Phytochemical and antimicrobial screening of medicinal plants for the treatment of acne

Abstract

Extracts of Camellia sinensis Linn. leaves, Glycyrrhiza glabra Linn. roots and rhizome and Calendula officinalis Linn. flowers were screened for their in vitro antimicrobial activity using agar disc diffusion method. The antimicrobial activity of petroleum ether, dichloromethane and methanolic extracts of different parts of these plants were studied against acne causing bacteria, namely Staphylococcus aureus (MTCC 96), Staphylococcus epidermidis (MTCC 2639) and Propionibacterium acnes (MTCC *1951). Methanolic extract of C. sinensis leaves possessed highest antibacterial activity against S. epidermidis. Lowest minimum inhibitory concentration (0.625 mg/mL) and minimum bactericidal concentration (2.5 mg/mL) against S. epidermidis were also observed for methanolic extract of C. sinensis leaves. Phytochemical screening revealed the presence of alkaloids, flavonoids, glycosides and terpenoids which indicates that these phytoconstituents may be responsible for their anti-acne activity.

Full text

Indian Journal of Natural Products and Resources
Vol. 3(1), March 2012, pp. 28-32
Phytochemical and antimicrobial screening of medicinal plants
for the treatment of acne
P Nand
1
, S Drabu
1
and R K Gupta
2
*
1
Maharaja Surajmal Institute of Pharmacy, Janakpuri, New Delhi-110058, India
2
University School of Biotechnology, Guru Gobind Singh Indraprastha University, New Delhi-110 075
Received 9 May 2011; Accepted 28 July 2011
Extracts of Camellia sinensis Linn. leaves, Glycyrrhiza glabra Linn. roots and rhizome and Calendula officinalis Linn.
flowers were screened for their in vitro antimicrobial activity using agar disc diffusion method. The antimicrobial activity of
petroleum ether, dichloromethane and methanolic extracts of different parts of these plants were studied against acne
causing bacteria, namely Staphylococcus aureus (MTCC 96), Staphylococcus epidermidis (MTCC 2639) and
Propionibacterium acnes (MTCC *1951). Methanolic extract of C. sinensis leaves possessed highest antibacterial activity
against S. epidermidis. Lowest minimum inhibitory concentration (0.625 mg/mL) and minimum bactericidal concentration
(2.5 mg/mL) against S. epidermidis were also observed for methanolic extract of C. sinensis leaves. Phytochemical
screening revealed the presence of alkaloids, flavonoids, glycosides and terpenoids which indicates that these
phytoconstituents may be responsible for their anti-acne activity.
Keywords: Antimicrobial activity, Calendula officinalis, Camellia sinensis, Disc diffusion method, Glycyrrhiza glabra,
Phytochemical screening.
IPC code; Int. cl. (2011.01)—A61K 36/00, A61P 31/10, A61P 17/10
Introduction
Camellia sinensis Linn. (Family-Theaceae) known
as green tea, is the second most common beverage
consumed worldwide next to water
1
. It is an evergreen
shrub or tree which is mainly cultivated in India and
China. Three main varieties of tea have been reported
namely green, black, and oolong. Green tea is made
from unfermented leaves and contains the highest
concentration of powerful antioxidants. Green tea has
a number of pharmacological activities such as
anticancer, lipid lowering, neuromuscular blocking
action, immunomodulatory effect, antiviral, anti-
bacterial
2
, antispasmodic, antioxidant
3
, anti-inflam-
matory
4
, etc. A large number of phytoconstituents like
alkaloids (caffeine, theobromine), proteins, enzymes,
carbohydrates, lipids, polyphenols, catechins
(epicatechin, epicatechin-3-gallate, epigallocatechin
and epigallocatechin-3-gallate), carbohydrates,
tannins, vitamins and minerals have been reported to
be present in this plant
5
.
Glycyrrhiza glabra Linn. (Family-Fabaceae)
known as liquorice, has been used in medicines for
more than 4000 years now. It is native to central and
south west Asia, Mediterranean basin of Africa, South
Europe and India. Various species of liquorice have
been reported namely Glycyrrhiza inflata Fisch.,
Glycyrrhiza uralensis Fisch. and Liquiritia officinalis
Moench
6
. In the traditional system of medicine,
liquorice is used as a demulcent, anti-tussive, laxative,
sweetener, diuretic, antiarthritic, antibacterial
7
, anti-
inflammatory
8
, anti-acne
9
, aphrodisiac, estrogenic,
antioxidant, antineoplastic, anticholinergic and
antiulcer
10
. Phytoconstituents isolated from this plant
are glycyrrhizin, glycyrrhizinic acid, glabrin A and B,
glycyrrhetol, glabrolide, isoglabrolide, isoflavones,
coumarins, triterpene sterols liquiritin, isoliquiritin,
flavones, chalcones and isoflavonoids, such as
glabridin
7-11
.
Calendula officinalis Linn. (Family-Asteraceae)
known as Pot marigold is an important medicinal
plant for the indigenous people of India, Europe, US
and China. Its leaves and flowers have been reported
to possess many pharmacological activities which
include antioxidant, anti-inflammatory, antibacterial
and antiviral activities
12-14
. Phytoconstituents isolated
from this plant are sitosterols, stigmasterols,
ψ-taraxasterol, lupeol, faradiol-3-O-palmitate,
_______________
*Correspondent author: E-mail: rkg67ap@yahoo.com;
Phone: +91 9871263252 (Mob.);
Fax: +91 11 23865941/2.

NAND et al.: ANTIMICROBIAL STUDY OF MEDICINAL PLANTS FOR ACNE
29
faradiol-3-O-myristate, faradiol-3-O-laurate, quercetin,
isorhamnetin, isoquercetin, calendoflaside, α-tocopherol
and essential oil rich in monoterpenes and
sesquiterpenes
15
.
Since the existing literature exhibits only a limited
research concerning the utilization of these extracts in
the treatment of acne, an attempt was made to
evaluate the antimicrobial activity of petroleum ether,
dichloromethane and methanolic extracts of leaves,
roots, stolons and flowers of these plants, respectively
by agar diffusion and broth microdilution methods.
Materials and Methods
Plant materials
Samples of C. sinensis leaves, G. glabra roots and
stolons and C. officinalis flowers were collected from
medicinal gardens and authorized herbal stores in
Delhi and authenticated by NISCAIR, Pusa Campus,
New Delhi with voucher specimen (NISCAIR/RHM/
consult/2008-09/978/09) and have been preserved in
our department for the future reference.
Extraction and preliminary phytochemical screening
Shade-dried plant leaves, roots, stolons and flowers
(200 g) were pulverized separately and subjected to
sequential solvent extraction by continuous hot
extraction (soxhlet) method. The extraction was done
with different solvents in their increasing order of
polarity such as petroleum ether (PE),
dichloromethane(DCM) and methanol(ME) and the
extracts were abbreviated as: Camellia sinensis
(CSPE, CSDCM, and CSME), Glycyrrhiza glabra
(GGPE, GGDCM, GGME) and Calendula officinalis
(COPE, CODCM, and COME). Every time, the marc
was dried in air at room temperature and later used for
extraction with other solvents. All the extracts were
evaporated using a rotary evaporator and the
percentage yield was thus recorded. Dried extracts
were stored at 4°C in airtight containers for further
studies. Concentrated extracts were subjected to
various chemical tests in order to detect the presence
of different phytoconstituents
16
.
Microorganism and media
Aerobic bacteria: Staphylococcus aureus (MTCC
96), Staphylococcus epidermidis (MTCC 2639) and
anaerobic bacteria: Propionibacterium acnes
(MTCC*1951) were obtained from the Microbial
Type Culture Collection Centre, Institute of Microbial
Technology, Chandigarh. Fresh cultures of the
isolates of aerobic and anaerobic bacteria were
suspended in nutrient broth and reinforced clostridium
medium, respectively. S. aureus and S. epidermidis
cultures were incubated for 24 h at 37 and 30°C,
respectively. P. acnes culture was incubated in an
anaerobic chamber at 37°C consisting of 10% CO
2,
10% H
2
and 80% N
2
for 48 h.
Antimicrobial screening using disc diffusion method
Antibacterial activity of extracts was tested using
agar disc diffusion method
17
. Fresh culture suspension
(100 µl) of test bacteria was evenly spread on nutrient
agar and reinforced clostridial agar plates. The
concentration of cultures was 5 × 10
5
CFU/mL. For
screening, 6 mm diam. filter paper disc, impregnated
with 20 µl of extract solution equivalent to 0.2 mg of
extract was placed on the surface of inoculated media
agar plates. Incubation was done at 37°C or 30°C for
24 and 48 h depending upon the type of bacteria
under optimum conditions. Clear zones of inhibition
were measured in mm, including the diameter of disc.
Zone measuring 10 mm or more was considered as
effective against test organisms. Clindamycin
(10 µg/disc) was used as positive control and the
respective solvents, which were used for extraction,
served as negative control.
Minimum inhibitory concentrations using microdilution
method
Minimum inhibitory concentration (MIC) of active
methanolic extracts (AME) was studied by using
broth microdilution method with slight modify-
cations
18
. Extracts were dissolved in (DMSO)
dimethyl sulfoxide (10% of total volume) and two
fold dilutions were done using pre-sterilized culture
broth to give final concentrations ranging from
5-0.078 mg/ml. 100 µl of each dilution was
distributed in 96 well plates. Sterility control
(sterilized nutrient broth) and growth control (culture
broth with DMSO) were also set up. Each test and
growth control well was inoculated with 5 µl of a
bacterial suspension (5x10
5
CFU/ml). All experiments
were performed in triplicate and the microdilution
plates were incubated under optimum conditions.
Bacterial growth was detected after the addition of
20 µl of 70% alcoholic solution of INT (0.5 mg/ml)
into each well followed by incubation for 30 minutes.
Colour change from yellow to purple indicated the
presence of microbial growth.
Minimum bactericidal concentration determination
The minimum bactericidal concentration (MBC) is
defined as the lowest concentration of a compound

INDIAN J NAT PROD RESOUR, MARCH 2012
30
that kills the microorganism. The MBCs of the
extracts were determined by plating 10 µl of samples
from each MIC well without visible growth onto
culture media plates
19
. Following the incubation for
optimum period, the plates were examined for colony
growth and MBCs were recorded.
Results and Discussion
In present investigation, percentage yield of nine
extracts indicated that GGME showed highest
percentage yield (20.11) followed by COME (11.22)
and CSME (9.55) (Figure 1). Phytochemical
screening showed the presence of the following
phytoconstituents: C. sinensis (alkaloids, flavonoids,
terpenoids and tannins), G. glabra (carbohydrate,
glycosides, flavonoids, saponins, terpenes and sterol)
and C. officinalis (flavonoids, saponins and
terpenoids). In vitro antimicrobial screening using
clindamycin phosphate as a positive control clearly
indicated that CSME, GGME and COME show
promising antimicrobial activity against all the three
organisms (Table 1). It was observed that all the
extracts of C. sinensis and G. glabra showed
significant antimicrobial activity against test
organisms except GGPE which did not exhibit
antimicrobial activity against S. epidermidis
(Figures 2-4).
Fig. 2—Antimicrobial activity of plant extracts against S.aureus
Fig. 3—Antimicrobial activity of plant extracts against
S. epidermidis
Fig. 1—Percentage yield of plant extracts
Table 1—Antimicrobial screening of plants against S. aureus (MTCC 96), S. epidermidis MTCC 2639) and P. acnes (MTCC *1951)
using disc diffusion method.
Zone of inhibition of extracts in mm
S. aureus* S. epidermidis* P. acnes*
PE DCM ME PE DCM ME PE DCM ME
Camellia
sinensis (Le)
7.5±0.28 9.0±0.06 14.4±0.27 7.4±0.05 10.0±0.06 17.8±0.16 7.6±0.18 7.8±0.1 13.8±0.2
Glycyrrhiza
glabra (R&S)
7.66±0.16 9.06±0.06 11.5±0.28 NA 8.0±0.12 11.8±0.15 7.1±0.05 13±0.05 13.9±0.15
Calendula
officinalis(Fl)
NA 9.06±0.12 NA NA 9.06±0.17 11.8±0.15 9.13±0.08 NA 12.4±0.18
Clindamycin
phosphate
14.94±0.08 18±0.11 18±0.05
Le = Leaves; R & St = Roots and Stolons; Fl = Flowers; PE = Petroleum ether extract; DCM = Dichloromethane extract;
ME = Methanolic extract; NA = No antibacterial activity. Values are Mean ± SEM (mm) of three measurements; *P< 0.05.

NAND et al.: ANTIMICROBIAL STUDY OF MEDICINAL PLANTS FOR ACNE
31
Fig. 4—Antimicrobial activity of plant extracts against P. acnes
CODCM and COME were found to be significantly
active against S. epidermidis, however, they did not
show inhibitory activity against S. aureus and
P. acnes. Highest zone of inhibition 17.8±0.016 mm
was observed for CSME against S. epidermidis. The
results, after preliminary antimicrobial screening,
revealed that active methanolic extracts have potential
in the treatment of acne hence, they were further
evaluated for their MICs and MBCs .The lowest
MICs against S. epidermidis (0.625 mg/ml), S. aureus
(1.25 mg/ml) and P. acnes (1.25 mg/ml) were
recorded in CSME (Table 2). Similarly lowest MBCs
against S. aureus (2.5 mg/ml), S. epidermidis
(2.5 mg/ml) and P. acnes (2.5 mg/ml) were also
observed in CSME (Table 2).
Though there is lack of literature to provide
evidence for the antimicrobial activity of these
medicinal plants against acne causing bacteria, yet
our findings could be based on the bactericidal action
of epigallocatechin gallate and epicatechin in
C. sinensis
20
. Antibacterial and anti-inflammatory
effect of G. glabra is attributed due to the presence of
glycyrrhizin and its hydrolysis product, glycyrrhetinic
acid
8
. Presence of triterpenoids in C. officinalis is
known to provide anti-inflammatory activity. It was
also reported that esters of faradiol-3-myristic acid,
faradiol-3-palmitic acid and 4-taraxasterol are the
three most active compounds to reduce edema and the
flavonoid, kaempferol, demonstrated antibacterial
activity against P. acnes
21
. Hence, the antimicrobial
activity of AMEs showed broad spectrum potential as
the active compounds are concentrated more in
ME fraction than in PE and DCM fractions which
supports phytochemical screening.
Conclusion
The results clearly indicated that scientific studies
carried out on these medicinal plants, possessing
traditional claims of effectiveness in skin disorders,
provided fruitful results. Therefore, methanolic
extracts of C. sinensis, G. glabra and C. officinalis,
possessing broad-spectrum activity could be utilized
in treating acne vulgaris and formulating anti-acne
herbal products. The main focus of our work is on the
anti-acne potential of herbs which we plan to study
further with the ultimate objective of providing
scientifically validated herbal remedies against acne.
Further in this regard, characterization is required to
determine the types of compounds responsible for the
antibacterial activity.
Acknowledgements
We are extremely grateful to Prof. Mullika Traidej
Chomnawang, Faculty of Pharmacy, Mahidol
University, Thailand for reviewing this manuscript
and for his invaluable comments and suggestions. We
are thankful to Prof. Rama Choudhary, HOD,
Microbiology, All India Institute of Medical Sciences
for providing facilities to carry out antibacterial
activity of plant extracts against anaerobic
microorganism. We acknowledge the financial
Table 2: Minimum inhibitory and minimum bactericidal concentrations of AMEs and Clindamycin
S. aureus (MTCC 96) S. epidermidis (MTCC 2639) P. acnes (MTCC*1951) Active methanolic extracts
MIC
(mg/ml)
MBC
(mg/ml)
MIC
(mg/ml)
MBC
(mg/ml)
MIC
(mg/ml)
MBC
(mg/ml)
CSME 1.25 2.5 0.625 2.5 1.25 2.5
GGME 2.5 >5 2.5 5 1.25 5
COME NA NA 2.5 >5 2.5 5
Clindamycin 0.312 0.63 0.078 0.156 0.078 0.156
CSME = Methanolic extract of Camellia sinensis; GGME = Methanolic extract of Glycyrrhiza glabra; COME = Methanolic extract of
Calendula officinalis; NA = No antibacterial activity. Results are average of three measurements.

INDIAN J NAT PROD RESOUR, MARCH 2012
32
support from All India Council for Technical
Education (8023/BOR/RID/RPS-27/2008-09) & UGC
for the SAP-DRS-1 funds (F. 3-23/2011).
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