Journal of Pharmacognosy and Phytochemistry 2018; 7(3): 536-541
E-ISSN: 2278-4136
P-ISSN: 2349-8234
JPP 2018; 7(3): 536-541
Received: 10-03-2018
Accepted: 12-04-2018
Jan Sher
Department of Botany, Garden
Campus, Abdul Wali Khan
University Mardan, Pakistan
Gul Jan
Department of Botany, Garden
Campus, Abdul Wali Khan
University Mardan, Pakistan
Cai zhiquan
Key Laboratory of Tropical
Plant Resources and Sustainable
Use, Xishuangbanna Tropical
Botanical Garden, Chinese
Academy of Sciences, Beijing,
China
Muhammad Israr
Department of Botany,
Government Post graduate
College Mardan, Pakistan
Farzana Gul
Department of Botany, Garden
Campus, Abdul Wali Khan
University Mardan, Pakistan
Siraj Khan
Department of Botany, Garden
Campus, Abdul Wali Khan
University Mardan, Pakistan
Muddaser Shah
Department of Botany, Garden
Campus, Abdul Wali Khan
University Mardan, Pakistan
Shakir Ullah
Department of Botany, Garden
Campus, Abdul Wali Khan
University Mardan, Pakistan
Correspondence
Jan Sher
Department of Botany, Garden
Campus, Abdul Wali Khan
University Mardan, Pakistan
Analgesic, anti-inflammatory, antioxidant activity
and phytochemical screening of Dryopteris
blanfordii plant
Jan Sher, Gul Jan, Cai zhiquan, Muhammad Israr, Farzana Gul, Siraj
Khan, Muddaser Shah and Shakir Ullah
Abstract
We investigated the scientific base for its traditional use in pain, inflammation, antioxidant and
phytochemicals potential of Dryopteris blanfordii. The powdered plant was extracted by the method of
cold maceration using (methanol, ethanol and aqueous) as solvents. The acetic acid induced writhing test
model was used for analgesic activity. Anti-inflammatory activity was evaluated by carrageenan-induced
mice paw edema. For antioxidant activity we used 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and reducing
power assay method respectively. In qualitative analysis, the phytochemical compounds such as
carbohydrates, alkaloids, phenols, flavonoids, saponins, tannins and quinine were screened by using
standard methods. In analgesic activity after the injection of ethanolic extract at a dose of 300mg/kg the
writhing was reduced to (21±0.13) /5 minutes and showed 45% inhibition, compared with standard drug
aspirin which reduced to (14±0.21) /5 minutes and having 40.43% inhibition. The anti-inflammatory
activity at a dose of 300 mg/kg during drug administration after four hours showed significant different
which compared with standard drug diclofenac sodium (10mg/kg) and 100, 200mg/kg. High dose at
300mg/kg reduced (1.04±0.03) after the 4th hours and standard drug diclofenac sodium reduced
(0.08±0.04) after 4th hours. The maximum rate of inhibition of antioxidant was observed in the
methanolic extract. In the qualitative analysis of the ethanol extract showed highest amount of phenols.
We concluded that the D.blanfordii plant showed high potential of inflammation, antioxidant and
biochemical compounds.
Keywords: Dryopteris blanfordii, antioxidant, phytochemicals, DPPH
1. Introduction
Pharmacology is the branch of biology which deals with the study of action of drug on living
tissue, where drugs is defined as any man-made, natural molecule which relates a biochemical
or physical effect on the cell, tissue, organ and organism. In drug discovery, medicinal herbs
has been considered the important source of pharmaceuticals, employed the action of human
diseases due to their excessive chemical variety and wide biological functionality (Nasrabadi
et al., 2012) [12]. Now a day, great cares have been rewarded to eco-friendly and bio-friendly
plants, which can checked and remedy several human diseases (Onsare et al., 2013) [15]. In
many cases, these man-made drugs cause side effects or adverse responses. As a result, more
attention was given to the study then use of medicinal plants has remained taken place through
the last two decades. The new isolation methods and pharmacological testing processes, new
plant drugs originate their way into new drug. Such use of single unadulterated compounds
with artificial medicines (Trease &Evans, 2003) [22]. Pteridophytes are the primitive land plants
group on earth and established large group of vascular cryptograms. The position of the
pteridophytes, between the lower cryptograms and higher vascular plants. Pteridophytes have a
long ecological history on earth planet. They were known as far back as 380 million years ago.
In our neighbor country India, Pteridophytes are mostly distributed in the Himalayan and
coastal regions (Upreti et al., 2009) [23]. Pteridophytes also showed pharmaceutical ability and
many of them are being used therapeutically (Kumar & Kaushik, 1999) [10]. The rural societies,
ethnic groups and traditional throughout the world are using plant parts like rhizome, stem,
fronds, pinnae and spores in various ways for the usage of several traditional since early time.
Many researches are working on taxonomy, ecology and distribution of pteridophytes has been
published from time to time but enough responsiveness has not been paid towards their
pharmaceutical useful aspects (Dixit, 2001) [5]. In the present study efforts have been made to
search medicinally important pteridophytes and properly recognized their useful feature.
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2. Materials and methods
2.1 Plant Collection and Identification
Dryopteris blanfordii was collected from District Dir (Upper)
in September 2017 and identified with the help of flora of
Pakistan and compared with the already specimen present in
the herbarium of Hazara University Mansehera.
2.2 Drying and powdering
The collected plant specimen was clean from dust and
damage parts were removed and then shade dried for 25 days.
The dried specimen was powdered with the help of electric
grinder and then the powdered material was stored in air tight
bottle and weighed in selected amounts (Kumaran, 2006) [11].
2.3 Preparation of extracts
The powdered was used for the preparation of extracts using
methanol, ethanol and aqueous in continuous method
respectively for 48 hrs. The extract was filtered through
watchman No.1 filter paper. The solvent was recovered by
rotary vacuum evaporator and the concentrated extracts were
further evaporated to get dry extracts. The dried extract was
stored in an airtight bottle (Ismail et al., 2012) [8].
2.4 Chemicals used
Methanol, ethanol, carrageenan, acetic acid and DPPH were
used.
2.5 Animal used
Young and healthy Swiss albino mice of 25-30 gm weight at
the age of one month were selected for the studies. The mice
were procured from animal house of NIH (National Institute
of Health) Islamabad. Mice were kept in hygienic condition in
polypropylene cages and feed at standard supplement
obtained from NIH. The room temperature were kept at 25-30
°C and acclimatized at 4 days.
3. Analgesic activity
For in vivo analgesic activity the acetic acid induced writhing
method was used.
3.1 Acetic acid induced writhing method
Acetic acid induced writhing test was carried out for analgesic
activity. The mice were divided into five groups. Mice of
group 1st (control group) was injected 1ml normal saline (1%
v/v). Then 1ml acetic acid was injected to all mice of group
2nd,3rd,4th and group 5th. Ten minutes after then injected acetic
acid solution, the writhing were count for /5 minutes. After
the writhing the group 2 mice were injected 1ml of aspirin
solution. The mice of group 3rd, 4th and 5th were injected 1ml
ethanolic extract solution at the dose of 100 mg/kg, 200
mg/kg and 300 mg/kg respectively. After the inhibition of
writhes were determined (Podder et al., 2011) [18]. Finally the
percentage (%) of analgesic activity was used by the
following formula;
𝑁𝑜. 𝑜𝑓 𝑤𝑟𝑖𝑡ℎ𝑖𝑛𝑔𝑠 𝑖𝑛 𝑡𝑒𝑠𝑡𝑒𝑑 𝑑𝑟𝑢𝑔
% inhibition = × 100
𝑁𝑜. 𝑜𝑓 𝑤𝑟𝑖𝑡ℎ𝑖𝑛𝑔𝑠 𝑖𝑛 𝑐𝑜𝑛𝑡𝑟𝑜𝑙
3.2 Anti-inflammatory activity
Carrageenan induced paw edema test model was carried out
for anti-inflammatory activity. Ethanolic extract of
D.blanfordii was tested for anti-inflammatory activity against
carrageenan paw edema in mice. Mice were divided into five
groups. Mice of group 1st (control group) was injected normal
saline (1% v/v). Then 1ml carrageenan was injected to
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group,3rd, 4th and 5th. Diclofenac sodium (dissolved in 10 ml
water) and then 1ml diclofenac sodium was injected to group
2nd. The mice of group 3rd, 4th and 5th were injected 1ml
ethanolic extract solution at the dose of 100 mg/kg, 200
mg/kg and 300 mg/kg respectively. The reduction of paw
edema of mice was compared with standard drug (Olajide et
al., 2000) [14]. The percent inhibition was calculated by the
following formula
Vc represent the edema value of control group, “Vt” represent
the edema volume of treated groups.
4. Antioxidant activity
The free radical scavenging activity of different extracts of
D.blanfordii were checked, using stable free radical, DPPH
(2, 2-diphenyl-1-picrylhydrazyl) or Reducing power or
Superoxide anion radical scavenging activity was determined
spectrophotometrically. The optical density of the oxidation
reaction were measured at 517 nm using spectrophotometer
with solvent and DPPH as blank (Ayoola et al., 2008) [8].
5. Phytochemical screening
The methanolic, ethanolic and aqueous extract of D.blanfordii
was screened by different chemical test for the identifying the
basic chemical constituents present in the extract. The
standard chemical tests for carbohydrates (Sharma et al.,
2016) [20], alkaloid (Kavitha & Gunavathy, 2014) [9], phenols
(Dahiru et al., 2006) [4], flavonoid (Zhao et al., 2007) [25],
quinine (Savithramma et al., 2011) [19] and tannin (Zohra et
al., 2012) [26] were performed by standard methods.
6. Statistical Analysis
The data were analyzed by one-way ANOVA (mean±SEM &
mean±SD) followed by application of Duncan test (Pro 8
SRO v8.0724 (B724)), Northampton, MA, USA. A 𝑃 value of
<0.05 was considered as a statistically significant.
7. Results
7.1 Analgesic activity
The result of acetic acid induced writhing test was mentioned
in a table 1. The mice of group 1(control group) showed
writhing i.e (27.4±0.10) /5 minutes. Group 2 nd mice
significantly reduced writhing i.e 14±0.21 / 5 minutes and
showed 40.43% inhibition. Among the group 3 rd, 4th and 5th,
the highest writhing counting was observed in Group 5th at the
concentration of 300mg/kg (21±0.13 in /5 minutes and
showed 45% inhibition reduction) followed by group 4 th mice
at a concentration of 200mg/Kg (28±0.32 /5 minutes) and
showed 52.44% inhibition reduction (Table 1; Fig 1).
7.2 Anti-inflammatory activity
The initial paw edema volume was noted in all the mice of 5
groups i.e G1 (0.65±0.05), G2 (0.45±0.03), G3 (0.62±0.05),
G4 (0.586±0.05), G5 (0.566±0.05) in mm³. After measuring
the initial paw edema volume,1ml carrageenan was injected to
all the mice of group 2 to 5.After one hour the paw edema
volume were recorded i.e G1(1.12±0.03), G2 (1.96±0.03), G3
(1.84±0.05), G4 (1.94±0.03) and G5 (1.76±0.04). After the
administration of carrageenan, the mice were injected
standard drug (Group 2nd) and ethanolic extract at selected
dose (G3 to G5). The results were noted at the interval of 1
hour. The group 2nd result was recorded 1.48±0.03, 1.11±0.03,
Journal of Pharmacognosy and Phytochemistry

1.01±0.03 and 0.08±0.04 (mm³) after 1 st, 2nd, 3rd and 4th hours of D.blanfordii was observed in the methanolic extract
respectively. The mice of group 3rd 1ml ethanolic extract was 66.08% (0.58±0.11) followed by the ethanolic extract 54%
injected at a dose of 100mg/kg and the paw edema volume (0.32±0.001) and aqueous 35.7% (0.63±0.01). The %
was noted 1.534±0.05, 1.33±0.03, 1.244±0.05 and 1.636±0.05 inhibition at 2 mg/ml concentration, the high % inhibition was
(mm³) after 1st, 2nd, 3rd and 4th hours respectively. The mice of observed in methanolic extract 67.5% (0.34±0.05) followed
group 4th 1ml ethanolic extract was injected at a dose of by ethanolic extract 45% (0.51±0.06) and aqueous extract
200mg/kg and the paw edema volume was recorded 25.9% (0.53±0.001) (Table 3; Fig 3).
1.69±0.03, 1.41±0.03, 1.37±0.03 and 1.22±0.03 (mm³) after
1st, 2nd, 3rd and 4th hours respectively. The mice of group 5 8. Qualitative detection of bioactive compounds of D.
was injected 1ml ethanolic extract at a concentration of blanfordii
300mg/kg and the paw volume was noted 1.65±0.03, Phytochemical detection of D.blanfordii in methanolic
1.32±0.03, 1.2±0.03 and 1.04±0.03 (mm³) after 1 st,2nd, 3rd and extracts showed the presence of carbohydrates, alkaloid,
4th hours respectively. The group 5th mice at the concentration flavonoid, phenol, saponin and tannin, but quinine was found
of 300mg/kg was observed a significant result at the 4th hours absent. Ethanolic extract showed the presence of
(1.04±0.03) followed by G3 and G4 (1.16±0.03) (Table 2; Fig carbohydrates, alkaloid, flavonoid, tannin phenol, saponin and
2). quinine. In aqueous extract, the detection of phytochemical
showed the presence of carbohydrates, alkaloid, flavonoid,
7.3 Antioxidant activity phenol, quinine and tannin, but saponine was found absent
(DPPH radical scavenging activity) (Table 4; Fig 4).
Antioxidant activity of D.blanfordii was carried out by using
methanolic, ethanolic and aqueous extracts. Extracts were Table 1: Analgesic activity of ethanolic extract of Dryopteris
subjected for the evaluation of antioxidant activity by using in blanfordii plant
vitro model systems. DPPH radical scavenging activity was Number of writhing in 5 Percent inhibition of
observed in all the extracts. Groups Dose
minutes (mean±SEM writhing
G1 10ml/kg 27.4±0.10 ----------
7.3.1 % inhibition antioxidant activity of the D.blanfordii 150mg/kg
G2 14±0.21 40.43
at concentration 1, 1.5 and 2mg/ml Aspirin
The antioxidant activity of D.blanfordii % inhibition was G3 100mg/kg 30** ±0.10 60.74
carried out at concentration of 1 mg/ml. The high % inhibition G4 200mg/kg 28* ±0.32 52.44
of D.blanfordii was observed in the methanolic extract 55.6% G5 300mg/kg 21** ±0.13 45
(0.31±0.03) followed by the ethanolic extract 48.7% ‘G’ stand for groups’ (Value are expressed in mean ±SEM) P* <
(0.542±0.05) and aqueous extract 30.58% (0.64±0.004). The 0.05 used as significant
% inhibition at 1.5mg/ml concentration the high % inhibition

Fig 1: Percentage (%) inhibition of writhing

Table 2: Anti-inflammatory activity in ethanolic extract of Dryopteris blanfordii plant.

Paw edema (mm3) Paw edema (mm3) after drug administration
Before carrageenin 1 hr. after carrageenin
Groups Dose 1r 2hrs 3hrs 4hrs
injection injection
(mean±SEM) (mean±SEM) (mean±SEM) (mean±SEM)
(mean±SEM) (mean±SEM)
G1 0.65±0.05 1.12±0.03 1.13*±0.05 1.13*±0.03 1.23**±0.03 1.13**±0.03
G2 Diclofenac sodium 10mg/kg 0.45±0.03 1.96±0.03 1.48**±0.03 1.11**±0.03 1.01**±0.03 0.08*±0.04
G3 100mg/kg 0.62±0.05 1.84±0.05 1.53*±0.05 1.33**±0.03 1.24*±0.05 1.13*±0.05
G4 200mg/kg 0.586±0.05 1.94±0.03 1.69**±0.03 1.41**±0.03 1.37**±0.03 1.22**±0.03
G5 300mg/kg 0.566±0.05 1.76±0.04 1.65**±0.03 1.32**±0.03 1.2**±0.03 1.04**±0.03
G’ stand for groups P*<0.05 is significant which compared with standard drug

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Fig 2: Percentage inhibition of anti-inflammatory activity

Table 3: Antioxidant activity of Dryopteris blanfordii Plant

Plant part used Concentration Extraction Mean±SD % inhibition
Methanol 0.31**±0.03 55.6
Whole part 1 mg/ml Ethanol 0.542*±0.05 48.7
Aqueous 0.64**±0.004 30.58
Methanol 0.58*±0.11 66.08
Whole part 1.5 mg/ml Ethanol 0.32**±0.001 54
Aqueous 0.63**±0.01 35.7
Methanol 0.34*±0.05 67.5
Whole part 2 mg/ml Ethanol 0.51*±0.06 45
Aqueous 0.53**±0.001 25.9
Value are expressed in mean ±SD) P* < 0.05 used as significant

Fig 3: Percentage (%) inhibition of Antioxidant activity

Table 4: Qualitative detection of bioactive compounds in Dryopteris Discussion

blanfordii Plant In the present study the analgesic, anti-inflammatory,
S.NO Phytochemical tests Methanol Ethanol Aqueous antioxidant activity and phytochemicals investigation of
1 Carbohydrates + + - Dryopteris blanfordii was carried out. The ethanolic extract of
2 Alkaloid + + + Dryopteris blanfordii showed significant results in analgesic
3 Flavonoid + + + and anti-inflammatory activities. In analgesic activity at a
4 Phenol + + + dose of 300mg/kg showed significant effect (21±0.13)
5 Saponin + + - writhing and 45% inhibition as compared to standard drug
6 Quanin - + + aspirin showed (14±0.21) writhing with 40.43% inhibition.
7 Tannin + + + The analgesic activity showed high potential due to the
(+) show presence and (-) show absence of phytochemical presence of alkaloids, flavonoids and phenolic contents
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Journal of Pharmacognosy and Phytochemistry
(Sultana et al., 2014) [21]. Alkaloids have been reported to
possess analgesic, antispasmodic and bactericidal,
antimalarial activities (Okwu and Okwu, 2004 [13]; Oomah,
2003) [16]. It was concluded that plant extract have high
flavonoids which showed high analgesic effect and without
flavonoids contents no analgesic effect (Oweyele et al.,
2005). Anti-inflammatory activity at a dose of 300 mg/kg
after four hour showed significant result followed by the dose
of 100mg/kg, 200mg/kg and standard drug diclofenac sodium
(10mg/kg). High dose of extract 300mg/kg reduce
inflammation (1.04±0.03) after 4th hours followed by group
3rd at the concentration of 200mg/kg at 4th hours (1.22±0.03).
Several reports are available on flavonoid groups which
exhibited high potential biological activities such as analgesic,
anti-inflammatory, antimicrobial, anti-angionic, anticancer
and anti-allergic reactions (Anyasor et al., 2010 [1]; Igbinosa
et al., 2009) [7]. Phytochemical detection in methanolic
extracts showed positive result except quinine. In ethanolic
and aqueous extract all test were positive but only
carbohydrates and saponine do not showed positive result in
aqueous extract. (Gracelin et al., 2013) [6] used methanolic
extract of five species of ferns in family Pteridaceae. Among
these selected ferns showed carbohydrates, alkaloids,
flavonoids and phenol, saponins, glycosides and tannins. The
antioxidant activity of D.blanfordii in 1mg/ml concentration
showed different result. High percent inhibition showed in
methanolic extract (55.6%) followed by the ethanolic extract
(48.7%) and aqueous extracts (30.58%), while the percentage
inhibition in 1.5mg/ml concentration mentioned in a table.
The high percent inhibition was observed in methanolic
extract (66.08%) followed by the ethanolic extract (45%) and
aqueous (35.7%). The percent inhibition in 2mg/ml
concentration were observed in methanolic extract (67.5%)
followed by the ethanolic (45%) and aqueous (25.9%).
Tannins and their derivatives are phenolic compounds
considered to be primary antioxidants or free radical
scavengers (Barile et al., 2007; Ayoola et al., 2008) [2, 3].
Flavonoids are known to possess antioxidant anticancer,
antiviral and anti- inflammatory properties (Valizadeh et al.,
2015). Phenolic compounds are well known to possess
biological activities such as antioxidant, antidiabetic,
hepatoprotective, anti-inflammatory, antimicrobial and
anticancer (Gracelin et al., 2013) [6].
Conclusion
In conclusion, the ethanolic extract of Dryopteris blanfordii
was proved a natural safe remedy for the treatment of
analgesia and inflammation. Our current findings
demonstrated scientific rationale for the folk use of the plant
as analgesic, anti-inflammatory and antioxidant potential.
Interestingly the D.blanfordii exhibited both peripheral as
well as central analgesic effect which might have been
attributed to the presence of such active principles, due to
which it has proven folk use in various nervous disorders.
Nevertheless, the isolation of pure secondary metabolites
from the plant will help us further in understanding the
mechanism of these activities and identification of lead
compounds of clinical utility.
Acknowledgments
The authors are very thankful to my lab fellow and my great
supervisor Dr. Gul Jan, my co-supervisor Muhammad Israr
and my seniors Muddaser Shah, Siraj Khan, Naimat Ullah
abid, Syed abid Ullah, Zahid Ullah for sharing the valuable
knowledge during current study.
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