, after which the zones of inhibition were
measured. The average values of replicates were reported.
2.10.2. Turbidimetric assay
The antimicrobial activity of the cow urine against the growth of
microorganisms was investigated using 2.2 mL of NB. Uninoculated NB
was used as a blank. The assay was conducted using 0, 50, 100, 200,
400 and 800 μL of cow urine diluted to 800 μL using distilled water.
Also, sterilized distilled specified above, please choose one form and
apply consistently. Samples were then cultured for 6 h, after which
they were analyzed using an ultraviolet–visible spectrophotometer
(Genesys 20).
3. Results and discussion
Previous studies have shown that cow urine has the potential
for medical applications because it possesses antimicrobial and lipase
activity. Toxins can be purged with the aid of cow urine and also serve
as anti-toxin in preventing all kinds of poisonous [13]. To determine if
cow urine contains antimicrobial properties, the minimum inhibitory
concentration (MIC) was measured, after which it was encapsulated
in nanoparticles to determine if this process could improve the
effectiveness with which cow urine prevents the growth of pathogens
(Fig. 1). In the presence of cow urine, it behaves like antibiotics and
preventing the cell wall formation, ultimately interfere with the micro-
bial cell division. Encapsulation of drugs enables control of their rate of
release against pathogenic bacteria [14]. Hence, cellulose nanoparticles
(CNPs) were used for encapsulation because they are believed to
enhance the effectiveness of antimicrobial agents delivered to the site
of interest. A previous study showed that drugs encapsulated with
CNPs were more effective due to their small size. Moreover, their
small size may impart favorable biological effects, making encapsulation
of CNPs a reasonable choice to increase the therapeutic potency of cow
urine [15,16]. In the current study, Disc diffusion and turbidimetric
assays were selected to study the zones of inhibition of pathogens.
3.1. Biological inhibition assays
3.1.1. Minimum inhibitory concentration (MIC) of cow urine
The MIC of cow urine was investigated by the disc diffusion assay.
Cow urine efficacy was assayed on agar plates swabbed with
B. subtilis, E. coli and A. niger (pin-point inoculum). No zones of
inhibition were observed against B. subtilis in response to 0, 1, 10
and 20 μL of cow urine. However, cow urine at 40 μL (8 ± 1.2 mm)
and 50 μL (8.5 ± 1.22 mm) produced inhibition zones of different
sizes and they increased with cow urine concentration. In contrast,
cow urine exerted no inhibitory effects against E. coli and A. niger at
any of the tested levels. This assay concluded that cow urine has
the potential to inhibit B. subtilis. Based on these findings, 50, 25
and 10 μL of cow urine were subjected to subsequent validation.
3.1.2. Turbidimetric assay
The turbidimetric assay was also used to evaluate the inhibitory
effects of cow urine against B. subtilis and E. coli [17], while the other
organism tested above was excluded because the turbidimetric assay
is not suitable to be tested upon fungi. Specifically, the efficacy of
0, 50, 100, 200, 400 and 800 μL was evaluated based on ultraviolet
Table 1
Turbidimetric method for determination of the inhibition of microorganisms.
Cow urine (μL) Absorbance (O.D.)
B. subtilis E. coli
0 0.110 ± 0.005 0.492 ± 0.021
50 0.130 ± 0.006 0.638 ± 0.029
100 0.175 ± 0.008 0.697 ± 0.029
200 0.233 ± 0.010 0.779 ± 0.031
400 0.062 ± 0.001 0.777 ± 0.030
800 0.056 0.096 ± 0.006
 K.H. Suk et al. / Powder Technology 328 (2018) 140–147
Table 2
Comparison on the effectiveness of cow urine with and without CNPs.
Microorganism Average inhibition zone (mm)
Cow urine alone Encapsulated cow urine
50 μL 25 μL 10 μL 50 μL 25 μL
B. subtilis 8 ± 1.1 6.85 ± 0.9 0 11 ± 1.5 10 ± 1.1 9 ± 1.1
E. coli 0 0 0 17 ± 2.2 0 0
A. niger Negative Negative Negative Positive Negative Negative
spectrophotometry (Table 1). For B. subtilis treated with 0 μL of cow
urine, the absorbance (A) was 0.110. However, the absorbance
increased to 0.130 and 0.175 in response to 50 and 100 μL of cow
urine, respectively, while it was 0.233 in response to 200 μL of cow
urine, nut decreased to 0.062 in response to 400 μL. Moreover, the
absorbance further decreased to 0.056 when the amount of cow urine
was further increased to 800 μL. A similar trend was observed in
response to treatment of E. coli with cow urine. Specifically, the
absorbance values were 0.492, 0.638, 0.697 and 0.779 in response to
treatment with 0, 50, 100 and 200 μL cow urine, respectively. However,
the absorbance decreased to 0.777 and 0.096 in response to 400 μL and
800 μL of cow urine, respectively. These findings indicate that the opti-
mum inhibitory effects of cow urine were obtained at 200 μL for
B. subtilis whereas 400 μL for E. coli. Moreover, the low amounts cow
urine used did not possess sufficient antimicrobial activity and therefore
failed to prevent the growth of the microorganisms. The positive results
only can be seen when at least 400 μL of cow urine was used for the
particular assay. Comparison of the results obtained for B. subtilis and
E. coli revealed that the inhibitory effects were greater against B. subtilis.
3.1.3. Disc diffusion assay
The efficacy of 10, 25 and 50 μL of cow urine in a total working vol-
ume of 50 μL against pathogenic bacteria was evaluated by disc diffusion
assay. In addition, the effects of ampicillin were evaluated as a control.
The results are shown in Table 2. As shown in Fig. 2A, 50 μL of cow
urine produced an inhibition zone of 8 mm against B. subtilis (a).
As the amount of cow urine used decreased, the inhibition zones were
reduced, with 25 μL producing a zone of 6.85 mm (b) and 10 μL having
no observable effect (c). By comparing to the ampicillin, the measured
inhibition zone is as 10 mm. Consider just specifying the size of the
zone of inhibition for ampicillin. Fig. 2B shows the effects of cow urine
against E. coli. In the initial tests, cow urine failed to produce any inhibi-
tion zones against E. coli. Also, above says 10, 25 and 50 μL used against
B. subtilis. Therefore, additional tests were conducted to investigate the
Fig. 2. Disc diffusion assay of the inhibitory effects of cow urine against (A) B. subtilis, (B) E. coli and (C) A. niger, where (a), (d) and (g) are 50 μL; (b), (e) and (h) are 25 μL and (c), (f) and
(i) are 10 μL. The control consisted of 50 μL ampicillin ((x), (y) and (z)).
143
effects of 50 (d), 25 (e) and 10 μL (f) of cow urine, which also produced
no inhibitory effects. Conversely, ampicillin produced an average zone
of inhibition of 17 mm (y). As shown in Fig. 2C, cow urine also had no
effects against A. niger, which grew across all portions of all plates,
except for the section containing 50 μL of ampicillin (z).
10 μL
3.2. Characterization of encapsulated cow urine in cellulose nanoparticles
3.2.1. Light and AFM microscopy images
Different images were captured on the CNPs encapsulated cow
urine. Fig. 3a & b represents the digital and light microscopy images of
the CNPs, show the intactness of the particles. Further, atomic force
microscopy was conducted to observe the morphology and roughness
of the samples. As shown in Fig. 3c, there were many particles present,
indicating the presence of cellulose nanoparticles in the samples [18].
It can be observed further that the cow urine has been encapsulated in
CNPs, and the sizes of nanoparticles formed were uniform. The spherical
nanoparticles had a diameter of approximately 50 nm, but the agglom-
erated particles were approximately 100 nm diameter. In this analysis,
the scanned samples yielded a root mean square (RMS) of 13.402 nm
to a maximum peak of 120.59 nm. The existence of the peaks was
mainly due to the degradation of lignin. Specifically, lignin in the wall
of cellulose is dissolved by acid or alkali solution, increasing the surface
of cellulose and resulting in a rough surface of the composite. Although
the water was used to dissolve the encapsulated cow urine, NaOH was
present during the initial phases of encapsulation, which led to the
rough surface.
3.2.2. Field emission scanning electron microscopy (FESEM)
The encapsulated cow urine sample was also observed under SEM to
ensure the uniformity of the particle size (Fig. 4). The size of the particle
was difficult to determine because of the strong bonds of the CNPs
agglomerate; however, the average was approximately 100 nm. Energy
Dispersive X-ray (EDX) analysis revealed that the sodium (Na) compo-
sition was discovered the most in the scanned sample at 1 keV energy.
It has been reported that Na is one of the major constituents of cow
urine [19]. Moreover, NaOH was used during the encapsulation of the
cow urine, which likely increased the amount of Na in the urine.
Therefore, the notable peak in the spectrum supplemental ratified that
the component coated with CNPs in actuality is cow urine.
3.2.3. Transmission electron microscopy (TEM)
Fig. 5 shows the TEM micrographs of the cow urine encapsulated in
CNPs. As shown in Fig. 5(a), the encapsulated cow urine was spherical,
144 K.H. Suk et al. / Powder Technology 328 (2018) 140–147
Fig. 3. Images of CNPs encapsulated cow urine. (a) Digital image; (b) light microscopy image; (c) atomic force microscopy image.
as expected. Moreover, the sample was found to have a rough surface,
confirming the results of AFM analysis above. These characteristics en-
abled agglomeration of the CNPs. Nevertheless, particle irregularity
has been reported to have a positive impact on emulsion stability [20].
The cow urine was encapsulated with CNPs using the microemulsion
technique; therefore, the prepared sample was able to resist changes
in its physicochemical properties with time. The cow urine was proven
additionally, to be encapsulated with CNPs when the size of it was
measured to be approximately about 30 to 40 nm, as in Fig. 5(b).
Further analysis by selected area electron diffraction (SAED) revealed
that the encapsulated cow urine had an amorphous nature (Fig. 5(c)).
The results presented herein will be useful to determining proper
materials for use in future applications. As shown in Fig. 5(d), the
energy-dispersive X-ray (EDX) spectrum indicated a high amount
of calcium (Ca) inside the sample, which is in accordance with the
results of a previous study that showed one of the major constituents
of cow urine is Ca [19]. Thus, the EDX spectrum indicated that the Ca
component present was actually cow urine that had been encapsulated
with CNPs.
3.2.4. X-ray photoelectron spectroscopy (XPS)
This analysis was conducted to determine the chemical characteris-
tics of encapsulated cow urine nanoparticle surfaces (Fig. 6). As shown
in Fig. 6 (a), the Si2p region revealed an obvious sharp peak at 104.26
eV, indicating the presence of silicon dioxide (SiO2) close to 103.5 eV.
These findings are reasonable given that SiO2 was used as the substrate
during analysis. Upon Cl2p analysis, Kr was found at 202.4 eV. As shown
in Fig. 6(b), the highest peak was at 200.51 eV, which was enough to
conclude that Kr is present at the peak. The highest K2p (c) peak was
found at 293.75 eV, which was very close to that of potassium bromide
(KBr) at 293.2 eV. Moreover, Na1s analysis (d) revealed that the highest
peak was at 1071.6 eV, which was close to sodium (Na) located
at 1071.5 eV. As shown in Fig. 6 (e), the highest peak value was
encountered at 533.11 eV.
These findings indicate that the organic carbonyl group (C_O) is
present because of its proximity to 533 eV. Moreover, nitridation
silicon oxide (NSi2O) was also found based on the detection of a peak
at 399.34 eV (f), which is close to the value of 399.9 eV. For C1s (g),
the highest peak was observed at 285.08 eV, which is similar to that of
a carbon carbon bond at 284.8 eV. Taken together, these findings
indicate that the sample has a single carbon bond composed of two
electrons, whereas the bond is actually a sigma bond formed between
one hybridized orbital from each of the carbon atoms that are present.
As shown in Fig. 6(h), O1s was found to be the most saturated in the
overall analysis. Indeed, the sample mostly contains organic carbonyl
groups with aldehydes and ketones as they are present in cow urine.
3.2.5. X-ray diffraction analysis
As stated elsewhere, XRD study was performed to reveal the crystal-
line nature of the nanoparticle. The XRD profile of the encapsulated cow
urine is presented in Fig. 7. The crystallinity of a composite is usually
determined by the highest peak, which indicates the strongest diffrac-
tion peak. According to a previous study, CNPs intensify the peak at
2θ = 22.5° [21]. On the attached Fig. 7, the most intense peak was
observed at 2θ = 32.14° whereby the composite was encapsulated
cow urine using CNPs. Shifting peaks occur commonly because of
changes in chemical compositions and temperature. The results of the
present study reflect that the chemical compositions had been altered
during the cow urine encapsulation process. Moreover, there were var-
ious temperature fluctuations during the encapsulation process, which
changed the lattice parameters of the CNPs, resulting in shifting peaks.
Additionally, the peak shifted to a higher diffraction angle, indicating
that the cellulose crystal lattice had been compressed. The 2θ angle
shows an increased pattern, further substantiating that the cow urine
had been successfully encapsulated with CNPs and indicating that this
occurred in compression instead of expansion mode. Overall, the
highest peak intensity was observed at 830 a.u., whereby encapsulated
cow urine compounds can be detected at that particular point. At high-
temperature usually cellulose may tend to be amorphous, in our case
we do not use any higher temperature. In addition to the state of
semi-crystalline, cellulose polymer may remain un-crystalline state
ultimately will be trapped among the growing crystals, that was not
happened in our study.
3.2.6. Fourier transform infrared spectroscopy
FTIR analysis was conducted on cow urine alone, encapsulated
cellulose nanoparticles (CNPs) and encapsulated cow urine to deter-
mine the infrared spectrum of the absorption of the samples in liquid
form. As shown in Fig. 8, the cow urine produced two noticeable
Fig. 4. FESEM analysis. EDS spectrum of encapsulated cow urine is displayed. FESEM
microphotograph of encapsulated cow urine is shown as an inset. Scale bar is indicated
the size 50 μm.
 K.H. Suk et al. / Powder Technology 328 (2018) 140–147
Fig. 5. TEM analysis. (a) 100 nm scale image of encapsulated cow urine. (b) Magnification
of encapsulated cow urine, scale bar indicates 20 nm. (c) SAED pattern of amorphous
structure of encapsulated cow urine. (d) EDS spectrum of encapsulated cow urine.
peaks at about 100.2 and 99.3% T. The samples produced a sinusoidal
wave-like pattern and possibly most of the data generated will be
following the same trend between 655.5 cm−1 to 660 cm−1 and
662.5 cm−1 to 672.5 cm−1. In the case of CNPs, no significant peaks
were observed. However, a broad valley was seen between 660 cm−1
and 665 cm−1 at approximately 98.7% T. For the encapsulated cow
urine, a small broad peak was seen at 99.9% T, in the range of 655 cm−1
and 662.5 cm−1. Comparison of the three graphs confirmed that the
cow urine had been encapsulated. The initial two peaks of cow urine
were combined into one broad peak at 660 cm−1. The highest peak,
reflecting encapsulated cow urine, was present at 99.9% T between the
first peak at 100.2% T and the next peak at 99.3% T. Further, the initial
Fig. 6. XPS high-resolution spectral analysis of encapsulated cow urine. The corresponding graphs are: (a) Si2p, (b) Cl2p, (c) K2p, (d) Na1s, (e) O1s, (f) N1s, (g) C1s and (h) overall survey
scan of chemical components.
145
Fig. 7. XRD pattern analysis of cow urine encapsulated within cellulose nanoparticles.
The position of the highest peak is indicated.
660 cm−1 came directly from the encapsulated CNPs, indicating that
the cow urine had been encapsulated with CNPs. However, the encapsu-
lated cow urine peak had smaller amplitude when compared to any two
peaks in the cow urine sample. The small amplitude indicated that the
encapsulated cow urine is in a stabilized resonance form; thus, the drug
content can be released in a controlled manner.
3.3. Biological activity of encapsulated cow urine
3.3.1. Disc diffusion assay
In this assay, cow urine encapsulated with cellulose nanoparticles
produced better results than cow urine alone (Table 2). Evaluation of
the effects against B. subtilis (Fig. 9A) revealed that, at 50 μL, encapsulat-
ed cow urine produced an average inhibition zone of 11 mm (a), which
was about 3 mm larger than that produced by the non-encapsulated
146 K.H. Suk et al. / Powder Technology 328 (2018) 140–147

Fig. 8. FTIR analysis of (a) cow urine only, (b) cellulose nanoparticles and (c) cow urine encapsulated in cellulose nanoparticles.

cow urine. Positive results were also observed for 25 μL and 10 μL of
encapsulated cow urine, which produced average inhibition zones of
10 mm (b) and 9 mm (c), respectively. Conversely, 10 μL of non-
encapsulated cow urine produced no visible inhibitory effect.
Additionally, 25 μL of encapsulated cow urine produced a zone of
inhibition 3.15 mm larger than that of non-encapsulated urine. The
average inhibition zone for the ampicillin control group was found
to be 15 mm (x) at 50 μL (1 mg/mL). Taken together, these results
indicate that encapsulated cow urine is much more effective when
applied to treat B. subtilis than cow urine alone, and that the effects
of the encapsulated urine are comparable to those of ampicillin.
Additional experiments were conducted to investigate the efficacy
against E. coli (Fig. 9B). Encapsulated cow urine produced no zone of
inhibition at 10 μL (e) and 25 μL (f), whereas a slight inhibition zone
was observed in response to 50 μL (d). The inhibition zone can be seen
quite obvious, but the clear zone was not as limpid enough as the con-
trol group. The average inhibition zone (for the 50 μL) was 17 mm,
which was approximately the same as that of the ampicillin control
group (y). Additionally, E. coli found in the intestine can produce
vitamin K2, helping to prevent invasion by pathogenic bacteria [22].
Therefore, the absence of a zone of inhibition indicates that cow urine
could exert a beneficial effect by not causing a harmful effect against
E. coli present in the intestine. To conclude, the slight inhibition zone
shown on the encapsulated cow urine using 50 μL prove that the effec-
tiveness of cow urine has been improvised minimally, and comparable
with ampicillin.
The effects of encapsulated cow urine on A. niger were also investi-
gated using the same amounts as for the other assays (Fig. 9C).
Treatment with 10 μL (h) and 25 μL (i) had no effect on A. niger,
and the organism was even able to grow over the infused zones.
Notwithstanding that it was a negative results, the fungus was managed
to be controlled unlike using cow urine only where the fungus grew
everywhere on the agar plate. However, growth was prevented by
treatment with 50 μL (g) of encapsulated urine. In comparison to the
cow urine only, encapsulated cow urine showed outstanding finding
which is about the same level with using ampicillin only. Comparison
of the levels of inhibition revealed that ampicillin exerted a somewhat
preferable result, but these differences were minor.
The application of cow urine in the medical field is welcomed by
researchers due to its therapeutic properties. The cow urine has been
proved its importance to treat different diseases and prevention.
It was proved that cow urine increases the secretion of phagocytic

Fig. 9. Disc diffusion method to test the inhibitory effects against (A) B. subtilis, (B) E. coli and (C) A. niger using encapsulated cow urine where (a), (d) and (g) are 50 μL; (b), (e) and (h) are
25 μL and (c), (f) and (i) are 10 μL. The control consisted of 50 μL ampicillin [(x), (y) and (z)].
 K.H. Suk et al. / Powder Technology 328 (2018) 140–147
activity, interleukin-1 and 2, which helps to control and prevent the dis-
eases. Moreover, the carbolic acid, phenols, magnesium, calcium in the
cow urine yields the strong antioxidant property. The plant-based origin
of cellulose nanoparticle is renewable, abundant, sustainable and nature
polymer, can be used for the encapsulation of cow urine. It makes the
encapsulated CNPs to become a potential use in the field of medicine
in particular with the drug release. The strategy of encapsulation
makes the delivery advantages for toxic, fragile and poorly soluble
compounds, and also possible to minimize the side effects. With these
advantages encapsulated CNPs can go for the potential antimicrobial
applications in medical field for the efficient production of implants,
face masks, bandages, skin replacements, artificial blood vessels,
supporting materials for enzyme immunization and nanoparticles
[23–30]. The cellulose obtained from other natural sources [31–33],
may use to imitate the present study and enhance the potential effect
of the encapsulated cow urine. Similarly, nanomaterials demonstrated
recently have potential to carry the biomolecules; can make use of
with cow urine [34–36].
4. Conclusion
In this study, the efficacy of cow urine encapsulated with cellulose
nanoparticles (CNPs) against pathogens was investigated. Cow urine
was encapsulated by the microemulsion method, then tested against
microbial growth by following the preliminary tests of cow urine with
disc diffusion and turbidimetric assays. Moreover, characterization of
the encapsulated cow urine by various types of microscopy and diffrac-
tion assays confirmed that encapsulation had occurred. On the B. subtilis
agar plate, both cow urine and encapsulated cow urine showed excel-
lent results based on the zones of inhibition. Conversely, cow urine
alone had no effect on the growth of E. coli, whereas the encapsulated
cow urine showed only a minor effect at the highest amount used.
However, the inability to prevent the growth of E. coli could be a positive
attribute because this particular microorganism is commonly found
in human intestines, where it produces vitamin B and K, thereby
preventing invasion by other harmful bacteria. Both cow urine and
encapsulated cow urine had positive results against A. niger, but encap-
sulated cow urine had much greater efficacy, and 50 μL of encapsulated
cow urine led to significant improvement. Further studies should be
conducted using different microorganisms and types of cellulose
for the cow urine loading to obtain greater insight into this potential
treatment modality.
Acknowledgement
Periasamy Anbu thanks Inha University for providing a research
grant for language editing support.
Conflict of interest
Authors have no conflict of interest.
References
[1] S. Fulendra, M.S. Kumar, N. Mahadevan, Nutraceuticals: uplift in health, Int. J. Recent
Adv. Pharma. Res. 2 (2012) 17–28.
[2] S. Raad, D. Deshmukh, S.N. Harke, M.S. Kachole, Antibacterial activity of cow urine
against some pathogenic and non-pathogenic bacteria, 4 (2013) 1534–1539.
[3] J.S. Sanganal, K. Jayakumar, G.M. Jayaramu, V.P. Tikare, K.L. Paniraj, R. Swetha, Effect
of cow urine on wound healing property in Wistar albino rats, Vet. World 4 (2011)
317–321, https://doi.org/10.5455/vetworld.4.317.
[4] I. Mohanty, M.R. Senapati, D. Jena, S. Palai, Diversified uses of cow urine, Int, J.
Pharm. Sci. 6 (2014) 20–22.
[5] T.F. Meyabadi, F. Dadashian, G.M.M. Sadeghi, H.E. Zanjani Asl, Spherical cellulose
nanoparticles preparation from waste cotton using a green method, Powder
Technol. 261 (2014) 232–240, https://doi.org/10.1016/j.powtec.2014.04.039.
[6] R.J. Moon, A. Martini, J. Nairn, J. Simonsen, J. Youngblood, Cellulose nanomaterials
review: structure, properties and nanocomposites, 40 (2011) 3941–3994,
https://doi.org/10.1039/c0cs00108b.
147
[7] M.C. Li, Q. Wu, K. Song, S. Lee, Q. Yan, Y. Wu, Cellulose Nanoparticles:
Structure-Morphology-Rheology Relationship, School of Renewable Natural
Resources, Louisiana State University AgCenter, Baton Rouge, Department of
Forest Products, Korea Forest Research Institute, Seoul, 2015 130–712 (K).
[8] L. Zhou, H. He, M.C. Li, K. Song, H.N. Cheng, Q. Wu, Morphological influence of
cellulose nanoparticles (CNs) from cottonseed hulls on rheological properties of
polyvinyl alcohol/CN suspensions, Carbohydr. Polym. 153 (2016) 445–454,
https://doi.org/10.1016/j.carbpol.2016.07.119.
[9] V. Nedovic, A. Kalusevic, V. Manojlovic, S. Levic, B. Bugarski, An overview of
encapsulation technologies for food applications, Procedia Food Sci. 1 (2011)
1806–1815, https://doi.org/10.1016/j.profoo.2011.09.265.
[10] S.H.A.M. Leenders, R. Becker, T. Kumpulainen, B. de Bruin, T. Sawada, T. Kato, et al.,
Selective co-encapsulation inside an M 6 L 4 cage, Chem. Eur. J. (2016) 1–8,
https://doi.org/10.1002/chem.201603017.
[11] S. Vrignaud, J.P. Benoit, P. Saulnier, Strategies for the nanoencapsulation of
hydrophilic molecules in polymer-based nanoparticles, Biomaterials 32 (2011)
8593–8604, https://doi.org/10.1016/j.biomaterials.2011.07.057.
[12] X. Li, Q. Zhao, L. Qiu, Smart ligand: aptamer-mediated targeted delivery of
chemotherapeutic drugs and siRNA for cancer therapy, J. Control. Release 171
(2013) 152–162, https://doi.org/10.1016/j.jconrel.2013.06.006.
[13] N. Jain, V. Gupta, R. Garg, N. Silawat, Efficacy of cow urine therapy on various cancer
patients in Mandsaur District, India - a survey, Int. J. Green Pharm. 4 (2010) 29,
https://doi.org/10.4103/0973-8258.62163.
[14] S. Labbaf, H. Horsley, M. Chang, E. Stride, J. Malone-lee, M. Edirisinghe, et al.,
An encapsulated drug delivery system for recalcitrant urinary tract infection, J. R.
Soc. Interface 10 (2013) 1–5.
[15] M. Mohan Yallapu, M. Ray Dobberpuhl, D. Michele Maher, M. Jaggi, S. Chand Chauhan,
Design of curcumin loaded cellulose nanoparticles for prostate cancer, Curr. Drug
Metab. 13 (2012) 120–128, https://doi.org/10.2174/138920012798356952.
[16] G. Maria, F. Calixto, J. Bernegossi, L.M. De Freitas, C.R. Fontana, M. Chorilli,
Nanotechnology-based drug delivery systems for photodynamic therapy of cancer
: a review, Molecules 21 (2016) 1–18, https://doi.org/10.3390/molecules21030342.
[17] N. Salgado, Analytical Methods, Development and validation of a microbiological
assay by turbidimetry to determine the potency of cefazolin sodium in the
lyophilized powder form, Anal. Methods (2014) 1391–1396, https://doi.org/10.
1039/c3ay41483c.
[18] S.C.B. Gopinath, C.S. Goh, M. Citartan, T. Lakshmipriya, M.K. Arshad, M.F. Fatin, et al.,
Micro-encapsulation of antibiotic in cellulose nanoparticle inhibits bacteria,
Micro Nanosyst. (2016) 1–6, https://doi.org/10.2174/18764029086661607201040.
[19] W.H.M. Saunders, Effects of cow urine and its major constituents on pasture
properties, N. Z. J. Agric. Res. 25 (1982) 61–68, https://doi.org/10.1080/00288233.
1982.10423373.
[20] S. Lam, K.P. Velikov, O.D. Velev, Pickering stabilization of foams and emulsions with
particles of biological origin, Curr. Opin. Colloid Interface Sci. 19 (2014) 490–500,
https://doi.org/10.1016/j.cocis.2014.07.003.
[21] G. Han, S. Huan, J. Han, Z. Zhang, Q. Wu, Effect of acid hydrolysis conditions on the
properties of cellulose nanoparticle-reinforced polymethylmethacrylate composites,
Materials (Basel). 7 (2014) 16–29, https://doi.org/10.3390/ma7010016.
[22] T. Li, F. Pu, R. Yang, X. Fang, J. Wang, Y. Guo, et al., Draft genome sequence of
Escherichia coli LCT-EC106, J. Bacteriol. 194 (2012) 4443–4444, https://doi.org/10.
1128/JB.00853-12.
[23] L. Fu, J. Zhang, G. Yang, Present status and applications of bacterial cellulose-based
materials for skin tissue repair, Carbohydr. Polym. 92 (2013) 1432–1442.
[24] Y. Zhang, T. Nypelö, C. Salas, J. Arboleda, I.C. Hoeger, O.J. Rojas, Cellulose nanofibrils:
from strong materials to bioactive surfaces, J. Renew. Mater. 1 (2013) 195–211.
[25] S. Dong, H.J. Cho, Y.W. Lee, M. Roman, Synthesis and cellular uptake of folic acid-
conjugated cellulose nanocrystals for cancer targeting, Biomacromolecules 15
(2014) 1560–1567.
[26] R. Kolakovic, T. Laaksonen, L. Peltonen, A. Laukkanen, J. Hirvonen, Spray-dried
nanofibrillar cellulose microparticles for sustained drug release, Int. J. Pharm. 430
(2012) 47–55.
[27] M. Jorfi, J. Foser, Recent advances in nanocellulose for biomedical applications,
J. Appl. Polym. Sci. 132 (2015) 1–19.
[28] J. Rojas, M. Bedoya, Y. Ciro, Current trends in the production of cellulose nanoparti-
cles and nanocomposites for biomedical applications, InTech Open Science/Open
Mind 2015, pp. 193–228.
[29] Y. Wang, P.B. Groszewicz, S. Rosenfeldt, H. Schmidt, C.A. Volkert, P. Vana, T.
Gutmann, G. Buntkowsky, K. Zhang, Thermoreversible self-assembly of perfluorinated
core-coronas cellulose-nanoparticles in dry state, Adv. Mater. 29 (2017), https://doi.
org/10.1002/adma.201702473.
[30] H.Q. Wang, H. Tan, S. Hua, Z.Y. Liu, W. Yang, M.B. Yang, High efficiency conversion of
regenerated cellulose hydrogel directly to functionalized cellulose nanoparticles,
Macromol. Rapid Commun. (2017), https://doi.org/10.1002/marc.201700409.
[31] T. Theivasanthi, F.L. Anne Christma, A.J. Toyin, S.C.B. Gopinath, R. Ravichandran,
Synthesis and characterization of cotton fiber-based nanocellulose, Int. J. Biol.
Macromol. (2018), https://doi.org/10.1016/j.ijbiomac.2017.11.054 (in press).
[32] K.H. Suk, S.C.B. Gopinath, Drug encapsulated nanoparticles for treating targeted
cells, Curr. Med. Chem. 24 (2017) 3310–3321.
[33] N.S. Ismail, S.C.B. Gopinath, Enhanced antibacterial effect by antibiotic-loaded starch
nanoparticle, J. Assoc. Arab Univ. Basic Appl. Sci. 24 (2017) 136–140.
[34] S. Anniebell, S.C.B. Gopinath, Polymer conjugated gold nanoparticles in biomedical
applications, Curr. Med. Chem. 25 (2018) 1–13.
[35] M.E. Foo, S.C.B. Gopinath, Feasibility of graphene in biomedical applications, Biomed
Pharmacother 94 (2017) 354–361.
[36] S. Ramanathan, S.C.B. Gopinath, Potentials in synthesizing nanostructured silver
nanoparticles, Microsyst. Technol. 23 (2017) 4345–4357.