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and Industrial Health

Antibacterial, antioxidant and cytotoxic studies of total saponin, alkaloid and sterols contents of
decoction of Joshanda: Identification of components identification through thin layer chromatography
Haroon Khan, Murad Ali Khan and Ab Dullah
Toxicol Ind Health published online 12 December 2012
DOI: 10.1177/0748233712468023

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Article
Toxicology and Industrial Health
1–7
Antibacterial, antioxidant and © The Author(s) 2012
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cytotoxic studies of total saponin, DOI: 10.1177/0748233712468023
tih.sagepub.com
alkaloid and sterols contents
of decoction of Joshanda:
Identification of components
through thin layer chromatography

Haroon Khan1, Murad Ali Khan2 and Abdullah2

Abstract
The current study was aimed to assess antibacterial, antioxidant and cytotoxic activity of total saponin, alkaloid
and sterol contents of Joshanda decoction followed by its constituent’s analysis via thin layer chromatography
(TLC). Saponins and alkaloids showed prominent antibacterial activity against Staphylococcus aureus, Bacillus
subtilis, Bacillus cereus and Klebsiella pneumoniae whereas sterols only against S. aureus. Saponin and alkaloid
contents of 97 and 108 mg/ml, respectively, showed prominent free radical scavenging activity against
1,1-diphenyl-2-picrylhidrazyl, with mild cytotoxicity in brine shrimp cytotoxic test. Under ultraviolet light at
254 nm, TLC of total saponins showed eight different compounds, total sterols comprising three while total
alkaloids two compounds of various polarities. It is concluded that the various contents of Joshanda decoction
possess outstanding susceptibility against bacteria implicating primarily upper respiratory tract infections
augmented by strong antioxidant activity.

Keywords
Joshanda, saponins, alkaloids and sterols, antibacterial, antioxidant

Introduction potential on scientific grounds (Khan et al., 2010,

Joshanda is a polyherbal formulation of Unani origin
(Greco-Arab). It is largely used against the inflamma-
tion of the mucous membranes of nose and air
passages (Azmi et al., 2010). Joshanda is one of the
leading household remedy against upper respiratory
infections, catarrh, cold and flu in Pakistan. Such rem-
edy is even practiced commonly in paediatric age
groups (Ashraf et al., 2010). This polyherbal formula-
tion consists of expectorant, respiratory demulcent
and anticatarrhal herbs, which assist in relieving the
enervating cough. It is also recommended for the
treatment of premenstrual syndrome (Akram et al.,
2011). The effect of the drug on the bronchial smooth
muscles of isolated tissues is already explored (Khe-
terpal et al., 1989). The anti-inflammatory activity
of the Joshanda decoction was strongly augmented
(Khan et al., 2012a). It is the continuation of our effort
to explore Pakistani medicinal plant for their therapeutic
2011a, 2011b, 2012b, 2012c; Saeed et al., 2010a).
While considering multiple traditional uses of
Joshanda, the current study was planned (1) to deter-
mine the total saponin, alkaloid and sterol contents of
Joshanda decoction (2) to evaluate their antibacterial,
antioxidant and cytotoxic activity and (3) to assess
their status for bioactive constituents over chromato-
graphic plat using thin layer chromatography (TLC).
1
Gandhara College of Pharmacy, Gandhara University, Peshawar,
Pakistan
2
Department of Chemistry, Kohat University of Science and
Technology, Kohat, Pakistan
Corresponding author:
Haroon Khan, Gandhara College of Pharmacy, Gandhara
University, Peshawar 251200, Pakistan
Email: harronkhan3@yahoo.com

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2 Toxicology and Industrial Health
Materials and methods
Sample collection
Joshanda (Hamdard Laboratories, Waqf, Pakistan)
was obtained in commercial pack from a herbal
medical store in Peshawar. Each commercial packet
contained the same composition of mentioned plants.
Each 100 g packet of Joshanda contains 3 g of Althaea
officinalis, 9 g of Cordia latifolia, 5 g of Glycyrrhiza
glabra, 3 g of Malva sylvestris, 5 g of Onosma bractea-
tum, 5 g of Viola odorata and 5 g of Zizyphus sativa.
Sample preparation
All the materials from the commercial packets were
taken out and ground to powder form using grinding
machine. The powdered materials were weighed. The
sample was taken in hot water at 70 C for 24 h to
make a decoction. Then it was filtered while hot on
Buckner funnel using vacuum pump. The filtrate was
centrifuged for 40 min at 5000 rpm to separate the
solid particles. The liquid mixture was concentrated
using vacuum rotary at 70 C (Khan et al., 2012a).
Subsequently it was dried in oven and finally ground
to powder using mortar and pistol. The percentage of
yield was calculated as 17.3%.
Quantitative screening
Total saponin contents. Twenty grams of ground sam-
ple were dispersed in 20 ml of 20% ethanol. The sus-
pension was heated on a water bath for about 4 h at
about 55 C with constant stirring. Then this mixture
was filtered and the residue was re-extracted with
another 20 ml of 20% ethanol. The volumes of the
combined extracts were reduced to 40 ml on a water
bath at about 90 C. The concentrate was shifted to a
250-ml separator funnel. Diethyl ether, 20 ml, was
added to this mixture and the separator funnel was
shaken vigorously. The aqueous layer was recovered.
Then 60 ml of n-butanol (n-BuOH) was added. The
combined n-BuOH extracts were washed twice with
10 ml of 5% aqueous sodium chloride. The remaining
solution was evapourated on a water bath. Then the
samples were dried in oven to a constant weight. The
percentage of saponin was calculated (Khan et al.,
2010).
Total sterol contents. Powder sample was extracted
with methanol three times and was concentrated.
Then it was suspended in 5% methanol and filtered.
Aqueous extract was exhaustively extracted with
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hexane. The resulting hexane-soluble extract was eva-
pourated and dried, which accounts for total sterol
contents.
Total alkaloid contents. Dry sample, 3.0 g, was added to
120 ml of 20% acetic acid in ethanol and was covered
to stand for 4 h. Then it was filtered, and the extract
was concentrated on a water bath to one quarter of the
original volume. Then concentrated ammonium
hydroxide (NH4OH) was added dropwise to complete
the precipitation. Solution was allowed to settle. The
precipitate was collected by filtration and weighed.
Percentage of alkaloids was calculated (Khan et al.,
2010).
Antibacterial assay. The crude extract and its subse-
quent solvent fractions of rhizomes were evaluated
against a variety of human pathogens by agar well-
diffusion method as described previously (Khan
et al., 2008). Briefly, the test material (3 mg/ml) was
dissolved in dimethyl sulphoxide (DMSO). Approxi-
mately 45 ml of molten nutrient agar was dispensed
on sterilized Petri plates and was permitted to harden.
Bacteria were dispersed on these nutrient agar plates
by preparing sterile soft agar accumulating 100 ml
of bacterial culture. Long sterile metallic borer of
6 mm was used for wells digging at suitable distance
and spotted for identification. Sample (100 ml) was
poured into each well, and the plates were incubated
at 37 C for 24 h. The antibacterial activity was esti-
mated in terms of inhibition zone. Imipenem (a
broad-spectrum b-lactam antibiotic) and DMSO were
used as positive and negative controls, respectively.
Minimum inhibitory concentration determination
(macrodilution method). For minimum inhibitory con-
centration (MIC) determination, contents (10 mg/
ml) were dissolved in DMSO. Sterile water was used
for dilution, consecutively in a 96-well microplate and
kept in a laminar flow cabinet. Similar concentration
of an actively growing culture of the test microbes
was added to different wells and cultures were grown
for 12 h in 100% relative humidity at 37 C. Later on,
wells were felt with tetrazolium violet. Violet colour
of the culture signifies growth. MIC was rated as the
least drug concentrations accountable for the preven-
tion of growth. Acetone was used as negative control,
whereas imipenem, amphotericin B and miconazole
were the positive controls in the assay (Khan et al.,
2012c).
Khan et al.
Bacterial and fungal strains. Assays were executed on
various pathogenic bacterial strains. Bacterial strains
were Bacillus subtilis ATCC 6633, Bacillus cereus
(clinical isolate), Shigella flexneri (clinical isolate),
Staphylococcus aureus ATCC 25923, Escherichia
coli ATCC 25922, Pseudomonas aeruginosa ATCC
27853 and Salmonella typhi ATCC 19430. The strains
were kept on agar slant at 4 C. Before any test, the
strains were activated at 37 C for 24 h on nutrient
agar (Khan et al., 2012c).
Free radical scavenging assay. To study the free radical
scavenging activity (RSA), Joshanda contents were
tested against the stable free radical 1,1-diphenyl-
2-picrylhidrazyl (DPPH; Khan et al., 2012d). The
solution of 10, 50 and 100 mg/ml of DPPH
(0.1 mM) was prepared by dissolving it in ethanol and
kept in dark. DPPH solution of 1 ml was added to 3 ml
of various drug concentrations. The absorbance was
taken at 517 nm in terms of colour change. The anti-
oxidant activity of the test materials was calculated by
comparison of the results with the ethanol. Ethanol
was used as negative control while vitamin C as a
standard antioxidant drug. All the analyses were
performed in triplicate. The percentage of free RSA
was calculated using the following formula:
ðcontrol absorbance  sample absorbanceÞ
RSA ð%Þ ¼  100
control absorbance
Brine shrimp cytotoxic assay
Brine shrimp cytotoxic assay of Joshanda contents
was determined using our previously reported method
(Saeed et al., 2010b). In brief, test samples were pre-
pared in respective solvents in the concentrations of
10, 100 and 1000 mg/ml. Brine shrimp (Artemia salina
Leach) nauplii were hatched in a specific tank at room
temperature. From stock solutions, 5, 50 and 500 mg/
ml were injected into nine vials (three vials for each
dilution). Each vial contained 10 shrimps and 5 ml
of brine solution. The vials were supplemented with
dry yeast suspension as their food and were incubated
for 24 h under illumination. For analysis, the live nau-
plii were counted using a 3 magnifying glass and the
percentage deaths at each dose were calculated. The
resulting data were processed using Finney programme
on a simple computer to estimate lethal dosage (LD50)
values.
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3
Thin layer chromatography
Alkaloids. The powder sample was soaked with a half
diluted NH4OH and lixiviated with ethyl acetate for
24 h at room temperature. The organic phase was
separated from the acidified filtrate and was basified
with NH4OH (pH 11–12). It was then extracted with
chloroform (3), condensed by evapouration and
used directly for TLC. Chloroform and methanol
(15:1) mixture was used for the separation of alkaloid
spots. The colour and retention factor (Rf) values
were recorded under ultraviolet (UV at 254 nm) as
well as visible light after spraying the TLC plates with
Dragndroff reagent (Wanger and Bladt, 1996).
Saponins
Two grams of powdered sample were extracted with
10 ml of 70% ethyl alcohol by refluxing for 10 min.
The filtrate was condensed and saturated n-BuOH,
in excess, was added to it with constant mixing. The
butanol portion was condensed and used for chroma-
tography. Chloroform, glacial acetic acid, methanol
and water (64:34:12:8) solvent mixture was used for
the separation of saponin spots. Chromatogram was
exposed to iodine vapours to visualize the colours.
The colours and Rf values of these spots were
recorded (Wanger and Bladt, 1996).
Sterols
Two grams of powdered sample was extracted using
10 ml methyl alcohol in water bath (80 C/15 min).
The condensed filtrate was directly used for TLC.
Chloroform, glacial acetic acid, methanol and water
(64:34:12:8) solvent mixture was used for the separa-
tion of sterol spots. The chromatograms were sprayed
with anisaldehyde–sulphuric acid reagent and heated
to 100 C/6 min then the colour and Rf values of the
separated spots were recorded under visible light
(Wanger and Bladt, 1996).
Results
Effect of quantitative analysis
The results of quantitative analysis of the total
saponin, alkaloid and sterol contents of Joshanda
decoction are shown in Table 1. The concentration
of different contents was 10.4%, 2.9% and 1% for
saponin, alkaloids and sterols, respectively.
4 Toxicology and Industrial Health
Table 1. Quantitative determination (%) of different phy-
tochemical groups of constituents of Joshanda decoctiona
S. no. Constituents Percentage of yield
1 Saponins 10.4 + 1.1
2 Sterols 2.9 + 1.3
3 Alkaloids 1.0 + 0.03
a
Data are mean of three different findings.
Effect of antibacterial activity
The details of bacterial susceptibility of Joshanda
contents are illustrated in Tables 2 and 3. When
exposed to total saponin and alkaloid contents, all the
tested four gram-positive bacteria showed profound
susceptibility. The MICs for saponins against S. aur-
eus, S. pneumoniae, B. subtilis and B. cereus were
1.40, 2.10, 4.70 and 3.40 mg/ml, respectively, whereas
the MICs for alkaloids against S. aureus, S. pneumo-
niae, B. subtilis and B. cereus were 9.20, 13.70,
1.10 and 2.60 mg/ml, respectively. Total sterols were
only effective against S. aureus (MIC: 1.90 mg/ml).
Of the tested four gram-negative bacteria, Kleb-
siella pneumoniae was the only bacterium responsive
to saponin and alkaloid contents with MICs of 6.50
and 8.20 mg/ml, respectively.
Effect of antioxidant activity
Against DDPH free radical, the scavenging activity of
the total contents of Joshanda decoction is present in
Table 4. The saponin contents were most potent with
half maximal inhibitory concentration (IC50) value of
97 mg/ml followed by alkaloid contents 108 mg/ml.
However, sterol content did not exhibit any antioxi-
dant activity against DDPH.
Effect of brine shrimp cytotoxicity
The effect of brine shrimp cytotoxicity in subse-
quently extracted contents of decoction is shown in
Table 5. Mild cytotoxicity was observed; saponin con-
tents (IC50: 1093 mg/ml) alkaloids (IC50: 947 mg/ml)
and sterols (IC50: 1104 mg/ml).
Effect of TLC studies
The Rf values of total saponin contents of Joshanda
decoction are present in Table 6. When observed
under UV lamp at 254 nm, it showed eight different
compounds of variable polarity. When the total sterol
and alkaloids contents of decoction were subjected to
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TLC, the UV lamp at 254 nm showed three and two
different compounds, respectively.
Discussion
The results of our study revealed considerable accu-
mulation of saponins, alkaloids and sterols in the
Joshanda decoction with prominent antibacterial
activity especially against pathogens that implicating
in upper respiratory tract infections (URIs) augmen-
ted by antioxidant activity strongly indicating towards
its therapeutic potential.
Gram-positive bacteria are primarily causative
agents of upper respiratory infections, especially Sta-
phylococcus and Bacillus species are well recognized
(Logan, 1988; Pettigrew et al., 2008). Over time, the
nasopharyngeal flora changes, but the overall bacterial
colonization has been noted to be significantly higher
during upper respiratory infection. However, the magni-
tude of effects in patients mostly fluctuated according to
the species of bacteria. Noteworthy, children are more
susceptible to secondary bacterial infections during and
after URI (Pettigrew et al., 2008).
Saponins occur extensively in various plant species
and exhibit a wide range of biological properties, both
beneficial and deleterious. The natural role of sapo-
nins in plants has been recognized as protection
against attack by pathogens and pets (Mert-Turk,
2006). In our findings, the saponin contents of
Joshanda demonstrated profound antibacterial activ-
ity selectively against gram-positive pathogens. It
could therefore be assumed that the chemical nature
behind our current finding may be of saponins. Addi-
tionally, the TLC studies of saponin contents con-
firmed the presence of eight different compounds.
The antibacterial potential of saponins has been
recognized (Hassan et al., 2010; Soetan et al.,
2006). Previously published research of individual
plants used in Joshanda also supported our findings
(Azmi et al., 2010; Walter et al., 2011).
Total alkaloidal contents of Joshanda also mani-
fested marked antibacterial activities like the saponin
contents. It was also specifically against gram-
positive bacteria. It is therefore suggested that the
alkaloidal contents of Joshanda strongly supported the
saponin contents in its activity. However, the TLC
studies identified only two compounds. Various stud-
ies are available consistent with our findings (Pfoze
et al., 2011; Tanaka et al., 2006).
In the presence of oxygen, bacterial adherence
induces colonization and supports various infections
Khan et al. 5

Table 2. Effect of total saponins, alkaloids, sterols and proteins extracted from Joshanda decoction against various patho-
genic bacterial strains in terms of the zone of inhibitiona
Zone of inhibition (ml)
Bacteria DMSO Saponin Alkaloids Sterols Ciprofloxacin Erythromycin
Gram-negative
K. pneumoniae – 19.0 + 0.02 18.5 + 0.02 – 27 + 0.02 –
S. typhi – 0.00 0.00 – 28 + 0.01 –
P. aeruginosa – 0.00 0.0 – 15 + 0.02 –
E. coli – 0.00 0.00 – 31 + 0.02 –
Gram-positive
S. aureus – 29.4 + 0.02 16.5 + 0.01 23 + 0.01 – 26.9 + 0.03
S. pneumoniae 22.3 + 0.02 19.6 + 0.02 – 27.1 + 0.01
B. subtilis – 18.00 + 0.02 25.5 + 0.03 – – 27 + 0.02
B. cereus – 19.0 + 0.03 25.2 + 0.01 – – 25 + 0.01
DMSO: dimethyl sulphoxide.
a
Data are SEM of three independent assays.

Table 3. Effect of total saponins, alkaloids, sterols and proteins extracted from Joshanda decoction against various patho-
genic bacterial strains in terms of MICa
MIC (mg/ml)
Bacteria DMSO Saponin Alkaloids Sterols Ciprofloxacin Erythromycin
Gram-negative
K. pneumoniae – 6.50 + 0.1 8.20 + 0.1 – 0.23 + 0.01 –
S. typhi – 0.00 0.00 – 0.19 + 0.01 –
P. aeruginosa – 0.00 0.0 – 2.1 + 0.01 –
E. coli – 0.00 0.00 – 0.12 + 0.00 –
Gram-positive
S. aureus  1.40 + 0.1 9.20 + 0.3 1.90 + 0.2 – 0.23 + 0.01
S. pneumoniae 2.10 + 0.2 13.70 + 0.02 – 0.23 + 0.01
B. subtilus – 4.70 + 0.2 1.10 + 0.1 – – 0.27 + 0.00
B. cereus – 3.40 + 0.1 2.60 + 0.1 – – 0.19 + 0.00
DMSO: dimethyl sulphoxide; MIC: minimum inhibitory concentration.
a
Data are SEM of three independent assays.

Table 4. Effect of total saponins, alkaloids, sterols and pro- contents of Joshanda exhibited potent free radical
teins extracted from Joshanda decoction on DPPH scavenging. Thus, it further strengthened the anti-
infectious potential of Joshanda contents. The antioxi-
Test organism Drugs IC50 (mg/ml) dant activities of saponins and alkaloids are already
DPPH Saponin 97 reported in the literature (Jie-Lun et al., 2012; Maiza-
Alkaloids 108 Benabdesselam et al., 2007). Additionally, the saponin
Sterols – and alkaloid contents showed mild cytotoxicity.
Vitamin C 51.3 The global recognition of the potential problems
IC50: half maximal inhibitory concentration; DPPH: 1,1-diphenyl-2-
associated with the use of antibiotics motivated
picrylhidrazyl. research efforts to identify alternatives to their use.
The outstanding antibacterial receptivity of saponin
and alkaloid contents specifically against bacteria,
and inflammation (Arita et al., 2007). In this context, implicating URI which was further augmented by
antioxidant plays a pivotal role by discouraging the their potent antioxidant potential, provided scientific
proliferation of infections. The saponin and alkaloid background to this household remedy against URI.

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6 Toxicology and Industrial Health

Table 5. Effect of total saponins, alkaloids, sterols and pro- activities of saponin-rich extracts from guar meal. Food
teins extracted from Joshanda decoction against Artemia Chemistry 119: 600–605.
salina leach Jie-Lun H, Shao-Ping N, Dan-Fei H, Chang L and Ming-
Test organism Drugs LD50 (mg/ml) Yong X (2012) Extraction of saponin from Camellia
oleifera cake and evaluation of its antioxidant activity.
Artemia salina leach Saponin 1093 International Journal of Food Science and Technology
Alkaloids 947 47: 1676–1687.
Sterols 1104
Khan H, Khan MA, Mahmood T and Choudhary M (2008)
Etoposide 07.4625
Antimicrobial activities of Gloriosa superba Linn (Col-
LD50: lethal dosage. chicaceae) extracts. Journal of Enzyme Inhibition and
Medicinal Chemistry 23: 855–859.
Table 6. TLC data of constituents of Joshanda in terms of Khan H, Saeed M, Gilani AH, Khan MA, Dar A and Khan I
Rf values (2010) The antinociceptive activity of Polygonatum ver-
ticillatum rhizomes in pain models. Journal of
Rf values (cm) Ethnopharmacology 127: 521–527.
S. no. Saponins Sterols Alkaloids Khan H, Saeed M, Gilani AH, Khan MA, Khan I and Ash-
raf N (2011a) Anti-nociceptive activity of aerial parts of
1 0.389 0.414 0.467 Polygonatum verticillatum: attenuation of both periph-
2 0.454 0.714 0.854 eral and central pain mediators. Phytotherapy Research
3 0.619 0.9 –
25: 1024–1030.
4 0.636 – –
5 0.688 – – Khan H, Tariq SA, Khan MA and Inayat-Ur-Rehman Ghaffar
6 0.753 – – R and Saifullah (2011b) Cholinesterase and lipoxygenase
7 0.831 – – inhibition of whole plant Withania somnifera. African
8 0.948 – – Journal of Pharmacy and Pharmacology 5: 2272–2275.
Khan H, Tariq SA and Khan MA (2011c). Biological and
TLC: thin layer chromatography; Rf: retention factor.
phytochemical studies on corms of Colchicum luteum
Baker. Journal of Medicinal Plants Research 5:
Moreover, it also encourages scientific community to
7031–7035.
combat bacterial resistance in a natural way.
Khan H, Khan MA, Muhammad N, Ashraf N, Gul F and
Funding Tariq SA (2012a) Antiinflammatory and antioxidant
This research received no specific grant from any funding activity of Joshanda partially mediated through
agency in the public, commercial, or not-for-profit sectors. inhibition of lipoxygenase. Phytopharmacology 3:
19–28.
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