International Journal of Pharmacy. Photon 106 (2015) 445-452
https://sites.google.com/site/photonjournals/home/international-journal-of-pharmacy
Research Article. ISJN: 8237-7516: Impact Index: 5.50
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International Journal of Pharmacy
The bark extracts of Himalayan gymnosperm Picea smithiana (Wall.): A
natural sources of antibacterial and antioxidant agent
S.C. Sati*, P. Kumar, S. Joshi
Department of Botany, D.S.B. Campus, Kumaun University, Nainital-263002, India
S.C. Sati, P. Kumar and S. Joshi receive Gregor
Mendel Research Award- 2015 in Pharmacy
Article history:
Received: 13 September, 2014
Accepted: 16 September, 2014
Available online: 23 March, 2015
Keywords:
Picea smithiana, phytochemical screening, antibacterial activity,
and gymnosperms plant extract
Abbreviations:
ZOI: Zone of Inhibition, MIC: Minimum Inhibitory
Concentration, MBC: Minimum Bactericidal Concentration
Corresponding Author:
Sati S.C.*
Professor
E-mail: [email protected]
Kumar P.
Research Scholar
Joshi S.
Assistant Professor
Abstract
The bark extract of Kumaun Himalayan
gymnosperm Picea smithiana was assessed for its
active principles. Flavonoids, terpenoid, tannins,
alkaloids,
saponin,
glycosides,
quinines,
carbohydrates, protein, starch, resin, volatile oil,
anthraquinone and phenol were found in both
methanol and ethanol bark extracts. The
antibacterial potential of methanol and ethanol
extracts of bark was investigated against
Agrobacterium tumefaciens, Bacillus subtilis,
Escherichia coli, Erwinia chrysanthemi and
Xanthomonas phaseoli using disc diffusion method.
The extracts were further evaluated for minimum
inhibitory concentration (MIC) and minimum
bactericidal concentration (MBC). Both methanol
and ethanol extracts of P. smithiana bark were
found effective by showing a mark zone of inhibition
(ZOI). Methanol extract showed the highest
inhibitory activity against A. tumefaciens (ZOI, 19
mm) while ethanol extract exhibited its highest
activity for E. chrysanthemi (ZOI, 12 mm). The MIC
and MBC values were recorded in the range 31.25250 µg/ml and 62.5-500 µg/ml, respectively. The
lowest value of MIC and MBC were recorded
against A. tumefaciens in methanol extract (31.25
µg/ml and 62.5 µg/ml respectively). The results of
bark extracts were also compared with leaf extracts
of P. smithiana for antimicrobial potential.
Citation:
Sati S.C., Kumar P., Joshi S., 2015. The bark extracts of
Himalayan gymnosperm Picea smithiana (Wall.): A natural
sources of antibacterial and antioxidant agent. International
Journal of Pharmacy. Photon 106, 445-452.
All Rights Reserved with Photon.
Photon Ignitor: ISJN82377516D749323032015
1. Introduction
It is a common view that the natural products are
healthier, harmless and more reliable when
compared to synthetic products. Nearly, about 80%
of the world’s inhabitants relying mainly on
traditional medicines for their primary health care
(Owolabi et al., 2007). The chemical constituents
of plant extracts are a part of the physiological
activities of living plants and hence they are
believed to have a better compatibility with the
human body. It is well known that there is paucity
of information on the phytochemical studies and
the fraction investigated for biology or
pharmacology is also quite meagre. Thus the
phytochemical investigation of a given plant is
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desirable as broad spectrum constituents in
bioactivities.
Healing potential of plant extracts is well known
and antimicrobial principal is one of the elements
besides other responsible for the healing.
Unfortunately, in the last one decade the pace of
development of new antimicrobial drug has slowed
down while the prevalence of resistance (especially
multiple) has increased tremendously (Hugo and
Russell, 1984). To increase the alternative
resources of antimicrobial drugs one approach is to
explore local ethnobotanicaly known medicinal
plants which have natural antimicrobial agents
445
(Khulbe and Sati, 2009). The search for novel
natural bioactive compounds, a foundation to new
drug discovery is receiving greater attention as
previously reliable standard new strains of multi
drug resistant pathogens (Muller, 2001).
and Erwinia chrysanthemi were obtained from G.
B. Pant University, Pantnagar, India.
Figure 1. Picea smithiana , a. leaves, b. close view of
bark
Ethnobotanical records and available literature
indicate that plants are the sleeping giants of
Pharmaceutical
industry
(Hamburger
and
Hostettmann, 1991). The drugs present in
medicinal plants are divided chemically into a
number of groups like alkaloids, carbohydrate,
protein,
glycosides,
terpenoids,
steroids,
flavonoids, tannins, saponins, starch, volatile oils,
phlobatannins,
resins,
fat,
quinone
and
anthroquinone (Parmer et al., 1999; Harborne and
Baxter, 2001). They may provide novel or lead
compounds which may be used as a natural source
of antimicrobial drugs in controlling various
diseases of plants and animals. Therefore, such
plants should be investigated to enrich the
knowledge and their properties, safety and efficacy
(Nascimento et al., 2000).
The most important bioactive compounds of plants
are alkaloids, flavonoids, tannins and phenolic
compounds that produce a definite physiologic
action on the human body. The phytochemical
research
based
on
ethno-pharmacological
information is generally considered an effective
approach in the discovery of new anti-infective
agents from higher plants (Duraipandiyan et al.,
2006).
1.1 Justification/ aim
Thus keeping the above account in mind the
present investigation was carried out on the
phytochemical and antibacterial assessment of stem
bark extracts of Himalayan spruce Picea smithiana
(Wall.).
2. Experimental
2.1 Materials and methods
2.1.1 Collection of plant material
Picea smithiana Wall. (Pinaceae) is a beautiful
ornamental big gymnospermous tree plant
commonly known as west Himalayan spruce, (Fig.
1.a). The bark of P. smithiana was collected from
Nainital, Kumaun Himalaya, India (Fig. 1.b) and
authenticated by the Department of Botany,
Kumaun University, Nainital. A voucher specimen
was deposited in the herbarium.
2.1.3 Microorganisms Used
Five (Gram +ve and −ve) bacteria Bacillus subtilis
MTCC 121, Escherichia coli MTCC 40,
Agrobacterium tumefaciens MTCC 609, were
obtained from Institute of Microbial Technology,
Chandigarh, India whereas Xanthomonas phaseoli
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2.1.2 Extraction procedure
The bark of P. smithiana was thoroughly washed
and dried at room temperature. The dried material
was powdered in an electric grinder. To prepare a
stock solution, 50 g of this powder was added to
200 ml of solvents (w/v). Solvents used for
extraction were methanol and ethanol. Powdered
bark was dissolved in solvent in 250 ml Erlenyer
flask and kept in a shaker for 6-10 hrs. The
prepared extract was filtered through Whatman
filter paper no.1. The final filtrate was concentrated
on a rotary evaporator under vacuum at 20° C and
utilized for antibacterial assessment (Mohanta et
al., 2007).
2.2 Phytochemical analysis of extract
The phytochemical analysis of methanol and
ethanol bark extracts were carried out by using
standard procedures following Harborne (1998) as
given below:
2.2.1 Tests for Carbohydrates
Molisch’s test: 1 ml of extract was treated with
few drops of Molisch’s reagent (α-naphthol, 20%
in ethyl alcohol). Then about 1ml of concentrated
sulfuric acid was added belatedly along the sides of
the tube. Formation of violet color indicates the
presence of carbohydrates.
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Fehling’s test: 1 ml of Fehling’s A (Copper
sulphate in distilled water) and 1ml of Fehling’s B
(Potassium tartarate and sodium hydroxide in
distilled water) reagents were mixed and boiled for
minute. Then equal volume of test solution was
added to the above mixture. The solution was
heated in a boiling water bath. Brick red precipitate
was observed, indicating the presence of
carbohydrates.
Iodine test: Crude extract was mixed with 2 ml of
iodine solution. A dark blue or purple coloration
indicated the presence of the carbohydrate.
2.2.2 Test for Proteins
Millon’s test: Crude extract when mixed with 2ml
of Millon’s reagent, white precipitate appeared
which turned red upon gentle heating that
confirmed the presence of protein.
Xanthoproteic test: The extracts were treated with
few drops of conc. Nitric acid. Formation of yellow
colour indicates the presence of proteins.
2.2.3 Tests for Glycosides
Keller- Killiani test: 1ml of glacial acetic acid
containing traces of ferric chloride and 1ml of
concentrated sulfuric acid, 1ml of extract was
added carefully. Appearance of brown ring at the
interface shows the presence of glycosides. A
violet ring may also appear below the brown ring.
Legal’s test: Extracts were treated with sodium
nitropruside in pyridine and sodium hydroxide.
Formation of pink to blood red colour indicates the
presence of cardiac glycosides.
Borntrager’s test: To 1ml of extract, 1ml of
benzene and 0.5 ml dilute ammonia solution were
added. A reddish pink color indicates the presence
of glycosides.
Baljet test: To 1ml of extract, 1ml of sodium
picrate is added. Appearance of yellow to orange
color detects the presence of glycosides.
2.2.4 Tests for Saponins
Foam test: 1 ml of extract was shaken vigorously
with 20 ml of distilled water for 5- 10 minutes in
graduated cylinders. Formation of one centimeter
layer of foam indicated the presence of saponins.
2.2.5 Test for Terpenoids
Salkowski test: 5 ml of each extract was mixed in
2 ml of chloroform, and concentrated sulphuric
acid (3 ml) was carefully added to form a layer. A
reddish brown coloration of the inter face was
formed to show positive results for the presence of
terpenoids.
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Trichloroacetic acid test: To 1ml of extract, 2 ml
of trichloroacetic acid was added. Formation of
colored precipitate showed the presence of
terpenoids.
2.2.6 Tests for Phenolic compounds
Ferric chloride test: Extracts were treated with 34 drops of ferric chloride solution. Formation of
bluish black colour indicates the presence of
phenols and tannin.
Lead acetate test: On addition of lead acetate
solution to the extract white precipitate appeared.
Dilute HNO3 test: On addition of dilute HNO3
solution to the extract reddish colour appeared.
2.2.7 Test for Alkaloids
Mayer’s test: Filtrates were treated with Mayer’s
reagent (Potassium Mercuric Iodide). Formation of
a yellow coloured precipitate indicates the presence
of alkaloids.
Wagner’s test: Filtrates were treated with
Wagner’s reagent (Iodine in Potassium Iodide).
Formation of brown/reddish precipitate indicates
the presence of alkaloids.
Dragendroff’s test: To 1 ml of extract, 2ml of
Dragendroff’s reagent is added. Orange red
precipitate is formed indicating the presence of
alkaloids.
2.2.8 Test for Flavonoids
Alkaline reagent test: Small quantity of each
extract sample was taken and added with lead
acetate solution. After few minutes appearance of
yellow colour precipitates which indicated the
presence of flavonoids.
Shinoda test: To 1 ml of extract, few drops of
concentrated HCl were added. To this solution 0.5
gram of magnesium turnings were added.
Observance of pink coloration indicated the
presence of flavonoids.
Lead acetate test: To the 1 ml of extract, lead
acetate solution was added. Formation of yellow
precipitate showed the presence of flavonoids.
Ferric chloride test: To 1ml of extract, 1ml of
ferric chloride (5% in water) was added. Formation
of brown color confirmed the presence of
flavonoids.
2.2.9 Test for Starch
To 1 ml of aqueous extract was added 10 ml of
NaCl saturated solution. After heating, starch
reagent was added a blue-purplish colour is a
positive test for the presence of starch.
447
2.2.10 Test for Volatile Oils
NaOH-HCl test: 2 ml of extract solution was
shaken with 0.1ml of dilute sodium hydroxide and
a small quantity of dilute HCl. A white precipitate
was formed with volatile oils.
2.2.11 Test for Phlobatannins
1 ml of extract was boiled with 1% aqueous HCl.
The formation of red precipitate indicated the
presence of phlobatannins.
2.2.12 Tests for Resins Turbidity test
Distilled water (5ml) was added to the extract. The
occurrence of turbidity showed the presence of
resins.
2.2.13 Test for Quinones
Few drops of concentrated Sulphuric acid was
added to 1 ml of extract appearance of red colour
indicate the presence of quinons.
2.2.14 Test for Tannins
In 1 ml of extract add few drops of 5% FeCl3.
Appearance of green colour indicates presence of
gallotannin, and brown colour indicates the
presence of tannins.
2.2.15 Test for Fat
Few drops of sudanIII added in to 1 ml of extract,
red colour appears with oil droplets.
2.2.16 Test for Anthraquinones
Few drops of concentrated Sulphuric acid were
added to 5 ml of extract. To this solution 1 ml of
ammonia was added. Appearance of rose-pink
colour indicates presence of anthraquinones.
2.3 Screening for Antibacterial Activity
Antibacterial tests of P. smithiana bark extract on
selected microorganisms were carried out using
disc-diffusion method (Bauer et al., 1966). A small
sterile cotton swab was dipped into the 24-hour-old
culture of bacteria and was inoculated by streaking
the swab over the entire agar surface. After
inoculation the plates were allowed to dry at room
temperature in laminar chamber. The filter paper
discs (5 mm) loaded with 40µl of extract were
placed on the surface of the agar plates. After 5 min
the plates were kept for incubation at 37±2° C for
24 h. Gentamycin (30 mg) was used as positive
controls and the respective solvent were taken as
negative control. After 24 h of incubation, the
dishes were observed for bioactivity (Plate- 1) and
the diameter was observed for zone of inhibition
(ZOI). All tests were performed in triplicate and
observed values of ZOI are expressed as mean
value with standard error of means (SEM).
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2.4 Determination of Minimum Inhibitory
Concentration (MIC) and Minimum Bactericidal
Concentration (MBC)
All the fractions were further tested for the
minimum inhibitory concentration and minimum
bactericidal concentrations. MIC was performed at
seven concentrations of extracts (500, 250, 125,
62.5, 31.25, µg/ml) following serial dilution
technique. All the discs showing no visible growth
of microorganisms were sub cultured and incubated
at 37°C for overnight. The highest dilution showing
100% inhibition was recorded as MBC.
3. Results
The results of phytochemical screening of the stem
bark of P. smithiana are listed in table-1. It is
evident from the test that the ethanolic and
methanolic extract of bark contain carbohydrates,
glycosides, proteins, saponins, terpenoids, tannins,
phenol, alkaloids, flavonoids, starch, volatile oils,
resins, quinines and anthraquinone, while it was
found negative for fat and phalobatanins acids
(Table 1).
The antimicrobial activity against 3 plants
pathogenic
and
2
animal
pathogenic
microorganisms at 1000 µg/ml concentration of P.
smithiana bark extract in the form of ZOI (Zone of
Inhibition) is presented in (Table 2). The
preliminary antibacterial screening indicated that
the methanol extract and ethanol extract of P.
smithiana are effective against the test organisms.
Both the extracts of P. smithiana showed a
significant zone of inhibition ranging from 9-19
mm (ZOI). The results showed that methanol
extract was more active against all the tested
bacterial strains than ethanol extract. The highest
ZOI was observed for A. tumefaciens at 1000
µg/ml concentration. Ethanol extract showed the
lowest activity among all other extract of P.
smithiana bark. Its highest activity was found
against E. chrysanthemi having ZOI 12 mm. The
range of MIC and MBC values of extracts were
62.5-500µg/ml and 125-500 µg/ml respectively
(Table. 3). The lowest MIC 31.25 µg/ml was
recorded against A. tumefaciens and B. subtilis for
methanol extract and against A. tumefaciens for
chloroform extract. The lowest MBC value was
observed against A. tumefaciens for methanol
extract. It is interesting to note that all the fractions
were found active against all the tested microbes
(Table 2-3).
4. Discussion
The use of medicinal plants as natural remedies is
considered more effective and safe alternative
treatment of various diseases because most of the
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Table 1: Phytochemical evaluation of P. smithiana bark
extracts
Plant
Observation
Metabolites
Ethanol
Methanol
extract
extract
Carbohydrates
+
+
Glycoside
+
+
Protein
+
+
Saponins
+
+
Terpenoids
+
+
Tannins
+
+
Phenol
+
+
Alkaloids
+
+
Flavonoids
+
Starch
+
+
Volatile oils
+
+
Phlobatannins
Resins
+
+
Quinones
+
+
Fat
Anthraquinone
+
+
Key; + = present; - = absent
Table 2: Antibacterial activity of different extracts of P.
smithiana bark
Microorganisms
E
M
G
A. tumefaciens
9±0.7
19±0.7
23±0.5
B. subtilis
11±0.7 16±0.6
25±0.6
E.
12±0.6 15±0.3
21±0.4
chrysanthemi
E. coli
10±1.2 14±1.4
23±0.6
X. phaseoli
11±0.7 14±1.4
24±0.6
*All the values are mean ± Standard Error of Mean
(SEM) of three determinations E- Ethanol, M- Methanol;
G – Gentamycin (+control)
Table 3: Antibacterial activity of different extracts of P.
smithiana leaves (Based on Sati and Joshi, 2013 for
comparison)
Microorganisms
A. tumefaciens
B. subtilis
E. chrysanthemi
E. coli
X. phaseoli
Diameter of Inhibition Zone
(mm)*
17±0.3
22±1.4
21±0.0
11±0.6
na
13±0.3
21±0.3
13±0.6
13±0.6
22±0.0
20±0.9
21±1.9
22±1.3
26±1.8
21±1.0
Table 4: MIC and MBC evaluation of P. smithiana bark
Concentration (µg/ml)
Microorganisms Ethan
Methanol
ol
MIC
MBC
MIC
MBC
A. tumefaciens
250
500
31.25
62.5
B. subtilis
125
250
31.25
125
E. chrysanthemi 62.5
125
62.5
125
E. coli
62.5
250
62.5
125
X. phaseoli
62.5
125
31.25
250
bacteria have developed resistance against
commercially available antibiotics. Moreover
antibiotics may cause some side effects like allergic
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reactions as well as, disturbances of normal fauna
of intestine. The available literature indicates that
Picea has been used for remedies of some ailments
(Karting et al., 1999; Canillac and Mourey, 2001;
Radulescu et al., 2011; Liu et al., 2011). Recently
Sati and Joshi (2013) reported antibacterial
potentialities of P. smithiana leaves. The present
study screened the phytochemical and antibacterial
properties of methanol and ethanol extract of stem
bark of P. smithiana. As evident from table-1 the
stem bark of studied plant contains various
phytochemical groups and methanol extracts
showed
the
presence
of
maximum
phytoconstituents as compared to other extracts. It
may be due the fact that methanol, was
comparatively more polar in nature. Thus the
preliminary phytochemical screening would be
useful in the detection of bioactive principles and
drug discovery from this studied plant material.
Antimicrobial properties of substances are
desirable tools in the control of infectious diseases
microorganisms. The active components usually
interfere with growth and metabolism of
microorganisms in a negative manner (Aboaba et
al., 2006). Several phenolic compounds like tannins
present in the cells of plants are potent inhibitors of
many hydrolytic enzymes such as proteolytic
macerating enzymes used by plant pathogens.
Similarly saponins and glycosides also have
antifungal as well as hydrolyzing properties to
release phenolics which are toxic to microbial
pathogens (Aboaba et al., 2001).
Due to stressful climatic and geophysical
conditions, Kumaun Himalaya plants offer greater
possibilities of having novel molecules and even
larger quantities of active compounds (Kaul, 2010,
Sati and Joshi, 2011). Phytochemicals may be
described as non-nutritive plant chemicals that
have protective or disease preventive properties.
They are regarded as non essential nutrients (Okwu
and Okwu, 2004).
The preliminary phytochemical analysis and
antibacterial assessment of P. smithiana bark
showed promising results. Thus the present
investigation would be a milestone in the
qualitative
phytochemical
analysis
and
antimicrobial potentiality of high altitude Kumaun
Himalayan gymnospermous plant. Recently Sati
and Joshi (2013) studied the antimicrobial activity
of P. smithiana leaf extracts. Their findings are
summarised in (Table-4) compared with results of
present study on bark extracts. As evident from Fig
2 and 3 the ethanol and methanol leaf extracts had
slightly better activity than bark but it should also
keep in mind that barks are considered composed a
waste products of plant. The results of the present
study also showed that the bark extracts of P.
449
smithiana
contain
many
phytochemical
components which are potentially significant
against many microbes responsible for various
diseases.
alkaloids,
saponin,
glycosides,
quinines,
carbohydrates, protein, starch, resin, volatile oil,
anthraquinone and phenol were found in both
methanol and ethanol bark extracts.
Graphical Abstract
The antibacterial potential of methanol and ethanol
extracts of bark was investigated against
Agrobacterium tumefaciens, Bacillus subtilis,
The bark extract of Kumaun Himalayan
gymnosperm Picea smithiana was assessed for its
active principles. Flavonoids, terpenoid, tannins,
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450
Escherichia coli, Erwinia chrysanthemi and
Xanthomonas phaseoli using disc diffusion
method. Both methanol and ethanol extracts of P.
smithiana bark were found effective by showing a
mark zone of inhibition (ZOI). Methanol extract
showed the highest inhibitory activity against A.
tumefaciens (ZOI, 19 mm) while ethanol extract
exhibited its highest activity for E. chrysanthemi
(ZOI, 12 mm)
The MIC and MBC values were recorded in the
range 31.25-250 µg/ml and 62.5-500 µg/ml,
respectively. The lowest value of MIC and MBC
were recorded against A. tumefaciens in methanol
extract (31.25 µg/ml and 62.5 µg/ml respectively).
The results of bark extracts were also compared
with leaf extracts of P. smithiana for antimicrobial
potential.
Justification of research
Kumaun Himalayan gymnosperm Picea smithiana
extracts have been used in traditional medicine
since time immemorial to control various types of
human diseases. Form the literature survey it is
revealed that no substantial work has been carried
out on the bark of P. smithiana. Hence, an effort
was made to investigate the phytochemical analysis
and antimicrobial activity. Antimicrobial activity
help to confirm the inhibitory potential of extracts
while phytochemical screening suggest the
presence of secondary metabolites in plant which
will be helpful to discover novel natural
compounds.
In the present study we focused the antibacterial
potential of different concentration of P. smithiana
bark extracts against gram positive and gram
negative bacterial strains.
Conclusion
Relying upon the results obtained it can be
concluded that the bark extracts of Kumaun
Himalayan gymnosperm Picea smithiana has
antimicrobial potentialities against the bacterial
strains. Out of the two extracts i.e. ethanol and
methanol, ethanolic extract showed greater
antibacterial activity than methanolic extract.
The phytochemical screening of the bark extracts
of P. smithiana also indicate that the presence of
some compounds i.e. alkaloids, flavonoids, tannins
and phenols which are not only useful for
antimicrobial but also as antioxidant.
The present results also suggest that Picea
smithiana bark extracts may be used to find
bioactive compounds from natural products
required for the development of new drugs against
plant and human pathogens.
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Acknowledgements
The study was supported by Uttarakhand Council
for Science and Technology (UCOST), Dehradun,
India and UGC (BSR) SAP, New Delhi. The
authors wish to thank Department of Plant
Pathology, G. B. Pant University of Agriculture
and Technology, Pantnagar and Microbial Type
Culture Collection (MTCC) for providing bacterial
strains.
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