You are on page 1of 11

This article was downloaded by: [Colorado College]

On: 06 April 2015, At: 15:53


Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered
office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Journal of Experimental Nanoscience


Publication details, including instructions for authors and
subscription information:
http://www.tandfonline.com/loi/tjen20

Formulation and characterisation of


nanosuspension of herbal extracts for
enhanced antiradical potential
a

Nazish Jahan , Saba Aslam , Khalil ur Rahman , Tuba Fazal ,


a

Fareeha Anwar & Rubab Saher


a

Department of Chemistry, University of Agriculture, Faisalabad,


Pakistan
b

Department of Biochemistry, University of Agriculture,


Faisalabad, Pakistan
Published online: 31 Mar 2015.

Click for updates


To cite this article: Nazish Jahan, Saba Aslam, Khalil ur Rahman, Tuba Fazal, Fareeha
Anwar & Rubab Saher (2015): Formulation and characterisation of nanosuspension of herbal
extracts for enhanced antiradical potential, Journal of Experimental Nanoscience, DOI:
10.1080/17458080.2015.1025303
To link to this article: http://dx.doi.org/10.1080/17458080.2015.1025303

PLEASE SCROLL DOWN FOR ARTICLE


Taylor & Francis makes every effort to ensure the accuracy of all the information (the
Content) contained in the publications on our platform. However, Taylor & Francis,
our agents, and our licensors make no representations or warranties whatsoever as to
the accuracy, completeness, or suitability for any purpose of the Content. Any opinions
and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content
should not be relied upon and should be independently verified with primary sources
of information. Taylor and Francis shall not be liable for any losses, actions, claims,
proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or
howsoever caused arising directly or indirectly in connection with, in relation to or arising
out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any
substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &

Downloaded by [Colorado College] at 15:53 06 April 2015

Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions

Journal of Experimental Nanoscience, 2015


http://dx.doi.org/10.1080/17458080.2015.1025303

Formulation and characterisation of nanosuspension of herbal extracts


for enhanced antiradical potential
Nazish Jahana*, Saba Aslama, Khalil ur Rahmanb, Tuba Fazala, Fareeha Anwara
and Rubab Sahera
a
Department of Chemistry, University of Agriculture, Faisalabad, Pakistan; bDepartment of
Biochemistry, University of Agriculture, Faisalabad, Pakistan

Downloaded by [Colorado College] at 15:53 06 April 2015

(Received 16 January 2015; final version received 26 February 2015)


Nanotechnology is a promising technique to increase the bioavailability of herbal
medicines. This paper presents the nanosuspension approach for increasing the
aqueous solubility and thereby bioactivity of important herbal extracts.
Nanosuspensions of the seeds of three plants extract (Silybum marianum,
Elettaria cardamomum and Coriandrum sativum) were prepared by using
polyvinyl alcohol (1.5% w/v) as a stabiliser. Prepared nanoparticles were
characterised by scanning electron microscope. Activity of nanosuspension
formulation was assessed by using four in vitro antioxidant assays. S. marianum,
E. cardamomum and C. sativum particle size was observed to fall in range of
446.1 112.6, 456.63 339.2 and 432.1 172.8 nm, respectively, most of the
particles were having spherical shape and smooth topology. These synthesised
nanoparticles were found to be more effective against quenching free radical than
their crude extracts and standards [butylated hydroxyl toluene and ascorbic acid].
C. sativum nanosuspension showed most free radical scavenging potential against
2,2-diphenyl-1-picrylhdrazyl (DPPH) and superoxide free radical scavenging
assays (IC50 0.59 0.01 and 0.81 0.11 mg/ml). S. marianum nanosuspension
was found to be most effective against DPPH radicals scavenging (IC50 0.34
0.02 mg/ml). It was concluded that nanosuspension of herbal medicines
potentiates the antioxidant potential.
Keywords: nanoparticles; plant extracts; scanning electron microscopy

1. Introduction
Nanotechnology is a promising approach of twenty-first century. It is an attractive area of
research related to production of nanoparticles of variable size and shape, in addition to
their potential advantages in clinical medicines.[1] It is on doorstep of providing a host of
new objects and approaches in modernising therapeutic and pharmaceutical field. In
present era, it significantly promotes the formation of biological medicine and
bioavailability enhancement of phytomedicines. Nanonisation of herbal drugs is opening
the new epoch of herbal drug discovery.[2]
Herbal medicines are extensively used all over the world since ages. The efficacy of
several species of therapeutic plants relies on the release of biologically active compounds.
The majority of the active components of extracts are incapable to pass the lipid
*Corresponding author. Email: nazishjahanuaf@yahoo.com
2015 Taylor & Francis

Downloaded by [Colorado College] at 15:53 06 April 2015

N. Jahan et al.

membranes of the cells because either they have markedly high molecular size or poor
water solubility, thereby suffers from low absorption and poor bioavailability.[3] Many
phytomedicines, thereby due to their poor absorption, exhibit least or no considerable
in vivo activity despite of their amazing in vitro potential. Due to these obstacles, some
extracts are not used clinically. It has been extensively suggested to incorporate herbal
drugs with nanotechnology, as nanostructured systems might be capable to strengthen the
action of herbal extracts decreasing the necessary dosage and side effects, and improving
bioactivity.[4] Numerous nano-oriented approaches are being projected with the aim of
optimising the technological features of drugs.[5,6] Nanosuspension technology is
introduced as a result of countless efforts made by formulation scientists and has been
evolved as a potent candidate for the delivery of the poorly soluble drugs in a more
efficient and pronounced manner.[7] Because by considering the limitations of previously
used technologies of particle size reduction, pharmaceutical scientists have been waiting
for such a universal approach that would be able to address the formulation-related
problems and barriers of poorly soluble drugs up to their desired level.[8,9]
Silybum marianum commonly known as milk thistle is a member of Asteraceae family
and genus Silybum. Silymarin is the major bioactive compound isolated from its seed
which has innumerable applications. Silymarin is employed for the oral therapy of chronic
liver disorder but it has poor aqueous solubility thereby poor bioavailability.
Pharmacokinetic studies have revealed that after oral administration just 23% 47% of
silymarin enters the systemic circulation.[10] Elettaria cardamomum generally known as
cardamom has well-known culinary value. Seeds of this plant are used in folk medicines
for the treatment of cardiac and gastrointestinal disorders.[11] Coriandrum sativum
commonly known as coriander has various pharmacological properties. Coriander is used
as anti-inflammatory, antihypertensive, antiedemic, antiseptic, antidiabetic, lipolytic and
myorelaxant.[12] This project had been designed to prepare the nanosuspension of three
poor aqueous soluble plants extract to enhance their bioactivities. Nanosuspensions of
S. marianum, E. cardamomum and C. sativum were prepared by nanoprecipitation method.
Prepared nanosuspensions were characterised for their particle size and morphology by
using scanning electron microscopy (SEM).

2. Experimental section
2.1. Chemicals and reagents
Polyvinyl alcohol (PVA), 2,2-diphenyl-1-picrylhdrazyl (DPPH), ascorbic acid, butylated
hydroxyl toluene (BHT), nitroblue tetrazolium regent, hydroxylamine hydrochloride,
ammonium thiocyanate and linoleic acid were purchased commercially from SigmaAldrich (St. Louis, MO, USA). All other chemicals were of analytical grade of standard
Merck (Darmstadt, Germany).

2.2. Collection of plant material


The seeds of S. marianum, E. cardamomum and C. sativum were collected from an
authentic medical store.

Journal of Experimental Nanoscience

2.3. Preparation of plants extract


Seeds of S. marianum, E. cardamomum and C. sativum were grinded into powder form.
Powder seeds (30 g) were extracted with n-hexane (300 ml) in Soxhlet apparatus for 6 h to
remove fatty substances. The marc was further extracted with ethanol (95%) for 6 h in
Soxhlet apparatus. The extract was concentrated on rotary evaporator (Rotavapor R-II
Buchi, Switzerland).[13]

Downloaded by [Colorado College] at 15:53 06 April 2015

2.4. Formulation of nanosuspension


Nano-precipitation method was followed with a slight modification for the preparation of
nanosuspensions. Plant extract (2.5 g) was dissolved in 15 ml of acetone and ethanol (3:1)
by sonication for 60 seconds. The resultant solution was then gradually injected
(1 ml min1) with a syringe connected to a thin teflon tube, into 25 ml water containing
PVA 1.5% w/v with continuous magnetic stirring at 1000 rpm. The resulting emulsion
obtained was then diluted in 50 ml PVA solution (0.2% w/v in water) in order to minimise
coalescence and the mixture was continuously stirred (500 rpm) for 6 h at room
temperature to allow solvent evaporation and nanoparticle formation. The resultant
nanosuspension was consequently cooled down to 18  C and lyophilised using
lyophiliser to obtain dry powder.[14]

2.5. Particle size analysis and morphological characterisation


Particle size and morphological features of nanosuspension were studied by using SEM at
varied magnification. Nanosuspensions powder was coated in a sputter coater. After that,
the images were taken on SEM and the scale bar was calibrated accurately.

2.6. Antioxidant activities


Antioxidant activities of plants crude extract and its nanosuspension were evaluated by
using different assays and compared with antioxidant activity of standard compounds,
BHT and ascorbic acid [15]:
(1)
(2)
(3)
(4)

DPPH scavenging assay,


superoxide radical scavenging assay,
nitric oxide radical scavenging assay and
ferric reducing antioxidant power assay.

Different concentrations (0.02 0.1 mg/ml) of plant crude extracts, nanosuspensions


and standard compounds were prepared and assessed for DPPH, nitric oxide and
superoxide free radical scavenging potential spectrophotometrically. Decrease in
absorbance with increase in concentration reflected high quenching potential of plant.
Results were reported in term of inhibitory concentration 50 (IC50) value. Antioxidant
potential against lipid peroxidation was evaluated in linoleic acid system by ferric reducing
antioxidant power assay after different time intervals and results were reported in terms of
percentage inhibition of lipid peroxidation.

N. Jahan et al.

3. Results and discussion

Downloaded by [Colorado College] at 15:53 06 April 2015

3.1. Particle size analysis and morphological characterisation


Nanosuspensions of three plant extracts were prepared by nanoprecipitation method to
increase bioactivities as they were associated with slow absorption leading to insufficient
and inconsistent bioavailability. The particle size distribution is the principal factor
influencing the in vivo fate of many herbal extract. These prepared nanosuspensions were
characterised for its particle size and morphological features which were observed with
SEM (Figures 1 3). Their SEM images revealed the particle size of suspension within the
accepted range of nanoparticles (10 1000 nm). Mean particle size of the S. marianum,
E. cardamomum and C. sativum nanosuspenion was found to be 446.1 112.6 nm
(Figure 1), 456.63 339.2 nm (Figure 2) and 432.1 172.8 nm (Figure 3), respectively.
The entire suspension of S. marianum, E. cardamomum and C. sativum had the particle size
range 287 614.7, 93.5 775.1 and 309.8 554.4 nm, correspondingly. Nanonisation of
herbal extracts can lead towards increase in dissolution velocity, wetting, particle surface
area and saturation solubility. This may in turn bring about more bioavailability due to
enhanced in vivo release as only solubilised particles can be absorbed through lipophilic
cellular membranes.[16] These nanostructured systems might be able to potentiate
required action of herbal extracts, reducing the necessary dosage, side effects and
improved activity. Nanosystems can distribute the bioactive component at adequate
concentration throughout the whole treatment period, directing it to the preferred site of
action. Traditional medications do not fulfil these requirements. Thereby, nanonisation of
plant extracts could be a valuable aid to increase the efficiency of plant extract and
minimise their side effects.[17] Scanning electron images of surface morphology of
prepared nanosuspension revealed their smooth texture. Their pictures showed that most
of the particles were having spherical shape and smooth topology.

3.2. Determination of antioxidant potential


Four different antioxidant assays were used to determine the antioxidant activity of plants
formulation. Results of three assays (DPPH, superoxide and nitric oxide) were reported in

Figure 1. SEM image of particle size (a) and morphology (b) of Silybum marianum nanosuspension.

Downloaded by [Colorado College] at 15:53 06 April 2015

Journal of Experimental Nanoscience

Figure 2. SEM image of particle size (a), (b), (c), (d) and morphology (e) of Elettaria cardamomum
nanosuspension.

Downloaded by [Colorado College] at 15:53 06 April 2015

N. Jahan et al.

Figure 3. SEM image of particle size (a) and morphology (b) of Coriandrum sativum
nanosuspension.

terms of IC50 value (Table 1) that indicates the concentration of antioxidant required to
neutralise 50% free radicals. Lower value of IC50 thereby indicates high inhibitory effect.
DPPH scavenging activity is a well-known method to determine the antioxidant potential
of medicinal plants in short time period.[18] Among plants extract, lowest IC50 value for
DPPH free radical scavenging assay was shown by E. cardamomum (5.21 0.1 mg/ml).
Nanosuspension of C. sativum (0.59 0.01 mg/ml) exhibited minimum IC50 value. Nitric
oxide radicals overproduction is also said to be related to different types of
neurodegenerative and muscles diseases. Nitric oxide radical scavenging potential of
different plants and their nanosuspension was determined by the generation of nitric
oxides radical in vitro by sodium nitroprusside. These free radical lead to the production of
nitrite ions by reacting with oxygen. Nitric oxide scavengers compete with oxygen and
thereby diminish the formation of nitrites ions.[19] S. marianum nanosuspension showed
Table 1. IC50 value of different plants formulations and standards.
IC50 value (mg/ml)
Plants formulation
and standards

DPPH radical
scavenging

Nitric oxide
radical scavenging

Superoxide
radical scavenging

Silybum marianum extract


Silybum marianum nanosuspension
Elettaria cardamomum extract
Elettaria cardamomum nanosuspension
Coriandrum sativum extract
Coriandrum sativum nanosuspension
BHT
Ascorbic acid

13.62 1.02
1.95 0.09
5.21 0.1
1.74 0.07
16.51 1.22
0.59 0.01
2.43 0.2
3.49 0.8

7.73 0.11
0.34 0.02
7.99 0.1
2.14 0.4
5.63 0.23
1.32 0.03
1.22 0.01
2.69 0.2

8.15 0.4
1.19 0.31
4.0 0.1
1.63 0.08
4.81 0.22
0.81 0.11
2.6 0.07
2.02 0.11

Note: Data are mean (n D 3) SD.

Downloaded by [Colorado College] at 15:53 06 April 2015

Journal of Experimental Nanoscience

lowest IC50 value (0.34 0.02 mg/ml) for this assay. Superoxide radical is regarded as a
powerful biological cause of reactive oxygen species because it leads to formation of
potent and hazardous hydroxyl radicals.[20] For this assay, lowest IC50 value was also
shown by C. sativum nanosuspension (0.81 0.11 mg/ml). Result depicted in Table 1
clearly reflected that nanosuspensions of plants showed more scavenging of free radicals
than crude plant extracts. Nanosuspensions of plants even show better results than
standard compounds BHT and ascorbic acid because when particle size is reduced up to
nano range, not only surface area but concentration gradient is also increased which
results in dramatic increase of the dissolution velocity as compared to a micronised
product and activity of extracts was improved by formulating their nanoparticles.[21]
Moreover, the use of surfactant and continuous stirring helped the nanosuspension
particles to avoid agglomeration, thus avoid the Ostwalds ripening [22] and led to
enhanced activity of drug. In the earlier work, same relation of particle size reduction and
drug activity was demonstrated by some scientists.[23,24] Thus, it can be stated that
particle size reduction of plants extract significantly increased their in vitro antiradical
potential which might lead towards their better in vivo activity. Lipid peroxidation that is
generally known as primary toxicological event is caused by the formation of free radicals
from a diversity of sources. It leads to oxidative degradation of lipids thereby damaging
cellular membranes. Ammonium thiocyanate method was used to evaluate the anti-lipid
peroxidative effects of plants extract [25] and nanosuspensions and results were reported
in terms of percentage inhibition (Figure 4). Nanosuspensions of all plants exhibited more
percentage inhibition of lipid peroxidation than their crude extracts. Percentage inhibition
for S. marianum nanosuspension was found to be 83.76 1.1% as compared to its crude
extract (39.76 5.01%). Nanosuspenion of C. sativum was found to be more effective
towards inhibition of lipid peroxidation after 72 h (91.75 2%) among all plants extract,

Figure 4. Percentage inhibition of lipid peroxidation of plant extracts, nanosuspensions and


standards. Data are mean (n D 3) SD.

N. Jahan et al.

Downloaded by [Colorado College] at 15:53 06 April 2015

nanosuspensions and standards. BHT and ascorbic acid exhibited considerable amount of
percentage inhibition but not more than plants nanosuspension. Appreciable inhibition of
peroxidation might be ascribed to the presence of well-known antioxidants, for instance
flavonols, xanthones, di-anthraquinones and flavans potentially responsible for the
considerable activity of the extracts. Due to nanosizing, surface area was increased and
molecules interacted very rapidly with solvent molecules which resulted in increased
solubility. Therefore, results of inhibition of lipid peroxidation might be based on
enhanced solubility of the samples. The crude extract of plants and standards due to larger
particle size were failed to scavenge free radicals in manner similar to that of
nanosuspension.

4. Conclusion
It is concluded that nanosuspensions of the selected plants S. marianum, C. sativum and
E. cardamomum significantly enhanced the antiradical potential as compared with their
crude extracts.

Disclosure statement
No potential conflict of interest was reported by the authors.

References
[1] Chingunpituk J. Nanosuspension technology for drug delivery. Walailak J Sci Technol.
2011;4:139 153.
[2] Bhadoriya SS, Mangal A, Madoriya N, Dixit P. Bioavailability and bioactivity enhancement of
herbal drugs by Nanotechnology: a review. J Curr Pharm Res. 2011;8:1 7.
[3] Saraf S. Applications of novel drug delivery system for herbal formulations. Fitoterapia.
2010;81:680 689.
[4] Bailey MM, Berkland CJ. Nanoparticle formulations in pulmonary drug delivery. Med Res
Rev. 2009;29:196 212.
[5] Chaudhary A, Nagaich U, Gulati N, Sharma V, Khosa R, Partapur MU. Enhancement of
solubilization and bioavailability of poorly soluble drugs by physical and chemical
modifications: a recent review. J Adv Pharm Educ Res. 2012;2:32 67.
[6] Modi A, Jain S, Kumar V. Zizyphus xylopyrus (Retz.) Willd: a review of its folkloric,
phytochemical and pharmacological perspectives. Asian Pac J Trop Dis. 2014;4:S1 S6.
[7] Ravichandran R. Nanoparticles in drug delivery: potential green nanobiomedicine applications.
Int J Green Nanotechnol. 2009;1:B108 B130.
[8] Pandya VM, Patel JK, Patel DJ. Formulation, optimization and characterization of simvastatin
nanosuspension prepared by nanoprecipitation technique. Der Pharmacia Lettre.
2011;3:129 140.
[9] Bonifacio BV, da Silva PB, dos Santos Ramos MA, Negri KMS, Bauab TM, Chorilli M.
Nanotechnology-based drug delivery systems and herbal medicines: a review. Int J Nanomed.
2014;9:1 3.
[10] Yang KY, Du Hyeong Hwang AMY, Kim DW, Shin Y-J, Bae O-N, Kim YI, Kim JO, Yong
CS, Choi H-G. Silymarin-loaded solid nanoparticles provide excellent hepatic protection:
physicochemical characterization and in vivo evaluation. Int J Nanomed. 2013;8:3333 3343.

Downloaded by [Colorado College] at 15:53 06 April 2015

Journal of Experimental Nanoscience

[11] Verma S, Jain V, Katewa S. Blood pressure lowering, fibrinolysis enhancing and antioxidant
activities of cardamom (Elettaria cardamomum). Indian J Biochem Biophys. 2009;46:503 506.
[12] Wangensteen H, Samuelsen AB, Malterud KE. Antioxidant activity in extracts from coriander.
Food Chem. 2004;88:293 297.
[13] Anwar S, Jahan N, Rahman K, Ali S, Aslam S. Microwave-assisted extraction and nitric oxide
and superoxide attenuation potential of polyphenolics from Bauhinia variegata. Asian J Chem.
2013;25:7125 130.
[14] Mishra SB, Pandey H, Pandey AC. Nanosuspension of Phyllanthus amarus extract for
improving oral bioavailability and prevention of paracetamol induced hepatotoxicity in
Sprague Dawley rats. Adv Nat Sci. 2013;4:035007.
[15] Aslam S, Jahan N, Ali S, Rahman K. An innovative microwave-assisted extraction and
antioxidant potential of polyphenols from different parts of Ocimum basilicum. J Med Plants
Res. 2012;6:2150 2159.
[16] Gupta S, Kesarla R, Omri A. Formulation Strategies to improve the bioavailability of poorly
absorbed drugs with special emphasis on self-emulsifying systems. ISRN Pharmaceutics.
2013;2013:1 16.
[17] Bhattacharya S. Phytosomes: the new technology for enhancement of bioavailability of
botanicals and nutraceuticals. Int J Health Res. 2009;2:225 232.
[18] Adithya ES, Lakshmi MS, Hephzibah P. In vitro antioxidant, anti-lipid peroxidation activities
and HPLC analysis of methanol extracts from bark and stem of Mahonia leschenaultia takeda.
Asian J Plant Sci Res. 2013;3:116 126.
[19] Rana M, Katbamna R, Padhya A, Dudhrejiya A, Jivani N, Sheth N. In vitro antioxidant and
free radical scavenging studies of alcoholic extract of Medicago sativa l. Rom J Biol.
2010;55:15 22.
[20] Jananie R, Priya V, Vijayalakshmi K. In vitro assessment of free radical scavenging activity of
Cynodon dactylon. J Chem Pharm Res. 2011;3:460 466.
[21] Chidambaram M, Krishnasamy K. Development of dimethylaminoethyl methacrylate based
nanoparticulate drug delivery system using nanoprecipitation method and optimization of
process parameters using Plackett-Burman factorial design. Nano Biomed Eng. 2014;6:19 30.
[22] Chahal AS, Madgulkar A, Kshirsagar S, Bhalekar M, Dikpati A, Gawli P. Amorphous
nanoparticles for solubility enhancement. J Adv Pharm Sci. 2012;2:167 178.
[23] Tan W, Wang S. Advances in the nanoparticle drug delivery systems of silymarin. J Chin Pharm
Sci. 2011;20:442 446.
[24] Hui Z, Li H. Preparation of silymarin nanosuspension by microemulsion dilution. Chin J
Pharm. 2011:1 5.
[25] Yen GC, Duh PD, Chuang DY. Antioxidant activity of anthraquinones and anthrone. Food
Chem. 2000;70:437 441.