www.ijpsonline.

com

TABLE 3: DETERMINATION OF RUGGEDNESS PARAMETERS

Area under curve % Label claim Mean S.D. R.S.D.
(Different analyst)
NSD TZN NSD TZN NSD TZN NSD TZN NSD TZN
180.6288 11.4562 99.83 98.26
179.9668 11.7171 99.37 100.37 99.58 99.72 ±0.231 ±1.267 0.002 0.013
179.7792 11.7040 99.56 100.53
(Different days)
NSD TZN NSD TZN NSD TZN NSD TZN NSD TZN
178.1429 11.7764 98.23 100.73
179.6027 11.7052 99.37 100.57 99.12 100.54 ±0.799 ±0.207 0.008 0.002
181.3239 11.7526 99.77 100.32
NSD stands for nimesulide and TZN stands for tizanidine

containing methanol and water (65:35), pH adjusted to 4.15 for their kind cooperation. They are also thankful to
with orthophosphoric acid, and detection was done at 307 m
Shri. Dattaji Meghe, Chairman and other office bearers
nm. The retention time for nimesulide was 6.84 min and
for tizanidine was 2.09 min. The average content of
rf o
of Nagpur Yuwak Shikshan Sanstha for providing
necessary facilities.
nimesulide and tizanidine found were 99.79 mg/tab and
ad ns
o
nl atio
2.01 mg/tab, respectively. As per USP-XXIV, system REFERENCES
suitability tests were carried out on freshly prepared
solution of nimesulide and tizanidine and parameters
ow lic 1. Zarapkar SS, Bhandari NP, Halkar UP. Simultaneous determination of
obtained with 20 µl solution are shown in Table 2. The
d ub nimesulide and chlorzoxazone in pharmaceutical dosage by RP-HPLC.
Indian Drugs 2000;37:467.
percentage recoveries of nimesulide (100.67%) and
e
rf e w P m).
2. Nagoji KE, Vijaysrinivas S, Kumar KM, Mathivanan N, Kumar SM,
tizanidine (100.50%) reveal no interference of excipients. Rao ME. Simultaneous RP-HPLC estimation of nimesulide and
Ruggedness and robustness studies were carried out, as diclofenac sodium. Indian J Pharm Sci 2003;65:407.
r
fo kno .co
3. Rajput SJ, Randive G. Assay of nimesulide in pharmaceutical dosage
they are the measures to indicate the degree of formulation. Eastern Pharmacist 1997;475:113.
reproducibility. It was observed that the method was 4. Nagoji KE, Rao SS, Rao ME, Rao KV. New Spectrophotometric
le ed ow
rugged and robust as after carrying out the analysis by
b
method for the estimation of Nimesulide in Pharmaceutical Dosage

la M dkn
formulation. Eastern Pharmacist 1999;496:117.
different analysts and on different days the results 5. Reddy MN, Murthy TK, Sankar DG. Simultaneous estimation of
i
showed RSD well within permissible limits. The results are
a by e Nimesulide and Tizanidine Hydrochloride in their combined Dosage
showed in Table 3.
v
a d .m 6.
Form. Eastern Pharmacist 2001;521:121.
Shingare MS, Naidu KR, Kale UN. RP HPLC Method for
Simultaneous estimation of Tizanidine HCl and Nimesulide in Tablets.
ACKNOWLEDGEMENTS s
i ste w Indian J Pharm Sci 2003;65:315.

F Drug Ltd.,
The authors are thankfulDto Indian o
wNagpur
P h w
( the pure
Accepted 6 April 2007
Revised 8 July 2006
and Wallace Pharmaceuticals,
i e
Goa for providing
Dr. K. P.itBhusari (Principal), Mr. A.
drug sample and to s
Received 7 November 2005

P. Mehare, Mr. h s and Mr. Dinesh Wanjari

Krishna Gupta
Indian J. Pharm. Sci., 2007, 69 (2): 281-283

T a

Wound Healing Activity of Lycopodium serratum

serratum

B. K. MANJUNATHA*, V. KRISHNA 1 , S. M. VIDYA, K. L. MANKANI 2 , AND Y. N. MANOHARA 2

Department of Botany and Biotechnology, S. R. N. M. N. College of Applied Sciences, Shimoga - 577 201, India,
1
Department of Biotechnology, Kuvempu University, Shankaraghatta - 577 451, India, 2Department of Pharmacology,
National College of Pharmacy, Shimoga - 577 201, India.

*For correspondence
E-mail: doctor_bkm@Yahoo.com

March - April 2007 Indian Journal of Pharmaceutical Sciences 283

CMYK283
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Wound healing activity of aqueous and ethanol leaf extracts of L. serratum was studied by excision, incision and
dead space wound models on rats. As compared to aqueous and control group of animals, ethanol extract showed
significant wound healing activity which was evidenced by significant decrease in the period of epithelialisation
(17.84±0.06) and increase in wound contraction rate, skin breaking strength (581.45±4.98), granulation tissue
breaking strength (512.80±5.08), dry weight of granulation tissue (47.23±0.10) and elevated concentration of
hydroxyproline (2322.83±8.49). Histopathalogy of the granulation tissue of the ethanol extract treated animals
showed few macrophages with increase in collagenation indicating the potency of the ethanol extract in promoting
the process of wound healing. The present finding provides a scientific base to the ethno medicinal use of L.
serratum.

Lycopodium serratum is a pteridophyte (Lycopodiaceae) ointment was prepared in 2% sodium alginate. For oral
commonly known as Club moss. The plant is found administration, suspensions of 30 mg/ml and 20 mg/ml of
distributed all along the moist deciduous forests of the aqueous and ethanol extracts were prepared in 1% gum
Western Ghats. The plant is used in the homeopathic tragacanth.
o m
preparations to increase efficiency of learning and
fr
memory, in treating Alzheimer’s disease1, cancer2 and oitis
media in children3. The plant is reported to contain d
Wistar rats of either sex weighing 150-200 g were
procured fromathe National
n s College of Pharmacy,
o and were maintained at standard
nl atioThe animals were fed with
alkaloids like serratezomines A-C4, lycoposerramine-A5, Shimoga, Karnataka
lycoposerramine F-O6, quinolizine or pyridine and alpha- housing conditions.
pyridone type alkaloids which are the potent inhibitors of
o
commercialw dietlic(Hindustan Lever Ltd., Bangalore) and
acetylcholineesterase7 and triterpenoids8. The tribal waterd ad libitum
u bthe Institutional
during the experiment. The study was
groups of western Ghats of Chikamagalur region use this
e
approved by
P . Animal Ethical Committee,
plant for treating wounds. The whole plant is ground in
frestudyw was conducted
National College
m
) for bothShimoga.
of Pharmacy, Acute toxicity
hot water and the thick paste thus obtained is applied
r ethanol
o bycostair case method following OECD 2002
and aqueous

fo knnorms.
10
externally to sores, cuts, wounds and burns (Personal extracts
communication). This paper reports the wound healing
e w . LD of aqueous and ethanol extracts were
The
efficacy of the pteridophytic plant Lycopodium serratum.
b d
l e o be 300 and 200 mg/kg. One tenth of the dose
found to
50

Plants were collected from the WesterniGhat la rangeMof dkwas n selected for the evaluation of wound healing activity
i.e., 30 and 20 mg/kg, respectively .
11

v
Chikmagalur district of Karnataka State, aduring bSeptember
y e
a (BKM-456) .was m The
s tetodSri Krishna
to October 2003 and voucher specimen rats were inflicted with excision wounds as described
authenticated by referring the ispecimen
s w 12
by Morton and Malone , under light ether anesthesia. A
F
Devaraya University herbaria and Kuvempu
o w
University 2
circular wound of about 500 mm was made on depilated
herbaria and a voucher D
h
specimen was
(w
deposited
departmental herbaria,PSRNMN College, Shimoga for
in the ethanol sterilized dorsal thoracic region of rats. The

future reference. s
i ite
h s
a
T shade dried and powdered mechanically and treated with 1% w/w framycetin sulphate cream
The plants were
(sieve size 10/45). About 250 g of powdered material was
exhaustively extracted with 70% ethanol for about 48 h
in a Soxhlet extractor. The extract was filtered and
concentrated in vacuum under reduced pressure using
rotary flash evaporator (Buchi, Flawil, Switzerland) (yield
20.8% w/w). For aqueous extract, 250 g of powdered
material was macerated with 1000 ml of distilled water for
three days with intermittent stirring, filtered and
concentrated (yield 15% w/w). Both the extracts were
subjected to preliminary phytochemical tests9.
Two types of drug formulations were prepared from each
of the extracts. For topical administration, 5% w/w
animals were divided into 4 groups of 6 each. The
animals of group I were left untreated and considered as
the control, the group II served as reference standard
(FSC), the group III and IV animals were treated with 50
mg of ointment prepared from aqueous and ethanol
extracts of L. serratum. The ointment was topically
applied once a day till epithelialisation was complete,
starting from the day of the operation. The wounds were
traced on graph paper on 4 th, 8th, 12th and 16th post
wound days and thereafter daily until healing was
complete. The parameters studied were percentage of
wound closure and period of epithelialisation.
In the incision wound model, 6 cm long paravertebral
incisions were made through full thickness of the skin on
either side of the vertebral column of the rat as

284 Indian Journal of Pharmaceutical Sciences March - April 2007

284 CMYK
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TABLE 1: EFFECT OF TOPICAL APPLICATION OF AQUEOUS AND ETHANOL EXTRACTS OF LYCOPODIUM

SERRATUM ON EXCISION WOUND MODELS
Groups (N) 4th day 8th day 12th day 16th day Mean time of epithelialisation in days
Control 13.91±0.31 26.35±0.19 58.58±0.18 76.52±0.19 22.61±0.15
Framycetin sulphate cream 38.58±0.15* 75.29±0.26* 83.58±0.16* 95.15±0.27* 17.37±0.17*
Aqueous extract 32.27±0.23* 62.01±0.22* 75.22±0.15* 86.30±0.12* 19.28±0.01*
Ethanol extract 35.55±0.09* 69.50±0.14* 80.23±0.07* 92.47±0.14* 17.84±0.06*
ANOVA 2708.0 1.137 5791.0 1941.0 386.8
F, df 3,20 3,20 3,20 3,20 3,20
N = 6 animals in each group. *P≤0.01 indicates significant when compared to control. Values are expressed as mean±SE.

described by Ehrlich and Hunt13. The wounds were
closed with interrupted sutures of 1 cm apart. The
grouping of the animals was similar to excision wound
model. The ointment was topically applied once in a day.
The sutures were removed on 8th post wound day. The
skin breaking strength of the wounds were measured on
10th day as described by the method of Lee14.
Under light ether anesthesia, dead space wounds were
created by subcutaneous implantation of sterilized
cylindrical grass piths (2.5 cm × 0.3 cm), one on either
side of the dorsal paravertebral surface of rat . The 15 d ub
e P
group. The group I served as control, which received 1 re
animals were divided into 3 groups of 6 rats in each
f w m
ml of 1% gum tragacanth/kg, p.o. The animals of group II
and III received oral suspensions of aqueous fand
r
o no .co
e p.o., k
the grassed
l
ethanol extracts, respectively (30, and 20 mg/kg
respectively). The granulation tissues formed onb n o
piths were excised on 10th post wound dayland
i a the tissue
M k
treated with ethanol and aqueous extracts 581.45±4.98 and
488.62±6.33, respectively when compared to control
(408.98±7.39) (Table 2).
m
rf o
In dead space wound model also, ethanol extract treated
animals showed significant increase in dry weight of
d ns
granulation tissue (47.23±0.10) and tissue breaking
a
n lo tio
strength (512.80±5.08) followed by aqueous extract treated
group of animals (Table 3). Estimation of hydroxyproline
o w lica content in the granulation tissue revealed that the higher
concentration of hydroxyproline was noticed in ethanol
and aqueous extract treated groups (2322.83±8.49 and
.
)
1871.00±17.23, respectively). The histological profile of
granulation tissue in control group of animals revealed
clumping of macrophages with poor collagenation is
observed (fig. 1), while in aqueous extract treated animals
w
moderate collagen deposition with scattered macrophages
have been noticed (fig. 2). However in the animals
treated with ethanol extract revealed increased collagen

v a subjected
breaking strength was measured. Simultaneously,
b y to ed fibers with few macrophages (fig. 3) indicating the effect
granulation tissue so harvested was
a thedmethod.mof of ethanol on collagen maturation.
s
hydroxyproline estimation following
Woessner and histopathologicali study was
16
tecarriedwout to Collagen is a major protein of the extracellular matrix and
s w
F onocollagenwformation.
evaluate the effect of the extracts is the component that ultimately contributes to wound
The data were subjected D h
P te test and the( values of strength.
to ANOVA followed by Increase in breaking strength of granulation
Turkey’s multiple comparison tissue indicates the enhanced collagen maturation by
is statistically
P ≤0.01 were considered
h si significant. increased crosslinking. In addition, increase in dry
T a
Preliminary phytochemical analysis of aqueous extract
granulation tissue weight indicates the presence of higher
protein content17. Breakdown of collagen liberates free
revealed the presence of flavonoids, saponins, tannins,
and glycosides where as ethanol extract showed positive TABLE 2: EFFECT OF TOPICAL APPLICATION OF
test to alkaloids, flavonoids, saponins, tannins, glycosides AQUEOUS AND ETHANOL EXTRACTS OF
and triterpenoids. LYCOPODIUM SERRATUM ON INCISION WOUND
MODELS.
In excision wound model, the ethanol extract treated Group (N) Tissue breaking strength (g)
animals showed faster epithelialisation of wound Control 408.98±7.39
Framycetin sulphate cream 655.70±13.03*
(17.84±0.06) which is more or less similar to the values of Aqueous extract 488.62±6.33*
standard drug treated group (17.37±0.17). While the Ethanol extract 581.45±4.98*
period of epithelialisation was 22.61±0.15 in case of ANOVA
F 160.2
control and 19.28±0.01 in case of the animals treated with df 3,20
aqueous extract (Table 1). In incision wound model, the N = 6 animals in each group.*P≤ 0.01 indicates significant when compared
mean skin breaking strength was significant in animals to control. Values are expressed as mean±SE.

March - April 2007 Indian Journal of Pharmaceutical Sciences 285

CMYK285
 www.ijpsonline.com

TABLE 3: EFFECT OF TOPICAL APPLICATION OF AQUEOUS AND ETHANOL EXTRACTS OF LYCOPODIUM

SERRATUM ON DEAD SPACE WOUND MODELS.
Groups (N) Granulation tissue dry weight (mg/100g) Breaking strength (g) Hydroxyproline (µg/100mg)
Control (1 ml of 1% gum 33.45±0.15 399.20±8.39 1285.00±21.98
tragacanth / kg b.w.)
Aqueous extract 39.36±0.13* 478.18±6.04* 1871.00±17.23*
Ethanol extract 47.23±0.10* 512.80±5.08* 2322.83±8.49*
ANOVA
F 2960.0 76.6 137.9
Df 2,15 2,15 2,15
N = 6 animals in each group. *P≤0.01 indicates significant when compared to control. Values are expressed as mean±SE.

m
rf o
a d ns
o
nl atio
o w lic
d ub
e
rf e w P m).
Fig. 1: Histological section of granulation tissue of control Fig. 3: Histological section of granulation tissue of the animal
animal treated with ethanol extract showing increased collagenation
and few macrophages.
r
fo kno .co
The sections were stained with haematoxylin and eosin. They
were observed under the magnification of 100 X. Arrow head in
the figure indicates macrophages and arrows indicates the

ble ed ow collagen fibers.

i n
la M dkphytoconstituents
a y like flavonoids which are known to

av d b .me slowing the onset of cell necrosis but also by improving

reduce lipid peroxidation not only by preventing or

is ste w vascularity.
F o ww
D
P te h ( Hence, any drug that inhibits lipid peroxidation is
Fig. 2: Histological section of granulation tissue of the aqueous
believed to increase the viability of collagen fibrils by
extract treated animal showing moderate collagen deposition
is si
and lesser macrophages increasing the strength of collagen fibers, by increasing

T h a the circulation, by preventing the cell damage and by

promoting the DNA synthesis . Hence, the wound
hydroxyproline and measurement of the hydroxyproline
could be used as an index for collagen turnover18. The
results revealed that, animals treated with aqueous and
ethanol extracts showed faster rate of epithelialization in
excision wound model which may be attributed to the
phytoconstituents like tannins 19, flavonoids 20 and
triterpenoids21 which are known to promote the wound
healing process mainly due to their antimicrobial
property. Increase in skin breaking strength and tissue
breaking strength in incision and dead space wound
model respectively indicated enhanced collagen
maturation. Increase in the granulation tissue dry weight
and hydroxyproline content indicated the high collagen
turnover which may be due to the activity of some
22
healing promoting activity of L. serratum may also be
attributed to the antioxidant and antibacterial potency of
the active constituents present in it. The present study
revealed that ethanol extract possesses significant wound
healing promoting activity compared to aqueous extract.
The present finding provides scientific evidence to the
ethnomedicinal property of Lycopodium serratum in
healing the wounds.
ACKNOWLEDGEMENTS
The authors are grateful to Sri. Girimaji N. Rajgopal, Sri.
S. V. Thimmaiah, Prof. Darmananda Rao (National

286 Indian Journal of Pharmaceutical Sciences March - April 2007

286 CMYK
 www.ijpsonline.com

Education Society) and Prof. T. S. Ramkumar (Principal,
S. R. N. M. N. College of Applied Sciences, Shimoga) for
financial support and to Prof. B. Abdul Rahiman
(Department of Biotechnology, Kuvempu University) Dr.
Y. N. Manohara (National College of Pharmacy, Shimoga)
for their support and encouragement.
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1. Xiao, X.Q., Zhang, H.Y. and Tang, X.C., J. Neurosci. Res., 2002, 67, 30.
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Pharmacol. Ther., 1997, 35, 296.
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16. Woessner, J.F., Archiv. Biochem., 1963, 93, 440.
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Hyderabad, 2002, 1.
18. Madhura, M.R. and Sushma, A.M., Fitoterapia, 2003, 74, 553.
19. Ya, C., Gaffney, S.H., Lilley, T.H., Haslam, E., In; Hemingway, R.W.
and Karchesy, J., Eds., Chemistry and significance of condensed
tannins, Plenum Press, New York, 1988, 553.
20. Tsuchiya, H., Sato, M., Miyazaki, T., Fujiwara, S., Tanigaki, S.,
Ohyama, M., Tanaka, T. and Iinuma M., J. Ethnopharmacol.,
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21. Scortichini, M. and Pia Rossi, M., J. Appl. Bacteriol., 1991, 71, 109.
N., Org. Lett., 2002, 4, 1243. 22. Getie, M., Gebre Mariam, T., Reitz, R. and Neubert, R.H.,
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Pharmazie, 2002, 57, 320.

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Tang, X.T., Tan, C.H., Ma, X.Q., Wang, B.D., Jiang, S.H. and Zhu,

a d ns
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D.Y., Planta Med., 2003, 69, 576.

8. Tsuda, Y., Fujimoto, T., Isobe, K., Sano, T. and Kobayashi, M.,
Revised 10 July 2006
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Nirali Prakashan, Pune, 1990, 120.
o w lic Indian J. Pharm. Sci., 2007, 69 (2): 283-287

d ub
e
rf e w P m).
r
fo kno .co
Design and Evaluationle ofedSilymarin-HP w
Silymarin-HP-ß-CD
o -ß-CD Solid
b
Dispersion T ablets ila M dkn
Tablets
v a by e
a d .m
s
i ste w
P. D. NAKHAT*, R. A. NAIDU, I. B. BABLA, S. KHAN AND P. G. YEOLE

F o ww
Department of Pharmaceutics, Institute of Pharmaceutical Education and Research Borgaon (Meghe),

D
Wardha - 442 001, India.

P te h (
h isis the main
Since solubility s i constraint for oral bioavailability of silymarin, an attempt has been made to design tablet
T a
formulations of silymarin-HP-β-CD solid dispersion in order to improve oral bioavailability. Tablet formulations
were prepared by direct compression technique using superdisintegrants such as crosscarmellose sodium, sodium
starch glycolate and polyplasdone XL in different concentrations. Developed formulations were evaluated for
various pharmaceutical characteristics viz. hardness, % friability, weight variation, drug content, disintegration
time and in vitro dissolution profiles. Amongst different batches, formulations containing crosscarmellose sodium
showed superior disintegration and dissolution profiles compared to other formulations. However all the formulations
showed improved dissolution over marketed formulation reflecting vital role of HP-β-CD dispersion in promotion
of silymarin oral bioavailability. Moreover, optimized formulation showed stability at varying temperature and
relative humidity.

*For correspondence
E-mail: premdnakhat@rediffmail.com

March - April 2007 Indian Journal of Pharmaceutical Sciences 287

CMYK287