Journal of Ethnopharmacology 155 (2014) 1300–1305

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

Research Paper

Antibacterial and anti-inflammatory activities of extract and fractions

from Pyrrosia petiolosa (Christ et Bar.) Ching
Dandan Cheng a, Yingying Zhang b, Demin Gao a,n, Hongmeng Zhang c
a
School of Pharmacy, Shandong University of Traditional Chinese Medicine (TCM), 250355 Jinan, China
b
School of Basic Medical Science, Shandong University of TCM, 250355 Jinan, China
c
Experimental Center of Shandong University of TCM, 250355 Jinan, China

art ic l e i nf o a b s t r a c t

Article history: Ethnopharmacological relevance: Pyrrosia petiolosa is commonly used as a traditional Chinese medicine
Received 12 December 2013 for treatment of acute pyelonephritis, chronic bronchitis and bronchial asthma. This study aims to
Received in revised form evaluate the antibacterial activity of the ethanol extract and its derived fractions of Pyrrosia petiolosa
24 June 2014
obtained with solvents of different polarities and to perform the anti-inflammatory screening.
Accepted 16 July 2014
Materials and methods: The powdered aerial parts of Pyrrosia petiolosa were used to extract various
Available online 28 July 2014
fractions with ethanol, petroleum ether, ethyl acetate, N-butanol and aqueous. Qualitative phytochemical
Keywords: screening was performed on the ethanol extract, petroleum ether fraction, ethyl acetate fraction, N-butanol
Antibacterial fraction and aqueous fraction. The agar diffusion method, minimum inhibitory concentration (MIC) and
Anti-inflammatory
minimum bactericidal concentration (MBC) were employed to evaluate antibacterial activity of the ethanol
Pyrrosia petiolosa
extract and fractions. The in vitro cytotoxicity of ethanol extract and fractions was determined using MTT
assay. The anti-inflammatory activity was analyzed using the mouse ear swelling induced by xylene.
Results: The phytochemical screening revealed the presence of anthraquinones, flavonoids, terpenoids,
steroids, saponins, phenols and reducing sugars in the extract and fractions. Antibacterial results showed
that petroleum ether fraction and N-butanol fraction inhibited all the tested microorganisms with the
maximum inhibition zone of 15.2570.35 mm. Ethyl acetate fraction also exhibited good antibacterial
activity except Pseudomonas aeruginosa ATCC 27853, while extract and aqueous fraction inhibited 8 out of
13 (61.5%) of the tested microorganisms. The MIC values of ethanol extract and fractions ranged from 1.25
to 10.00 mg/mL and most of the MBC values were equal or twice as high as the corresponding MIC values.
The in vitro cytotoxicity showed the ethanol extract and fractions exhibited non-toxic or low toxic activity
against lung cancer cell lines A549 and mouse spleen cells. In anti-inflammatory experiment, ethanol
extract at 5.0 and 10.0 mg/kg exhibited significant anti-inflammatory activity against the mouse ear
swelling induced by xylene and the maximum inhibition rate reached as high as 67%.
Conclusions: Pyrrosia petiolosa could be a potential candidate for future development of a novel
antibacterial and anti-inflammatory agent.
& 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Pyrrosia petiolosa (Christ et Bar.) Ching belongs to Pyrrosia
genus in the family Polypodiaceae, which is a perennial herb fern
mainly distributed in the regions of China, North Korea and Russia.
The aerial parts of Pyrrosia petiolosa have been used as a tradi-
tional Chinese medicine for treatment of acute pyelonephritis,
chronic bronchitis and bronchial asthma (National Pharmacopoeia
Commission (NPC), 2010; Wang et al., 2011). In addition, Pyrrosia
petiolosa has been widely used as diuretic drug (Jones et al., 2006).
n
Corresponding author. Tel.: þ 86 531 87587962; fax: þ86 531 89628081.
E-mail address: gdm0115@163.com (D. Gao).
It has also been reported that ethanol extract of Pyrrosia petiolosa
has antioxidant activity (Banerjee and Sen, 1980; Lai et al., 2003;
Hsu, 2008; Rivera et al., 2011).
Previous studies showed that the chemical components of Pyrrosia
petiolosa included flavonoids, polysaccharides, chlorogenic acid, luteo-
lin, quercitin and saponin (Takemoto and Hikiao, 1963; Yang et al.,
2003; Wang et al., 2006 ), which were related to antibacterial and
anti-inflammatory activities. Therefore, we hypothesized that Pyrrosia
petiolosa should have antibacterial and anti-inflammatory activities.
However, detailed antibacterial activities have not been reported.
Moreover, no information available about anti-inflammatory activity
for Pyrrosia petiolosa can be found.
The aim of this study was to evaluate the in vitro antibacterial
potency of the ethanol extract and its derived fractions of Pyrrosia

http://dx.doi.org/10.1016/j.jep.2014.07.029
0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
 D. Cheng et al. / Journal of Ethnopharmacology 155 (2014) 1300–1305
petiolosa and its anti-inflammatory activity. The in vitro antibac-
terial activity was determined by the agar diffusion method,
minimum inhibitory concentration (MIC) and minimum bacterici-
dal concentration (MBC), while anti-inflammatory activity was
analyzed using the mouse ear swelling induced by xylene.
2. Materials and methods
2.1. Materials
The plants of Pyrrosia petiolosa obtained from mountain Meng-
shan (Shandong, China) were authenticated by author and the
voucher specimen (No. SDCM 201010) was deposited at the
Herbarium of the Shandong University of TCM, China (SDCM).
Ethanol, petroleum ether, ethyl acetate and N-butanol were
purchased from Tianjin chemical reagent Co., LTD., China. Anti-
biotics were bought from Shandong Lukang Pharmaceutical
Group, China.
2.2. Preparation of extract and fractions
The dried, powdered materials were extracted twice in 60%
ethanol (v/v) at the boiling temperature for 4 h, followed by
filtration using filter paper. Two percolates were mixed, con-
centrated and subsequently freeze-dried. The freeze-dried sample
was sequentially extracted with petroleum ether, ethyl acetate,
N-butanol and aqueous. The respective fractions were centrifuged,
filtered and lyophilized. The dried residues were dissolved in
DMSO and adjusted to 10.00 mg/mL, and then were filtered using
0.22 mm micro filter. The filtrate was stored in labeled sterile
bottles in a freezer at 4 1С for further use in a week.
2.3. Phytochemical screening
Phytochemical screenings of extract and fractions were per-
formed for the qualitative detection of reducing sugars, anthra-
quinones, flavonoids, pigmentum, volatile oil, terpenoids,
saponins, steroids, alkaloids, phenols and organic acid using
standard procedures (Trease and Evans, 1983; Sofowora, 1993;
Mishra et al., 2009).
2.4. Test microorganisms and experimental animals
The microorganisms used in the study were obtained from
College of Basic Medical Sciences, Shandong University of TCM,
China. The reference bacterial strains were Pseudomonas aerugi-
nosa ATCC 27853, Staphylococcus aureus ATCC 25923, Escherichia
coli ATCC 25922, Shigella fiexneri ATCC 12022, Proteus vulgaris
ATCC 13315, Bacillus subtilis ATCC 21616, Sarcina lutea ATCC 9341
and fungi was Saccharomyces cerevisiae yeast ATCC 20602. The
following isolated strains were used: Pseudomonas aeruginosa,
Staphylococcus aureus, Escherichia coli, Proteus vulgaris and Shigella
fiexneri.
Experimental animals for anti-inflammatory activity were
Kunming mice purchased from Experimental Animal Center,
Shandong University of TCM, China. The animals were fed and
handled in strict accordance with Animal Care and Use Committee
of Shandong University of TCM, China.
2.5. Antibacterial activity assay
Antibacterial activities were carried out by in vitro the agar
diffusion method (Rios et al., 1988). Autoclaved Mueller-Hinton
agar (MHA) was poured into sterile Petri dishes and solidified into
plate. The inoculated plates were incubated at 37 1C for 24 h.
1301
Sterile 6 mm diameter paper discs (Whatman no. 4) saturated
with 10 μL of ethanol extract or fractions were aseptically placed
on top of the agar layer, and the plates were incubated at 37 1C for
24 h. The antibacterial activity was evaluated by measuring the
diameter of inhibition zone surrounding disc. Standard antibiotic
discs [Gentamicin (10 μg/disc), Erythromycin (15 μg/disc), Cipro-
floxacin (25 μg/disc) and Streptomycin (15 μg/disc)] were used as
positive controls. Discs containing 10 μL DMSO solution were used
as a negative control. All the antimicrobial tests were performed
in triplicate and the results were reported as mean 7standard
deviation of three replicates.
2.6. Determination of the minimum inhibitory concentration (MIC)
The MIC values were determined by a broth microdilution
method as described previously (Peng et al., 2011) and the clinical
and Laboratory Standards Institute (CLSI) reference broth micro-
dilution methods (CLSI, 2012) with a little modification. In brief, a
series of ethanol extract and fraction concentrations were pre-
pared with the broth and 100 mL of these solutions were added to
sterile 96-well plates. Then 100 mL aliquots of culture containing
approximately 106 CFU/mL of the tested microorganisms were
added to set the final concentration of ethanol extract or each
fraction at 0.25, 0.50, 0.625, 1.25, 2.50, 5.00, 10.00, and 20.00 mg/
mL. The well only containing broth and inocula was used as a
negative control. The inoculated 96-well plates were incubated at
37 1C for 24 h, and the optical density (OD) of each well was
measured at the wavelength of 600 nm.
2.7. Determination of the minimum bactericidal concentration
(MBC)
The MBC values were defined as the lowest ethanol extract or
fraction concentration killing at least 99.9% of the bacterial
inoculum within 24 h (Raafat et al., 2008). In brief, the contents
in the MIC well and those above the MIC for each microorganism
were plated sterile Petri dishes containing MHA (Jones et al.,
2005). After incubation at 37 1C for 24 h, the bacterial colonies on
the Petri dishes were counted. In this assay, each experiment was
performed in triplicate.
2.8. Stability of antibacterial activity
At a regular interval of three days, the stability of antibacterial
activity against Escherichia coli was performed in a period of one
month. The antibacterial activities of ethanol extract and fractions
were evaluated by the agar diffusion method as previously
described (Holder and Boyce, 1994).
2.9. Toxicity analysis
2.9.1. Clinical symptoms
Kunming mice were grouped and treated as described below. In
the course of intraperitoneal administration of the drug for seven
consecutive days, each mouse was carefully observed daily its
clinical features, including vomit, secretions, muscle crump, ner-
vous symptoms and heart rate.
2.9.2. In vitro cytotoxicity assay
The in vitro cytotoxicity of Pyrrosia petiolosa ethanol extract and
fractions was determined using MTT assay (Mosmann, 1983).
Briefly, lung cancer cells A549 and mice spleen cells in a 96-well
plate (106 cells per well) were cultured in RPMI containing 10%
fetal bovine serum (FBS). After an overnight incubation at 37 1C in
5% CO2, the ethanol extract and fractions were diluted with growth
1302 D. Cheng et al. / Journal of Ethnopharmacology 155 (2014) 1300–1305

medium to obtain serially desired concentration. 100 μL of ethanol components in the ethanol extract and reducing sugar in the
extract or fraction (100 μg/well) was added to the cells. The cells aqueous fraction.
were then incubated at 37 1C in 5% CO2 and the cytotoxicity of the
ethanol extract and fractions was assayed using MTT assay. 50 μL
3.2. Antibacterial activity
of MTT was added to each well and incubated for 1 h. Suitable
blank and positive control were included. The optical densities
The antibacterial activity is shown in Table 2. Our results showed
of the wells were measured at 482 nm. The LC50 values were
Pyrrosia petiolosa ethanol extract and fractions exhibited varying
calculated as the concentration of ethanol extract and fractions
degrees of antibacterial activity against the tested microorganisms.
resulting in a 50% reduction of absorbance compared to untreated
Ethanol extract showed high inhibitory activity against
cells.
Pseudomonas aeruginosa ATCC 27853 (16.60 70.57 mm) and
Saccharomyces cerevisiae ATCC 20602 (16.05 71.34 mm). Aqueous
2.10. Mouse ear swelling assay fraction inhibited 8 out of 13 (61.5%) of the tested microorganisms
and its maximum inhibition zone of 17.20 71.70 mm for Sacchar-
Anti-inflammatory activity was evaluated using mouse ear omyces cerevisiae ATCC 20602. It was particularly mentioned that
swelling assay (Gad, 1994). Fifty Kunming mice were randomly petroleum ether fraction and N-butanol fraction showed broad
divided into five groups (10 mice per group) and given intraper- inhibitory activity against all tested microorganisms. For Bacillus
itoneal administration of the aspirin (reference control, 2.0 mg/kg subtilis ATCC 21616 and Sarcina lutea ATCC 9341, ethanol extract
in phosphate-buffered saline), normal saline (untreated control, and fractions showed moderate antibacterial activity or no activity.
2.0 mL/kg) and ethanol extract (2.5, 5.0 and 10.0 mg/kg, respec-
tively) for seven consecutive days. After 30 min in the final
3.3. MIC and MBC analyses
treatment, 0.2 mL xylene was evenly spread on both sides of left
ear of each mouse in the anesthetized state, while right ear was
The MIC and MBC values are shown in Table 3. The MIC values
used as control. Four hours later, the mice were sacrificed and both
of all the ethanol extract and fractions ranged from 1.25 to
ears were cut off along ear base. On which, 9 mm round ear pieces
10.00 mg/mL. Overall, petroleum ether fraction showed maximum
in diameter was hit in the same area of each ear and then weighed
activity with MIC 2.50 mg/mL for Escherichia coli ATCC 25922 and
accurately. Ear swelling inhibition rate was determined by the
Shigella fiexneri ATCC 13315. Ethyl acetate fraction exhibited good
following formula: ear swelling inhibition rate (%) ¼(A  B)/A 
activity with the MIC 2.50 mg/mL against most of tested micro-
100, where A and B denote ear swelling of the negative group and
organisms. Ethanol extract and aqueous fraction had the same MIC
ear swelling of the drug groups, respectively.
values for 8 out of 13 tested microorganisms. In all cases, most of
the MBC values were found to be equal or twice as high as the
2.11. Statistical analysis corresponding MIC values. Considering inhibition zone, MIC value
and MBC value, petroleum ether fraction and N-butanol fraction
Each experiment was repeated thrice. The results were were regarded as the promising antibacterial candidates.
expressed as the mean 7SD of 10 mice per group and evaluated
with Student's t-test. P o0.01 was considered as significant
difference. 3.4. Stability analysis

The stability of antibacterial activity of ethanol extract and

3. Results fractions is shown in Fig. 1. The antibacterial activities of
ethanol extract and fractions were relatively stable in the first six
3.1. Phytochemical screening of Pyrrosia petiolosa days, followed by a rapid decline, while antibacterial activity of
N-butanol fraction gradually declined to the lowest.
Phytochemical constituents are shown in Table 1. Anthraqui-
nones, flavonoids, terpenoids, steroids and phenols were observed 3.5. Toxicity
in all the extract and fractions, while reducing sugars and saponins
were only present in ethanol extract and aqueous fraction. Further In all groups of Kunming mice, no death occurred within 7 days
analysis showed that petroleum ether fraction contained rich after a dose of 10.0 mg/mL ethanol extract or fractions. Symptoms
anthraquinone and pigmentum. Ethyl acetate fraction enriched associated with toxicity such as vomit, secretions, muscle crump,
steroids and terpenoids. Flavonoids and phenols were the main rapid heartbeat and convulsion were not observed.
The in vitro cytotoxicity against lung cancer cells A549 and
Table 1 mouse spleen cells of the ethanol extract and fractions of Pyrrosia
Phyto-constituents of the extract and fractions of Pyrrosia petiolosa.
petiolosa was evaluated using MTT assay and the results are shown
Phytochemical Ethanol Petroleum Ethyl N-butanol Aqueous in Table 4. Apart from the N-butanol fraction with IC50 value
parameters extract ether fraction acetate fraction fraction 0.33 mg/mL, ethanol extract and other fractions could be regarded
fraction as non-toxic against lung cancer cell lines A549. However, all the
extract and fractions showed a certain degree of toxicity against
Reducing sugars þ    þ
Anthraquinones þ þ þ þ þ spleen cells.
Flavonoid þ þ þ þ þ
Pigmentum þ þ þ þ 
Volatile oil þ þ þ þ  3.6. Xylene-induced ear edema in mice
Terpenoids þ þ þ þ þ
Saponins þ    þ As shown in Fig. 2, ethanol extract at 5.0 and 10.0 mg/kg
Steroids þ þ þ þ þ indicated significant inhibition of the xylene-induced ear edema
Alkaloids     
and the maximum inhibition rate reached as high as 67%. How-
Phenols þ þ þ þ þ
ever, no obvious anti-inflammatory activity appeared at 2.5 mg/kg
þ ¼ present,  ¼absent. ethanol extract.
 D. Cheng et al. / Journal of Ethnopharmacology 155 (2014) 1300–1305 1303

Table 2
Antibacterial activity of the extract and fractions of Pyrrosia petiolosa.

Test strains Ethanol extract Petroleum ether fraction Ethyl acetate fraction N-butanol fraction Aqueous fraction

Pseudomonas aeruginosa ATCC 27853 16.60 7 0.57 12.75 7 0.35 NA 11.90 7 0.57 15.157 0.21
Staphylococcus aureus ATCC 25923 13.75 7 1.06 14.75 7 1.06 15.25 7 0.35 15.25 7 0.35 11.25 7 1.06
Escherichia coli ATCC 25922 11.50 7 0.71 13.007 0.71 12.75 7 0.35 14.60 7 0.85 12.75 7 1.06
Proteus vulgaris ATCC 12022 NA 12.25 7 0.35 12.50 7 0.71 11.50 7 0.71 NA
Shigella fiexneri ATCC 13315 NA 12.25 7 1.06 11.007 0.01 13.50 7 0.71 NA
Saccharomyces cerevisiae ATCC 20602 16.05 7 1.34 11.007 0.01 12.50 7 0.71 12.75 7 0.35 17.20 71.70
Bacillus subtilis ATCC 21616 NA 11.007 0.71 11.75 7 1.07 11.007 0.71 NA
Sarcina lutea ATCC 9341 12.25 7 0.35 11.50 7 1.41 14.25 7 1.06 10.50 7 0.01 11.007 0.01
Pseudomonas aeruginosa 15.30 7 0.38 12.25 7 0.25 NA 10.60 7 1.07 13.25 7 0.33
Staphylococcus aureus 12.50 7 0.86 13.50 7 0.25 14.00 70.25 14.50 7 0.46 11.007 1.12
Escherichia coli 11.007 0.79 12.50 7 0.28 12.007 0.75 14.007 0.74 11.50 7 0.46
Proteus vulgaris NA 12.25 7 0.35 12.007 1.08 11.50 7 0.22 NA
Shigella fiexneri NA 11.50 7 0.16 11.007 0.55 13.007 0.43 NA

NA: no activity.
The antibacterial activity is expressed as inhibition diameter (mm) of the extract and fractions of Pyrrosia petiolosa.
Values are mean7 SD or results obtained from three separate experiments.

Table 3
The MIC (mg/mL) and MBC (mg/mL) of the ethanol extract and fractions of Pyrrosia petiolosa.

Test strains Ethanol extract Petroleum ether fraction Ethyl acetate fraction N-butanol fraction Aqueous fraction

MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC

Pseudomonas aeruginosa ATCC 27853 5.00 5.00 2.50 5.00 ND ND 5.00 5.00 5.00 5.00
Staphylococcus aureus ATCC 25923 5.00 10.00 2.50 10.00 5.00 5.00 1.25 2.50 5.00 ND
Escherichia coli ATCC 25922 5.00 10.00 2.50 5.00 5.00 5.00 1.25 2.50 2.50 2.50
Proteus vulgaris ATCC 12022 5.00 10.00 2.50 5.00 2.50 5.00 1.25 2.50 ND ND
Shigella fiexneri ATCC 13315 10.00 10.00 5.00 5.00 2.50 2.50 1.25 2.50 ND ND
Saccharomyces cerevisiae ATCC 20602 5.00 5.00 5.00 ND 2.50 5.00 2.50 5.00 2.50 5.00
Bacillus subtilis ATCC 21616 ND ND 2.50 5.00 2.50 5.00 2.50 5.00 ND ND
Sarcina lutea ATCC 9341 1.25 5.00 5.00 10.00 2.50 5.00 2.50 5.00 2.50 5.00
Pseudomonas aeruginosa 5.00 5.00 5.00 10.00 ND ND 2.50 5.00 2.50 5.00
Staphylococcus aureus 2.50 5.00 5.00 10.00 2.50 ND 2.50 ND 5.00 ND
Escherichia coli 5.00 5.00 2.50 5.00 2.50 5.00 5.00 5.00 5.00 5.00
Proteus vulgaris ND ND 2.50 2.50 2.50 5.00 1.25 5.00 ND ND
Shigella fiexneri ND ND 2.50 5.00 5.00 5.00 1.25 5.00 ND ND

ND: not determined.

Table 4
In vitro cytotoxicity of the extract and fractions of Pyrrosia petiolosa.

Samples LC50 (mg/mL)

Lung cancer cell A549 Spleen cells

Petroleum ether fraction – 0.53

Ethyl acetate fraction – 0.59
N-butanol fraction 0.33 0. 21
Ethanol extract – 0.36
Aqueous fraction – 0.32

“–”means no cytotoxicity.

Fig. 1. The stability of antibacterial activity of ethanol extract and fractions against
Escherichia coli. The X axis represents the number of days after preparation of
ethanol extract and fractions and Y axis represents zone of inhibition (mm) by the
agar diffusion method.
4. Discussion
The phytochemical analysis revealed the presence of anthra-
quinones, flavonoid, terpenoid, steroid, phenol and organic acid in
the extract and fractions. As shown in Table 1, each extract or
fraction contains at least six types of secondary metabolites, and
some of the extract and fractions exhibited substantial antibacter-
ial activities as shown by low MIC and MBC values against the
tested microorganisms (Table 3).
It is known that secondary metabolites such as reducing
sugar, anthraquinones, flavonoid, terpenoid, steroid, phenol and
organic acid have good antimicrobial properties (Dorman and
Deans, 2000; Kuete, 2010). Reducing sugars have been found to
show antibacterial properties (Mabeku et al., 2007; Dhale
and Markandeya, 2011). Anthraquinones are reported to have
antibacterial activity by causing bacteria membrane perturbation
(Coenye et al., 2007). Flavanoids and phenols are effective anti-
bacterial substances due to their ability to form complexes with
extra cellular and soluble proteins and to complex with bacterial
cell walls leading to the death of the bacteria (Cowan, 1999).
Terpenoids are known to have antibacterial property by affecting
the synthesis of cell membranes components, prenylation of
proteins and the use of carbon source (Nayak et al., 2010). The
1304 D. Cheng et al. / Journal of Ethnopharmacology 155 (2014) 1300–1305
Fig. 2. Effect of ethanol extract on mouse ear swelling response. The bar chart
represented ear swelling inhibition rate of aspirin group (2.0 mg/kg) and ethanol
extract groups (2.5, 5.0 and 10.0 mg/kg, respectively). Ear swelling inhibition rate¼
(ear swelling of the negative group ear swelling of the drug groups)/ear swelling
of the negative group  100. *P o0.01 significantly different from the negative
control as determined by the Student's t-test.
activities of these phytochemical components may be responsible
for the antibacterial activities observed in the study.
Overall, non-polar and weakly polar fractions of Pyrrosia
petiolosa like petroleum ether and N-butanol, showed a broad-
spectrum antimicrobial activity against the tested bacteria, and
polar fraction exhibited better bacterial activity against some
tested bacteria like Pseudomonas aeruginosa ATCC 27853. It should
be mentioned that the bioactive phytochemical constituents in the
ethanol extract or fraction is not a guarantee for antibacterial
activity. Bacterial susceptibility to the extract or fraction is both
due to the types, concentration of the bioactive compounds and
their mechanism of the action (Ono et al., 2004) and to bacterial
cell wall composition and its plasmids content (Karaman et al.,
2003).
The ethanol extract and aqueous fraction inhibited the growth
of eight out of 13 (61.5%) tested strains and the inhibition of
Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus
ATCC 25923 to some extent justified the use of the plant in the
treatment of acute pyelonephritis and chronic bronchitis. The
remaining five tested microbes, including Proteus vulgaris ATCC
12022, Shigella fiexneri ATCC 13315, Bacillus subtilis ATCC 21616,
Proteus vulgaris (isolated) and Shigella fiexneri (isolated), were not
susceptible to the extracts. It was inferred that the active compo-
nents in the ethanol extract or aqueous fraction were not sufficient
to inhibit all the tested microorganisms.
Petroleum ether fraction showed 100% inhibitory activities
against all the tested microorganisms with different diameters of
inhibition. It was thought the fraction contained more non-polar
components such as terpenoids and volatile oil as shown in
Table 1. Their synergy with flavanoids and phenols may be a
plausible explanation for this phenomena observed.
The MIC/MBC was not determined for extract and some of
the fractions, such as ethyl acetate fraction against Pseudomonas
aeruginosa, ethanol extract and aqueous fraction against Proteus
vulgaris and Shigella fiexneri. Some of fractions, even at higher
concentration (20 mg/mL), could not inhibit or kill most of the
bacteria. We speculated these fractions or extract might have
undiscovered inhibitory mechanism, or they only have bacterios-
tasis and no bactericidal effect.
Most antibacterial studies have focused on activity of plant
extracts, but few on the time-dependent stability of the activity
(Marathe et al., 2013). Herein, we reported that the activities of
ethanol extract and fractions were stable over a period of six days
after extraction. This indicated the active principles in the ethanol
extract and fractions had a relative short shelf life. Considering the
phytochemical components in the fraction, the principal active
components were thought to be hydroxyl compounds such as
flavonoids, anthraquinones. These compounds are susceptible to
oxidation (Malterud et al., 1993; Zhang et al., 2005) and their
activities rapidly declined. For antimicrobial agents, it is manda-
tory that the extract and fractions should be non-toxic or low
cytotoxic. In the present study, ethanol extract and fractions
exhibited non-toxic or low cytotoxic effect against lung cancer
cells A549 and spleen cells. If these results were confirmed by
more cells like monkey kidney Vero cells, hepatocyte, and accord-
ingly, the possibility of Pyrrosia petiolosa extract or fraction could
be considered as an alternative of antimicrobial agents.
Mouse ear swelling induced by xylene is a suitable experi-
mental animal model for evaluation of anti-inflammatory effect of
nature products. Mouse ear swelling was accompanied by the
release of pro-inflammatory substances, which increased capillary
permeability and inflammatory cells infiltration (Richardson,
Vasko, 2002). In our study, ethanol extract at 5.0 and 10.0 mg/kg
exhibited marked anti-inflammatory activity in mouse ear edema
induced by xylene. This may be due to its flavone and/or
mangiferin, as the main active constituents of Pyrrosia petiolosa,
could inhibit the release of proinflammatory substances and
decrease capillary permeability (Bhatia et al., 2008; Rivera et al.,
2011).
In conclusion, our present results provided evidence that
Pyrrosia petiolosa had good antibacterial and anti-inflammatory
activities, and therefore suggested that the traditional use of this
medicinal plant for the treatment of acute pyelonephritis nephritis
and chronic bronchitis effect might primarily attribute to both its
antibacterial and anti-inflammatory properties, which help us to
further explore the bio-active mechanism and develop the relative
new drugs.
Acknowledgment
This work was financially supported by China germplasm bank
of wild species (WGB-1204).
References
Banerjee, R.D., Sen, S.P., 1980. Antibiotic activity of pteridophytes. Economic Botany
34, 284–298.
Bhatia, H.S., Candelario-Jalil, E., de Oliveira, A.C., Olajide, O.A., Martínez-Sánchez, G.,
Fiebich, B.L., 2008. Mangiferin inhibits cyclooxygenase-2 expression and
prostaglandin E2 production in activated rat microglial cells. Archives of
Biochemistry and Biophysics 477, 253–258.
CLSI, CLSI Document M07-A9, 32, Clinical and Laboratory Standards Institute,
Wayne, PA, 2012: 16–19.
Coenye, T., Honraet, K., Rigole, P., Nadal, Jimenez, P., Nelis, H.J., 2007. In Vitro
Inhibition of streptococcus mutans biofilm formation on hydroxyapatite by
subinhibitory concentrations of anthraquinones. Antimicrobial Agents and
Chemotherapy 51, 1541–1544.
Cowan, M., 1999. Plant products as antimicrobial agents. Clinical Microbiology
Reviews 12, 564–582.
Dhale, D.A., Markandeya, S.K., 2011. Antimicrobial and phytochemical screening of
Plumbago zeylanica Linn. (Plumbaginaceae) Leaf. Journal of Experimental
Medicine 2, 4–6.
Dorman, H.J., Deans, S.G., 2000. Antimicrobial agents from plants: antibacterial
activity of plant volatile oils. Journal of Applied Microbiology 88, 308–316.
Gad, S.C., 1994. The mouse ear swelling test (MEST) in the 1990s. Toxicology 93,
33–46.
Holder, I.A., Boyce, S.T., 1994. Agar well diffusion assay testing of bacterial
susceptibility to various antimicrobials in concentrations non-toxic for human
cells in culture. Burns 20, 426–429.
Hsu, C.,Y., 2008. Antioxidant activity of pyrrosia petiolosa. Fitoterapia 79, 64–66.
Jones, R.N., Fritsche, T.R., Ge, Y., Kaniga, K., Sader, H.S., 2005. Evaluation of PPI-
0903M (T91825), a novel cephalosporin: bactericidal activity, effects of mod-
ifying in vitro testing parameters and optimization of disc diffusion tests.
Journal of Antimicrobial Chemotherapy 56, 1047–1052.
Jones, W.P., Chin, Y.W., Kinghorn, A.D., 2006. The role of pharmacognosy in modern
medicine and pharmacy. Current Drug Targets 7, 247–264.
 D. Cheng et al. / Journal of Ethnopharmacology 155 (2014) 1300–1305
Karaman, İ., Şahin, F., Güllüce, M., Ogütçü, H., Şengül, M., Adıgüzel, A., 2003.
Antimicrobial activity of aqueous and methanol extracts of Juniperus oxycedrus
L. Jourjnal of Ethnopharmacology 85, 231–235.
Kuete, V., 2010. Potential of cameroonian plants and derived products against
microbial infections: a review. Planta Medica 76, 1479–1491.
Lai, L., Lin, L.C., Lin, J.H., Tsai, T.H., 2003. Pharmacokinetic study of free mangiferin in
rats by microdialysis coupled with microbore high-performance liquid chro-
matography and tandem mass spectrometry. Journal of Chromatography, A
987, 367–374.
Mabeku, L.B.K., Penlap, B.V., Ngadjui, B.T., Fomum, Z.T., Etoa, F.X., 2007. Evaluation
of antimicrobial activity of the stem bark of cylicodiscus gabunensis (Mimosa-
ceae). African Journal of Traditional, Complementary and Alternative medicines
4, 87–93.
Malterud, K.E., Farbrot, T.L., Huse, A.E., Sund, R.B., 1993. Antioxidant and radical
scavenging effects of anthraquinones and anthrones. Pharmacology 47, 77–85.
Marathe, N.P., Rasane, M.H., Kumar, H., Patwardhan, A.A., Shouche, Y.S., Diwanay,
S.S., 2013. In vitro antibacterial activity of Tabernaemontana alternifolia (Roxb)
stem bark aqueous extracts against clinical isolates of methicillin resistant
Staphylococcus aureus. Annals of Clinical Microbiology and Antimicrobials 12,
26.
Mishra, A.K., Mishra, A., Kehri, H.K., Sharma, B., Pandey, A.K., 2009. Inhibitory
activity of Indian spice plant Cinnamomum zeylanicum extracts against
Alternaria solani and Curvularia lunata, the pathogenic dematiaceous moulds.
Annals of Clinical Microbiology and Antimicrobials 8, 9.
Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays. Journal of Immunological
Methods 65, 55–63.
National Pharmacopoeia Commission (NPC), 2010. Chinese Pharmacopoeia. Chinese
medicine science and technology press, Beijing, China, pp. 83–84.
Nayak, B.S., Ramdath, D.D., Marshall, J.R., Isitor, G.N., Eversley, M., Xue, S., Shi, J.,
2010. Wound healing activity of the skin of the common grape (Vitis vinifera)
Variant, Cabernet Sauvignon. Phytotherapy Research 24, 1151–1157.
Ono, T., Kashimura, M., Suzuki, K., Oyauchi, R., Miyachi, J., Ikuta, H., Kawauchi, H.,
Akashi, T., Asaka, T., Morimoto, S., 2004. In vitro and in vivo antibacterial
1305
activities of the tricyclic ketolide te-802 and its analogues. Journal of Antibiotics
(Tokyo) 57, 518–552.
Peng, Z.X., Tu, B., Shen, Y., Du, L., Wang, L., Guo, S.R., Tang, T.T., 2011. Quaternized
chitosan inhibits icaA transcription and biofilm formation by Staphylococcus on
a titanium surface. Antimicrobial Agents and Chemotherapy 55, 860–866.
Raafat, D., von Bargen, K., Haas, A., Sahl, H.G., 2008. Insights into the mode of action
of chitosan as an antibacterial compound. Applied and Environmental Micro-
biology 74, 3764–3773.
Richardson, J.D., Vasko, M.R., 2002. Cellular mechanisms of neurogenic inflamma-
tion. Journal of Pharmacology and Experimental Therapeutics 302, 839–845.
Rios, J.L., Recio, M.C., Villar, A., 1988. Screening method for natural products with
antimicrobial activity: a review of the literature. Journal of Ethnopharmacology
23, 127–149.
Rivera, D.G., Hernández, I., Merino, N., Luque, Y., Álvarez, A., Martín, Y., Amador, A.,
Nuevas, L., Delgado, R., 2011. Mangifera indica L. extract (Vimang) and mangi-
ferin reduce the airway inflammation and Th2 cytokines in murine model of
allergic asthma. Journal of Pharmacy and Pharmacology 63, 1336–1345.
Sofowora, E.A., 1993. Medicinal Plants and Traditional Medicine in Africa, 2nd
edition. John Wiley & Sons, London, UK.
Takemoto, T., Hikiao, H., 1963. Isolation of diploptene from Pyrrosia lingua Farwell.
Chemical and Pharmaceutical Bulletin 11, 409–410.
Trease, E.A., Evans, B., 1983. Medicine and Plants, 3rd edition Pitman, London, UK.
Wang, N., Wang, J.H., Li, X., Ling, J.H., Li, N., 2006. Flavonoids from Pyrrosia petiolosa
(Christ) Ching. Journal of Asian Natural Products Research 8, 753–756.
Wang, Q., Deng, J., Yang, K., Xu, L., 2011. Effects of mangiferin on cytokines in rats
with chronic bronchitis and expression of macrophage COX-2 in mice. Zhong-
guo Zhong Yao Za Zhi 36, 1348–1352.
Yang, C., Shi, J.G., Mo, S.Y., Yang, Y.C., 2003. Chemical constituents of Pyrrosia
petiolosa. Journal of Asian Natural Products Research 5, 143–150.
Zhang, X., Thuong, P.T., Jin, W., Su, N.D., Sok, D.E., Bae, K., Kang, S.S., 2005. Anti-
oxidant activity of anthraquinones and flavonoids from flower of Reynoutria
sachalinensis. Archives of Pharmacal Research 28, 22–27.