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Original Article

Insignificant anti-acne activity of Azadirachta

indica leaves and bark

Pratibha Nand, Abstract

Sushma Drabu,
Rajinder K. Gupta1
Maharaja Surajmal Institute of
Pharmacy, Janakpuri, 1University
School of Biotechnology, Guru
Gobind Singh Indraprastha
University, New Delhi, India
Background and Aim: Azadirachta indica has a long history of medicinal usage in skin
ailments due to its antibacterial and anti-inflammatory properties. The present study
was undertaken to explore the potential of the leaves and bark of Azadirachta indica
in the treatment of acne. Materials and Methods: Monographic analysis of the plant
was carried out, followed by the phytochemical screening of the sequential extracts
of leaves and bark of Azadirachta indica. The results indicated presence of alkaloids,
flavonoids, saponins, terpenes, and tannins. Gas chromatography–mass spectrometry
(GC/MS) analysis of the test extracts indicated the presence of many phytoconstituents
in dichloromethane and methanolic extracts namely stigmasterol, nimbiol, sugiol,
4-cymene, α-terpinene terpinen-4-ol, and vitamin E. The test extracts were evaluated
against acne causing bacteria, namely Staphylococcus aureus (MTCC 96), Staphylococcus
epidermidis (MTCC 2639), and Propionibacterium acnes (MTCC *1951), for their in vitro
antimicrobial activity, using agar disc diffusion method. Results: Dichloromethane and
methanolic extracts of Azadirachta indica showed very little activity against S. aureus and
S. epidermidis, but did not show any antimicrobial activity against P. acnes. Conclusion:
Despite possessing antibacterial properties, Azadirachta indica showed insignificant
effect against acne causing bacteria.
Key words: Antibacterial activity, Azadirachta indica, GC/MS analysis, monographic
analysis, phytochemical screening

INTRODUCTION of research concerning the utilization of plant extracts

Plants have been used for thousands of years in the
treatment of skin ailments, as they have great therapeutic
potential as antimicrobial agents. Despite the availability of
a number of antibiotic and antibacterial agents, the resistant
strains of acne necessitate the search for a new anti-acne
agent of plant origin. It is also believed that plant-based
products are generally safe, have less side effects, and are
available at an affordable price. As the literature shows lack
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DOI:
10.4103/0976-9234.99650
in the treatment of acne, an attempt has been made to
evaluate the leaves and bark of Azadirachta indica, which
possess antibacterial and anti-inflammatory activity. [1] This plant
belongs to the family Meliaceae and grows widely in India,
Pakistan, and other tropical and subtropical regions of the
world. A. indica A. Juss (Indian neem), and M. azadirachta
(Persian lilac) are two closely related species of Meliaceae.[2]
Neem is an evergreen tree with fairly glabrous leaves. Every
part of the tree has some therapeutic value and has been
used since antiquity as a household remedy against various
ailments.[3,4] The thickness of the bark varies according to
age and the portion of the tree from where it has been
taken. Its external surface is rough and fissured in texture
with a rusty-gray colour, while the laminated inner surface
is yellowish and foliaceous with longitudinal furrows. The
leaves are lanceolated, acute, serrated, 7–8.5 cm long and
1.0–1.7 cm wide with slightly yellowish-green colour and

Address for correspondence:

Prof. Rajinder K. Gupta, Dean, University School of Biotechnology, G.G.S. Indraprastha University, Sector 16C, Dwarka, New Delhi, India.
E-mail: rkg67ap@yahoo.com

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Nand, et al.: Insignificant anti-acne activity of Azadirachta indica
possess a characteristic odour along with a bitter taste.[5] The
bark and leaf extracts have been used as folk medicine to
control diseases such as leprosy, blood morbidity, itching,
skin ulcer, burning sensation, and respiratory disorders.
This extract is also fungistatic, antibacterial, and a general
health promoter.[6] It has been used in the treatment of
rheumatism, chronic syphilitic sores, and ulcers.[7] Chemical
investigation, mentioned in the literature, highlighted the
presence of more than 135 compounds in the different
parts of the plant.[3] These compounds have been classified
into two categories :- isoprenoids and nonisoprenoids.
The isoprenoids include diterpenoids and triterpenoids
containing limonoids, azadirone, nimbin, nimbolide,
salanin, and azadirachtin. Nimbin and nimbiol are the bitter
principle constituents of this plant. The nonisoprenoids
include proteins (amino acids) and carbohydrates
(polysaccharides), sulphur compounds, polyphenolics
such as flavonoids and their glycosides, dihydrochalcone,
coumarin and tannins, and aliphatic compounds. [8]
Nimbolide has shown antibacterial activity against S. aureus
and S. coagulase.[9] Noticeable improvement was observed
when a paste of crushed neem leaves, which is a popular
home remedy, was applied on the part of face affected by
acne.[10] The bark provided an antibacterial effect due to the
presence of margolone, margolonone, isomargolonone,
and so on. Condensed tannins in the bark contain gallic
acid, (+) gallocatechin, (–) epicatechin, (+) catechin, and
epigallocatechin, of which gallic acid , (–) epicatechin,
and catechin are primarily responsible for inhibiting the
generation of chemiluminescence by the activated human
polymorphonuclear neutrophil (PMN),[11] indicating that
these compounds inhibit oxidative burst of PMN during
inflammation. Literature has also revealed the presence
of polyphenolic compounds in the bark, which have a
powerful antioxidant potential to neutralize the damaging
effects of free radicals, and these compounds provide anti-
inflammatory activity. The methanolic extract of the leaves
provided antipyretic and anti-inflammatory effects in male
rabbits.[12] Based on the beneficial effects of Azadirachta
indica, the present study is undertaken to ascertain the
antimicrobial potential of neem in the treatment of acne.
MATERIALS AND METHODS
Collection and identification of plant material
The leaves and bark of Azadirachta indica were collected
from the medicinal gardens in Delhi, authenticated by
the National Institute of Science Communication and
Information Resources (NISCAIR), Pusa Campus,
New Delhi with voucher specimen (NISCAIR/RHM/
consult/2008-09/978/09), preserved within the department
for future reference.
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Monographic analysis of herbs
Individual herbs were evaluated according to their standard
specifications mentioned in the Ayurvedic Pharmacopoeia
of India. Tests were carried out to check the presence
of foreign organic matter, loss on drying, total ash,
acid-insoluble ash, alcohol-soluble extractive and water-
insoluble extractive values.
Preparation of extracts
Shade-dried leaves and bark of the plant (200 g) were
pulverized separately and subjected to sequential solvent
extraction using the continuous hot extraction (soxhlet)
method. The extraction was carried out with different solvents
present in their increasing order of polarity, such as petroleum
ether (PE), dichloromethane (DCM), and methanol (ME).
These extracts were abbreviated as: AILPE, AILDCM,
AILME and AIBPE, AIBDCM, and AIBME respectively.
Each 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 recorded. Dried extracts were stored
at 4°C in airtight containers for further studies.
Phytochemical screening
Concentrated extracts of Azadirachta indica leaves and
bark were subjected to qualitative analysis for screening
of alkaloids, cardiac glycosides, saponins, tannins,
anthraquinones, terpenes, and flavonoids, by using the
methods described by Harborne.[13]
Gas chromatography–mass spectrometry analysis
Three sequential extracts were analyzed for volatile
components with an Agilent 6890N gas chromatograph
connected to a 5975B mass-selective detector. The
chromatographic separation was done on a capillary
column of fused silica HP-5 ms (0.25 mm × 30 m × 0.25
µm). 1 µl of each extract was injected in the split mode (1
: 50), with the injector temperature at 280 0C. The oven
temperature was programmed, starting from 70 ºC (1min)
at 25 ºC/min to 150 ºC (0 min) at 3 ºC/ min to 200 ºC
(1 min) at 8ºC/min, and finally reaching a temperature of
280 ºC (3 min). Helium was used as the carrier gas. The
detection was performed in the EI mode with ionization
energy of 70 eV, source at 230 ºC, and quadruple at 150
ºC. The relative percentage amounts of the separated
compounds were automatically calculated from the peak
areas of the total ion chromatogram. The identity of the
components was confirmed by comparing their retention
time and spectral data with the corresponding data from
the NIST’05 library.[14]
Antibacterial screening microorganism and media
Aerobic bacteria: Staphylococcus aureus (MTCC 96),
Staphylococcus epidermidis (MTCC 2639), and anaerobic
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Nand, et al.: Insignificant anti-acne activity of Azadirachta indica

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 °C and
30 °C, respectively. The P. acnes culture was incubated in
an anaerobic chamber at 37°C, consisting of 10% CO2,
10% H2, and 80% N2, for 48 h.
Evaluation of antibacterial activity
The antibacterial activity of extracts was tested using agar
disc diffusion method. [15] 100 μl of fresh culture suspension
of test bacteria was evenly spread on nutrient agar and
reinforced clostridial agar plates. The concentration of
cultures was 5 x 105 CFU/ml. For screening, a 6 mm
diameter filter paper disc, impregnated with 20 µl of extract
solution, equivalent to 0.2 mg of extract was placed on the
surface of the inoculated media agar plates. Incubation was
done at 37 °C and 30 °C for 24 h and 48 h respectively,
depending on the type of bacteria, under optimum
conditions. Clear zones of inhibition were measured in mm,
including the diameter of the disc. The zone measuring
10 mm or more was considered as an effective extract
against test organisms. Clindamycin (10 µg/disc) was used
as a positive control and the respective solvents used for
extraction, served as the negative control.
Statistical analysis
All the samples for antimicrobial screening were run in
triplicate and the mean values were used for the result
analysis. Data was expressed as mean ± SEM. Statistical
analysis using one way ANOVA was done with SPSS
software version 10.0.1 and P values less than 0.05 were
considered significant.
RESULTS
In the present study, the monographic analysis of A. indica
leaves and bark revealed that all the tested parameters
were within the pharmacopoeial limits [Table 1]. The
percentage yield of test extracts indicated that AIBME has
the highest percentage yield (11.87) followed by AILME
(7.93). Qualitative phytochemical analysis of crude plant
extracts revealed the presence of alkaloids, flavonoids,
saponins, terpenes, and tannins in the A. indica leaves as
well as bark [Table 2]. The previous findings had reported
the beneficial effects of flavonoids in fighting ailments
and improving human health. [16] The composition of
all the test extracts, after GC/MS analysis, was recorded
[Table 3]. Twenty-six important constituents were identified
in six test extracts, out of which AILME indicated
the presence of stigmasterol (4.55%), phytol(0.9%),
4-cymene(0.73%), α-terpinene(0.9%), and terpinen-4-ol
(2.19%).
Vitamin E was found in AILDCM extract. Furthermore,
AIBDCM revealed the presence of many active
phytoconstituents like stigmasterol (0.44%) and phenolic
components, namely, nimbiol (4.77%) and sugiol (1.48%).

Table 1: Monographic analysis of Azadirachta indica

Leaves Bark
Parameters %w / w Obtained value ± SD Pharmacopoeial Obtained value ± SD Pharmacopoeial
limit (%) limit (%)
Foreign organic matter 1.26 ± 0.057 NMT 2 0.087 ± 0.034 NMT 2
Total ash 8.47 ± 0.33 NMT 10 5.10 ± 0.24 NMT 7
Acid-insoluble ash 0.869 ± .0028 NMT 1 0.945 ± 0.065 NMT 1.5
Alcohol-soluble extractive % 14.19 ± 0.04 NLT 13 13.25 ± 0.40 NLT 6
Water-soluble extractive 22.24 ± 0.234 NLT 19 13.44 ± 0.19 NLT 5

Table 2: Percent yield and phytochemical screening

Scientific Extract (%) Alkaloid Cardiac Flavonoid Saponin Terpenes Tannin
name Yield glycoside
Azadirachta PE 1.4 + - + + ++ +
indica (le) DCM 2 + + + + + +
ME 7.93 ++ + + ++ + +
Azadirachta PE 0.765 + - + - ++ -
indica (b) DCM 2 + + + ++ + +
ME 11.87 ++ ++ + ++ + +
le: Leaves; b: Bark; PE: Petroleum ether extract; DCM: Dichloromethane extract; ME: Methanolic extract

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Nand, et al.: Insignificant anti-acne activity of Azadirachta indica

Table 3: GC/MS profiling of Azadirachta indica leaves and bark

Compound ID Retention time % Area
AILPE AILDCM AILME AIBPE AIBDCM AIBME
4-Cymene 7.78 1.37 - 0.73 - 1.06 2.86
α-Terpinene 8.96 2.05 - 0.9 2.34 2.51
Terpinen-4-ol 13.3 2.51 - 2.19 1.06 4.06 2.96
2-Methoxy-4- 18.24 - - - 0.45 -
vinylphenol
Lauric acid 26.43 0.61 - - - - -
Pentadecanal 30.87 1.64 - - - - -
Hexadecanal 30.88 1.64 - - - - -
Tetradecanal 30.89 1.63 - - - - -
Methyl palmitate 36.56 - - 2.39 - 0.75 1.69
Palmitic acid 37.80 9.89 - 1.33 - - -
Phytol 41.24 21.44 3.24 0.90 - - -
Methyl stearate 41.47 0.90 - - - - 0.50
Triacontane 47.37 12.82 13.61 - - - 43.88
Tricosane 47.41 - - - - 43.88
Tetracosane 47.49 - 13.61 - - 13.43 41.34
Pentacosane 49.5 1.78 2.27 - - 7.85 -
Heneicosane 49.57 - - - - 10.91 -
Nimbiol 50.04 - - - - 4.77 -
Hexacosane 51.6 2.38 - - - 10.91 41.34
Stigmasterol 51.62 - - 4.55 - 0.44 -
Sugiol 52.34 - - - - 1.48 -
Heptacosane 54.11 5.83 3.85 2.02 - 11.96 -
Vitamin E 56.06 - 7.44 - - - -
Octadecane 57.15 4.72 6.38 - 0.51 43.88
Tetratriacontane 61.00 - - - - 13.43 -
Nonacosane 61.21 32.69 4.99 - - - -
AIL: Azadirachta indica leaves; AIB: Azadirachta indica bark; PE: Petroleum ether extract; DCM: Dichloromethane extract; ME: Methanolic extract

Table 4: Mean ± SEM (mm) of zone of growth inhibitions, in mm , obtained by preliminary antimicrobial
screening of plants against Staphylococcus aureus (MTCC 96), Staphylococcus epidermidis (MTCC
2639) and Propionibacterium acnes (MTCC *1951) using the disc diffusion method
Scientific name Staphylococcus aureus Staphylococcus epidermidis Propionibacterium acnes
PE DCM ME PE DCM ME PE DCM ME
Azadirachta NA NA 8.1± 0.12 NA NA 8.13 ± 0.11 NA NA NA
indica (le)
Azadirachta NA 8.83 ± 0.15 NA NA 8.81 ± 0.14 NA NA NA NA
indica (b)
Clindamycin 12.96 ± 0.08 - - 18 ± 0.11 - - 18 ± 0.05 - -
Phosphate
(1%w / v)
le: Leaves; b: Bark; PE: Petroleum ether extract; DCM: Dichloromethane extract; ME:Methanolic extract; NA: No activity

It also showed the presence of 4-cymene (1.06%),
α-terpinene (2.34%), and terpinen-4-ol (4.06%). The
antimicrobial screening was carried out on the sequential
extracts, using clindamycin phosphate as a positive control
[Table 4]. Among these, the highest zone of inhibition was
observed in AIBDCM (8.83 ± 0.15 mm) against S. aureus.
AILME was found to be effective against the aerobic test
strains with inhibition zones of 8.10 ± 0.12 mm and 8.13 ±
0.12 mm against S. aureus and S. epidermidis, respectively.
This revealed that the extracts exhibited little activity against
test organisms, in comparison to clindamycin (18 ± 0.11
mm). Moreover, AILPE and AIBPE did not show any
kind of activity against the test organisms. Also, none of
the extracts were found to be effective against P. acnes.

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Nand, et al.: Insignificant anti-acne activity of Azadirachta indica
DISCUSSION
Neem is a versatile medicinal plant and is a unique source of
various compounds possessing diverse therapeutic benefits.
In the present study, the phytochemical and GC/MS analyses
have indicated the presence of bioactive compounds.
Literature has also revealed that the bark and leaves possess
polyphenolic compounds, which are responsible for
antibacterial, antioxidant, and anti-inflammatory activities.
[17]
Another study indicates that petroleum ether, water, and
ethanolic extracts of Azadirachta indica exhibit moderate
inhibitory activity against P.acnes.[18] In this study, although
the active components documented for anti-acne activity,
namely α-terpinene and terpinen-4-ol,[19] were found in
both AILME and AIBDCM, the results demonstrated an
insignificant anti-acne activity in AILME and AIBDCM
extracts, compared to clindamycin.
CONCLUSION
Hence, the present study does not support the usage of
this plant for the treatment of acne vulgaris because of
its insignificant activity against acne-causing organisms.
ACKNOWLEDGMENTS
We acknowledge the financial support from All India Council
for Technical Education 8023/BOR/RID/RPS-27/2008-09 &
UGC-SAP-3-23/2011. We also would like to thank Professor
Rama Choudhary, HOD, Microbiology, All India Institute of
Medical Sciences for providing anaerobic microbial facility to
carry out antibacterial activity of plant extracts.
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How to cite this article: Nand P, Drabu S, Gupta RK. Insignificant anti-
acne activity of Azadirachta indica leaves and bark. J Pharm Negative
Results 2012;3:29-33.
Source of Support: All India Council for Technical Education 8023/BOR/
RID/RPS-27/2008-09 & UGC-SAP-3-23/2011. Conflict of Interest: None
declared.
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