Quantification of reserpine content and antibacterial activity of Rauvolfia serpentina (L.) Benth. ex Kurz

Abstract

Reserpine is well known bioactive compound isolated from Rauvolfia serpentina. The aim of this study
was to quantify reserpine content and evaluate the antibacterial activity of methanol extracts of R.
serpentina against Salmonella typhimurium, Escherichia coli, Citrobacter freundii, Proteus vulgaris,
Enterococcus faecalis and Staphylococcus aureus. Roots of R. serpentina were collected from
Gadarpur and Uttarakhashi of Uttarakhand State, India. The antibacterial activity of the methanol
extracts was evaluated by determination of minimum inhibitory concentration (MIC) and the diameter of
zone of inhibition (ZOI) against both Gram positive and Gram negative bacteria using agar well diffusion
method. The study reveals that reserpine content was higher (0.37%) in the sample collected from
Gadarpur, whereas it was found to be 0.31% in sample collected from Uttarakashi. The highest zone of
inhibition (13 mm) with lowest MIC (625 μg) was observed against Staphylococcus aureus and highest
MIC (10 mg) was observed against Escherichia coli, whereas Proteus vulgaris was observed resistant to
tested extracts upto 10 mg. R. serpentina contain good amount of reserpine and exhibited strong
antibacterial activity against most of the tested human pathogenic bacteria. Therefore, the results of the
study support the folklore claim of the plant species.

Full text

Vol. 8(2), pp. 162-166, 8 January, 2014
DOI: 10.5897/AJMR2013.5847
ISSN 1996-0808 ©2014 Academic Journals
http://www.academicjournals.org/AJMR
African Journal of Microbiology Research
Full Length Research Paper
Quantification of reserpine content and antibacterial
activity of Rauvolfia serpentina (L.) Benth. ex Kurz
J. S. Negi1*, VK Bisht1, A. K. Bhandari1, D. S. Bisht1, P. Singh2 and N. Singh1
1Herbal Analytical Laboratory, Herbal Research and Development Institute, Mandal, Gopeshwar (Chamoli)- 246 401,
Uttarakhand, India.
2Department of Chemistry, HNB Garhwal University, Srinagar- 246 174, Uttarakhand, India.
Accepted 12 December, 2013
Reserpine is well known bioactive compound isolated from Rauvolfia serpentina. The aim of this study
was to quantify reserpine content and evaluate the antibacterial activity of methanol extracts of R.
serpentina against Salmonella typhimurium, Escherichia coli, Citrobacter freundii, Proteus vulgaris,
Enterococcus faecalis and Staphylococcus aureus. Roots of R. serpentina were collected from
Gadarpur and Uttarakhashi of Uttarakhand State, India. The antibacterial activity of the methanol
extracts was evaluated by determination of minimum inhibitory concentration (MIC) and the diameter of
zone of inhibition (ZOI) against both Gram positive and Gram negative bacteria using agar well diffusion
method. The study reveals that reserpine content was higher (0.37%) in the sample collected from
Gadarpur, whereas it was found to be 0.31% in sample collected from Uttarakashi. The highest zone of
inhibition (13 mm) with lowest MIC (625 µg) was observed against Staphylococcus aureus and highest
MIC (10 mg) was observed against Escherichia coli, whereas Proteus vulgaris was observed resistant to
tested extracts upto 10 mg. R. serpentina contain good amount of reserpine and exhibited strong
antibacterial activity against most of the tested human pathogenic bacteria. Therefore, the results of the
study support the folklore claim of the plant species.
Key word: High performance thin layer chromatography (HPTLC), reserpine, ciprofloxacin, zone of inhibition,
minimum inhibitory concentration.
INTRODUCTION
Many bacteria and fungi produce human diseases which
are currently controlled through the massive use of
synthetic bactericides and fungicides. Some of them are
resistance to synthetic drugs and caused therapeutic
problem (Guillemot, 1999). Plants extracts are one of the
options that have recently received attention and
expected that it will be active against synthetic drug
resistant pathogens. Therefore, the search for plant
based new antibacterial and antifungal agents are
imperative. Rauvolfia serpentina extract have been used
to treat infections for thousands of years in Indian system
of medicines. It is used for the treatment of fever, anxiety,
epilepsy, snake bite, rheumatism, insanity, eczema,
intestinal disorders, psychiatric disorders, nervous
disorders, cardiovascular disorder, bacterial infections
and in the management of hypertension schizophrenia
(Kirtikar and Basu, 1993; Gaur, 1999; Joshi and Kumar,
2000; Manuchair, 2002).
Reserpine has highly complex pattern of activity and is
the main biological active phytochemical of the com-
mercial drug Sarpgandha prepared from R. serpentina.
Indole alkaloids such as reserpine, ajmaline and ajma-
*Corresponding author. E-mail: negijs@yahoo.com. Tel: +911372 254210. Fax: +911372 254273.

licine were determined from R. serpentina and R.
vomitoria by high performance layer chromatography
(HPLC) and high performance thin layer chromatography
(HPTLC) (Klushnichenko et al., 1994; Srivastava et al.,
2006).
In pharmaceutical industries, reserpine is in great
demand and mainly extracted from Rauvolfia species.
Pharmacological studies demonstrate that Rauvolfia pos-
sesses cardiovascular (Anitha and Kumari 2006), antihy-
pertensive (Von Poser et al., 1990), antiarrhyth-
mic (Kirillova et al., 2001), antiinflammatory (Rao et al.,
2012), antipyretic (Amole and Onabanjo, 1999), antidia-
betic (Campbell et al., 2006), anticancer (Bemis et al.,
2006), hypoglycaemic and hypolipidemic (Qureshi et al.,
2009), hepatoprotective (Gupta et al., 2006a), sedative
(Weerakoon et al., 1998), antihistaminase (Sachdev et
al., 1961), mosquito larvicidal (Das and Chandra, 2012),
antibacterial (Ahmed et al., 2002) and antidiarrhoeal
(Ezeigbo et al., 2012) activities.
It is also reported that R. tetraphylla leaves have potent
antibacterial activity against Gram positive and Gram
negative bacteria which might be due to the presence of
alkaloids (Abubacker and Vasantha, 2011). But no
scientific investigation has so far been reported in
literature regarding antibacterial activity of R. serpentina
cultivated in Uttarakhand. Due to high market demand, R.
serpentina has been introduced for cultivation in the state
of Uttarakhand, India, in recent years and successfully
grown at farms field with excellent biomass and seeds
production capacity.
It is important to analyze the main contents of R.
serpentina before recommending them for large scale
cultivation and medicinal uses. Standardization of herbal
drug is also a scientific interest in the herbal drug
industry. Considering that, present study was designed to
quantify the reserpine content in R. serpentina roots and
also evaluate antibacterial activity.
MATERIALS AND METHODS
Roots of R. serpentina were collected from Gadarpur farm of Herbal
Research and Development Institute, Uttarakhand, India (desig-
nated as RS I) and farmer’s nursery located at Uttarakhashi (RS II).
The plant materials were washed with tap water, cut in small pieces
and spread over glass plate to dryness. The dried samples were
grinded through pulverizer and particles passed through sieve were
taken for extraction and analysis. Standard, reserpine was procured
from Sigma Aldrich (Germany), precoated silica gel 60 F254 TLC
plate from Merck and all other chemicals used were HPLC grade.
Extraction and preparation of samples solution
Hundred milligrams of powdered roots of R. serpentina were treated
with 1 ml of ammonia for 10 min and then extracted in 10 ml
methanol (MeOH). Solvent was removed to dryness under vacuum.
The dried extracts were dissolved with 2 ml methanol to make 50
mg/ml solution. Both the extracts were filter through 0.45 µm
syringe filter and used for analysis. Accurately 15 µl of each sample
was applied to TLC plate.
Negi et al. 163
Preparation of standard solutions
A stock solution of reserpine (0.1 mg/ml) was prepared in methanol.
Different volumes (2, 4, 6 and 8 µl) of the stock solution equivalent
to 200, 400, 600 and 800ng were applied to the TLC plates. The
calibration curve, correlation coefficient and regression equation
were obtained using WinCATS software.
Instrumentation and chromatographic conditions
The standard and sample solutions were applied on precoated 20
×10 cm silica gel 60 F254 plate in the form of bands with 100 µl
syringe using automatic sample applicator (Linomat 5). Samples
were applied to the plate as 6 mm band, 10 mm apart from Y and
15 mm from X axis using N2 gas. The slit dimension was 5 × 0.30
mm and scanning speed was 20 mm/s. The plate was developed in
a twin trough chamber saturated with mobile phase (chloroform:
toluene: ethylacetate: diethylamine). After development, the plate
was dried with the help of dryer and observed under UV chamber.
The well developed bands of reserpine in standard and R.
serpentina extracts were scanned at 254 nm in absorption mode
with CAMAG TLC scanner controlled by WinCATS software. The
source of radiation was deuterium lamp emitting a continuous UV
spectrum in the range of 190 to 400 nm.
Antibacterial activity
Antibacterial activity of R. serpentina extracts was determined by
well diffusion method according to Deshmukh et al. (2012) with
slight modifications. Bacterial cultures of Salmonella typhimurium,
Escherichia coli, Citrobacter freundii, Proteus vulgaris,
Enterococcus faecalis and Staphylococcus aureus were obtained
from Department of Microbiology, HNB Garhwal University,
Srinagar, India and used as test organism. All the bacteria were
maintained on nutrient agar No. 2 (Himedia, India) at 37°C. The
Gram positive bacteria (Enterococcus faecalis and Staphylococcus
aureus) and Gram negative bacteria (Salmonella typhimurium,
Escherichia coli, Citrobacter freundii and Proteus vulgaris) were pre
cultured in nutrient broth. The stock culture suspensions were
diluted with sterile saline water (0.85% NaCl). The Petri dishes were
flooded with Mueller Hinton Agar and after solidification of agar 0.1
ml of diluted inoculums were spread over Mueller Hinton Agar
(Himedia, India) in the dishes using sterile L spreader to achieve
confluent growth of test organism. Wells were then bored into the
agar using a sterile 6 mm diameter cork borer. Accurately 100 μl of
6.25, 12.5, 25, 50 and 100 mg/ml crude extracts were introduced
into the wells, plates were then incubated in refrigerator for about 2
h to allow the diffusion of solution in the medium. After that these
plates were incubated at 37°C in incubator for 24 h. Controls were
set up in parallel using the solvents that were used to dissolve the
extracts. The plates were observed for minimum inhibitory
concentration (MIC-lowest concentration of antibacterial that will
inhibit the visible growth of microorganism) and zones of inhibition
(ZOI). The effects were compared with those of 100 µl Ciprofloxacin
at a concentration of 100 µg/ml (10 µg) and the zone of inhibition
was measured using antibiotic zone scale.
RESULTS AND DISCUSSION
High performance thin layer chromatography (HPTLC)
was used for the estimation of reserpine (structure in
Figure 1) in R. serpentina. The standard and sample
solutions were spotted in the form of band on the TLC

164 Afr. J. Microbiol. Res.
N
N
H
OO
H3CO OCH3
OCH3
H3CO
OCH3
H3CO2C
Figure 1. Structure of reserpine.
Figure 2. HPTLC Chromatograms of (A) Reserpine, (B, C) Rauvolfia Serpentina
collected from Gadarpur (RS I) and Uttarakashi (RS II).
plates and run in different solvent systems. The mobile
phase consisting of chloroform: toluene: ethylacetate:
diethylamine (7:7:4:1) gave well defined bands and sharp
peaks. The rf value and correlation coefficient for reser-
pine was found 0.36 and 0.99, respectively. The chroma-
togram of standard and samples are shown in Figure 2.
The bands of reserpine in samples were confirmed by
comparing rf values with standard. The qualitative results

Negi et al. 165
Table 1. Antibacterial activity of Rauvolfia serpentina against different test organisms.
Bacterial test
organism
Strain no.
RS I
RS II
Standard (Ciprofloxacin)
MIC (µg/µl)
ZOI (mm)
MIC (µg/µl)
ZOI (mm)
MIC (µg/µl)
ZOI (mm)
Salmonella typhimurium
MTCC 3224
50
8
50
6
50
-
Escherichia coli
MTCC 443
100
12
100
10
25
18
Citrobacter freundii
MTCC 4221
25
9
25
7
12.5
16
Proteus vulgaris
MTCC 1771
-
-
-
-
25
8
Enterococcus faecalis
MTCC 439
25
9
25
7
6.25
11
Staphylococcus aureus
MTCC 3103
6.25
13
6.25
11
50
10
MIC= minimum inhibitory concentration, ZOI= zone of inhibition.
confirmed the presence of reserpine in both the samples
studied. R. serpentina collected from Gadarpur was
found to contain 0.37% reserpine (dry weight basis) while
R. serpentina collected from Uttarakashi contain 0.31%.
Reserpine has been estimated in Rauvolfia species by
HPLC and HPTLC. The total reserpine content in
Rauvolfia species were found 0.06 to 3.0% (Kokate et al.,
1998; Gupta et al., 2006b). Kumar et al. (2010) had also
quantified the reserpine content of R. serpentina
collected from different geographical locations of South
India. They observed that the reserpine content was
ranged from 0.0382 to 0.1442%. Baratto et al. (2012)
quantified reserpine content in the dried stem bark of R.
sellowii by HPLC and found 0.01% dry weight basis.
Comparison with previous studies clearly shows that the
R. serpentina cultivated in Uttarakhand has good
reserpine content. This may be attributed to the ambient
climatic and topographic conditions of Uttarakhand state.
The methanol extracts of R. serpentina exhibited
excellent antibacterial activity against tested bacterial
organisms as compared to the standard ciprofloxacin.
The results were summarized in Table 1. Zone of
inhibition are average of triplicate experiments. Sample 1
(RS I) of R. serpentina exhibited higher zone of inhibition
than Sample 2 (RS II). The highest zone of inhibition (13
mm for RS I and 11 mm for RS II) with lowest MIC (625
µg) was observed against Staphylococcus aureus and
highest MIC (10 mg) was observed against Escherichia
coli, whereas Proteus vulgaris was observed resistant
upto 10 mg of methanol extract of R. serpentina. It was
also observed that R. serpentina has similar effect
towards Citrobacter freundii and Enterococcus faecalis.
Deshmukh et al. (2012) reported antibacterial activity of
R. serpentina against S. typhii, S. aureus, E. coli and B.
subtilis.
The methanol extract of R. serpentina roots was
reported most effective (MIC 40 µg/µl) against S. typhii,
moderate against B. subtilis (MIC 80 µg/µl) and least
effective against S. aureus and E. coli (MIC 90 µg/µl).
The petroleum ether extract of R. serpentina has been
tested for antibacterial activity against Gram positive and
negative bacteria and observed 3.0 to 7.8 mm zones of
inhibition for Gram positive bacteria and 5.0 to 8.2 mm for
the Gram negative bacteria (Harisaranraj et al., 2009).
Comparison of our results with these findings clearly
shows that methanol extract of R. serpentina is more
effective than petroleum ether extract. Antimicrobial
activity of R. tetraphylla has also been reported, its
methanol extract showed 0.25 to 100 mg/ml minimum
inhibitory concentration against bacterial pathogens and
0.5 to 100 mg/ml against fungal pathogens (Shariff et al.,
2006). Our results demonstrated that methanol extract of
R. serpentina has concentration dependent antibacterial
activity against most of the tested organism.
Conclusion
R. serpentina cultivated in Uttarakhand has good
reserpine content and also exhibited moderate to strong
antibacterial activity against tested human pathogenic
bacteria. Therefore, the species is recommended for
large scale cultivation. The results of the study support
the folklore claim along with the development of new
antimicrobial drugs from the plant. The antibacterial
activity of R. serpentina may be attributed to the various
phytochemical constituents present in the crude extract.
Therefore, further work is needed to isolate the active
principle from the plant extract which may have even
more potency.
ACKNOWLEDGEMENTS
The authors are thankful to Agriculture and Processed
Food Products Export Development Authority, Ministry of
Commerce and Industry, Government of India, provided
financial support for equipments (Grant No.
FLR/059/2006-07/13692). We also wish to thank director,
HRDI for providing facilities.
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