Journal of Traditional and Complementary Medicine 7 (2017) 152e157

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Journal of Traditional and Complementary Medicine

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Original article

Antioxidant, antimicrobial activity and bioactive compounds of

Bergenia ciliata Sternb.: A valuable medicinal herb of Sikkim Himalaya
Mithilesh Singh a, *, Neha Pandey b, Vasudha Agnihotri b, K.K. Singh a, Anita Pandey b
a
G.B. Pant Institute of Himalayan Environment and Development, Sikkim Unit, Pangthang, Gangtok, Sikkim 737101, India
b
G.B. Pant Institute of Himalayan Environment and Development, Kosi-Katarmal, Almora, Uttarakhand 263643, India

a r t i c l e i n f o a b s t r a c t

Article history: Bergenia ciliata Sternb., commonly known as Paashaanbhed, is a well known herb of Sikkim Himalaya
Received 10 February 2016 with various pharmaceutical properties. However, scientific exploration of B. ciliata, growing in the
Received in revised form Sikkim Himalaya, for phytochemicals and pharmacological properties is in infancy. With this view, the
9 April 2016
present study was undertaken to investigate B. ciliata leaf extracts for antioxidant, antimicrobial activity
Accepted 14 April 2016
Available online 18 May 2016
and bioactive compounds. Three solvents viz., methanol, ethyl acetate and hexane were used for
extraction and the respective leaf extracts were analyzed for total phenolic and flavonoid contents along
with the antioxidant and antimicrobial activities. Amongst the tested solvents, methanol was found to be
Keywords:
Antioxidant
the best solvent for extraction with highest total phenolic contents and the lowest IC50 values for the 2,2-
Bergenia ciliata diphenyl-1-picrylhydrazyl (DPPH) and 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)
Phenolic content assays. Methanol extract also exhibited effective antimicrobial activity, particularly against bacteria and
Flavonoid content actinomycetes. Further, high performance liquid chromatography (HPLC) analysis revealed that meth-
Sikkim Himalaya anolic extract contains the highest amount of all the three analyzed bioactive compounds viz. bergenin,
catechin and gallic acid. The current study suggests that the methanol extract of B. ciliata is a potential
source of natural antioxidant and antimicrobial compounds that can be used in food and drug industries.
Copyright © 2016, Center for Food and Biomolecules, National Taiwan University. Production and hosting
by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction Extensive investigation of medicinal plants for biological activ-

ities and bioactive compounds is the crucial and the foremost step
Microbial contamination and side effects of synthetic antioxi- in development of effective alternative medications. In view of this,
dants are two important major concerns of food and pharmaceu- in the present study, Bergenia ciliata Sternb. (family Saxifragaceae),
tical industries. Increasing propensity for replacing synthetic a high value plant of the Sikkim Himalaya, has been investigated for
antioxidant by natural one on one side and development of mi- antioxidant, antimicrobial activity and bioactive compounds. It is
crobial resistance to existing antibiotics from the other has an important perennial medicinal herb that grows widely in the
encouraged researchers toward appraising medicinal plants for temperate Himalaya between 1500 and 3000 m asl.3 The plant
dual antioxidant and antimicrobial properties. Though, since im- species flourishes well in rocky areas and on the cliffs. The plant has
memorial time, medicinal plants have been used to treat and pre- been in use as folklore medicine since ancient times for dissolution
vent various human ailments and they are considered as reservoir of kidney and gall bladder stones.4,5 In Sikkim Himalaya, Bergenia
of bioactive compounds.1,2 Till date, biological properties and rhizome is used to treat fractured bones, fresh cuts, wounds, diar-
bioactive compounds of many medicinal plants are not studied. rhea, pulmonary infections, vomiting, fever, cough and boils by
locales.6e8 Bergenia is also used for the treatment of heart disease,
haemorrhoids, stomach disorders and ophthalmia. In addition, it is
accredited with analgesic, antiviral, anti-inflammatory and anti-
Abbreviation: ABTS, 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); DPPH, malarial properties.9,10 The plant's virtues, to a large extent, are
2,2-diphenyl-1-picrylhydrazyl; HPLC, high performance liquid chromatography.
attributed to its secondary metabolites such as bergenin, gallic acid
* Corresponding author. Tel.: þ91 3592 237328x237189; fax: þ91 3592 237415.
E-mail address: [email protected] (M. Singh). and catechin which are therapeutic and account for its use in
Peer review under responsibility of The Center for Food and Biomolecules, traditional medicine.11,12
National Taiwan University.

http://dx.doi.org/10.1016/j.jtcme.2016.04.002
2225-4110/Copyright © 2016, Center for Food and Biomolecules, National Taiwan University. Production and hosting by Elsevier Taiwan LLC. This is an open access article
under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
 M. Singh et al. / Journal of Traditional and Complementary Medicine 7 (2017) 152e157 153

Despite the widespread use of this plant in traditional medicine, 2.5. Determination of antioxidant activity
the scientific literature with respect to its biological properties is
scanty. To the best of our knowledge, so far, antioxidant, antimi- 2.5.1. DPPH radical scavenging assay
crobial properties and the bioactive compounds associated with the The effect of extracts on 2,2-diphenyl-1-picrylhydrazyl (DPPH)
leaf of Bergenia plant, growing in the Sikkim Himalaya, have not radical was determined using the method of Liyana-Pathiranan and
been investigated. Therefore, the present study was conducted Shahidi.15 A solution of 0.135 mM DPPH in methanol was prepared
with the objectives to (1) obtain the most effective solvent for and 1.5 mL of this solution was mixed with 1.5 mL of extract in
extracting the potent bioactive compounds, especially phenolics methanol. The reaction mixture was mixed thoroughly and left in
and flavonoids, (2) investigate different extracts for antioxidant and the dark at room temperature for 30 min. The absorbance of the
antimicrobial activities and, (3) quantify the amount of bergenin, mixture was measured spectrophotometrically at 517 nm. Butyl-
catechin and gallic acid present in the leaf extracts of B. ciliata. ated hydroxytoluene (BHT) was used as standard. The ability of
sample to scavenge DPPH radical was calculated by the following
equation:
2. Material and methods
DPPH radical scavenging activity (%)
2.1. Plant collection
¼ [(Abs control  Abs sample)]/(Abs control)  100
Leaves of B. ciliata were collected during flowering season
where Abs control is the absorbance of DPPH radical þ methanol;
(March 2014) from the plants growing in the arboretum of G.B. Pant
Abs sample is the absorbance of DPPH radical þ extract/standard.
Institute of Himalayan Environment and Development (GBPIHED),
The radical scavenging activity of extracts was determined by IC50
Sikkim Unit, Gangtok, India (latitude 27 210 35.700 N; longitude 88
value. IC50 value is the concentration of extracts at which DPPH
370 24.400 E), situated at the elevation of 2047 m asl. Voucher spec-
radicals are scavenged by 50%. The lower IC50 value indicates higher
imens (GBP3590) were deposited at the Herbarium of GBPIHED.
radical scavenging capacity and vice versa.

2.2. Extract preparation

2.5.2. ABTS radical scavenging assay
The leaves were washed thoroughly under running tap water The 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
and finally with distilled water and dried on blotting paper at room (ABTS) radical scavenging assay was done according to Re et al.16
temperature to get consistent weight. The dried leaves were later with slight modifications. The ABTS radical cation solution was
ground to powder using grinder. To prepare samples, 2 g powder prepared by mixing equal quantities of ABTS (7 mM) and ammo-
was soaked separately in 10 mL of different solvents (ethyl acetate, nium persulfate (2.45 mM) and allowing them to react for 16e20 h
methanol and hexane) for 24 h. The supernatant was transferred at room temperature, in dark. The working solution was then pre-
into a new tube and the residue was re-extracted twice with 10 mL pared by diluting the previous solution with methanol until the
solvent. Extracts were filtered using a Buckner funnel and What- absorbance at 734 nm was 0.706 ± 0.02. ABTS solution was freshly
man No. 1 filter paper. Each filtrate was concentrated to dryness in prepared for each assay. Plant extracts (1.5 mL) were allowed to
oven, maintained at 40  C. Each extract was resuspended in the react with equal volume of the ABTS working solution and the
methanol to yield a 50 mg/mL stock solution. The yield of the absorbance was taken at 734 nm after 7 min using the spectro-
extraction was calculated from {(W1/W2)  100}, where W1 is the photometer. The ABTS scavenging capacity of the extracts was
weight of extract after evaporation of solvent and W2 is the dry compared with that of BHT and the percentage inhibition was
weight of the plant sample. calculated as:

ABTS radical scavenging activity (%) ¼ [(Abs control  Abs sample)]/

2.3. Total phenolic content (TPC)
(Abs control)  100
Total phenolic content in Bergenia extracts was determined using
where Abs control is the absorbance of ABTS radical þ methanol;
FolineCiocalteu assay.13 Solutions of each extract (100 mL; 1 mg/mL)
Abs sample is the absorbance of ABTS radical þ extract/standard.
were taken individually in test tubes. To this solution, 2.5 mL of 10-
ABTS radical scavenging activity of extracts was determined by IC50
fold diluted FolineCiocalteu reagent was added, and the test tubes
value as mentioned above in DPPH assay.
were thoroughly shaken. After 3 min, 2.0 mL of 7.5 % Na2CO3 solution
was added and the mixtures were incubated for 30 min. The
absorbance of the reaction mixtures was measured at 760 nm by
2.6. Antimicrobial assay
using a spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan).
Gallic acid was used as a standard and TPC of Bergenia extracts was
Total eight strains (4 bacteria, 2 actinomycetes and 2 fungi) were
expressed in milligram gallic acid equivalents (mg GAE/g extract).
taken from Microbial Culture Collection established in the Micro-
biology laboratory of GBPIHED Institute, Almora, India. Initially test
2.4. Total flavonoid content (TFC) organisms were grown on the respective media i.e. bacteria and
actinomycetes in Tryptone Yeast extract (TY) and fungus on Potato
Total flavonoid content was determined by the aluminum Dextrose broth (PD) in conical flask, at 25 C for 24 h. Antimicrobial
chloride calorimetric method,14 with some modifications. Briefly, activity was performed on TY and PD agar plates following disc
the test samples were individually dissolved in methanol. Then, the diffusion assay at 25 C, 2 days for bacteria and 6 days for actino-
sample solution (2 mL) was mixed with 2 mL of 2% AlCl3. After mycetes and fungus. Minimum inhibitory concentration (MIC) of
10 min of incubation at ambient temperature, the absorbance of the extracts was determined following the protocol of Clinical and
solution was measured at 435 nm by using a spectrophotometer Laboratory Standards Institute (CLSI).17,18 All of the experiments
(UV-1800, Shimadzu, Kyoto, Japan). The flavonoid content was were performed in triplicate, and results were expressed as di-
expressed as milligram quercetin equivalent (mg QE/g extract). ameters (mm) of inhibition.
154 M. Singh et al. / Journal of Traditional and Complementary Medicine 7 (2017) 152e157

2.7. High performance liquid chromatography (HPLC) analysis content of extractable phenolic compounds was determined
through a linear gallic acid standard curve (y ¼ 2.271x þ 0.18;
Detection and quantification of catechin, gallic acid and berge- R2 ¼ 0.995). The highest content of total phenolic compounds was
nin were carried out using Shimadzu 20 AD, HPLC system (Shi- detected in the Bergenia methanolic extract (473.4 mg GAE/g
madzu, Japan) consisted of UV detector, a binary pump, a 10 mL extract) followed by ethyl acetate extract (249.7 mg GAE/g extract)
injection loop, and reverse phase C-18 column of dimensions (p < 0.05). In the hexane extract, phenolic compounds could not be
4.6  250 mm. The mobile phase used for catechin was 75% A detected (Table 1). These results demonstrate clearly that the
(water þ 0.1% trifluoroacetic acid) and 25 % B (methanol) with a content of phenolic compounds is dependent on the polarity of the
flow rate of 0.8 mL/min. The eluted samples were detected by the solvent used; higher the polarity of the solvent, higher the content
UV detector at 280 nm. For gallic acid, mobile phase used was 90 % of phenolic compounds. These results are in agreement with the
A (water þ 1% acetic acid) and 10% B (acetonitrile) with a flow rate previous study of Singh et al.20 that reported methanol as an
of 1.0 mL/min. The eluted samples were detected by UV detector at effective solvent for antioxidant extraction, phenolic compounds in
271 nm. particular.
For analysis of bergenin, elution was carried out with solvent
75% A (water þ 0.1% trifluoroacetic acid) and 25% B (acetonitrile) as 3.3. Total flavonoid content (TFC)
mobile phase having flow rate of 1.0 mL/min. Detection was carried
out at 248 nm. Calibration curve was constructed by plotting the Flavonoids are common secondary metabolites present in plants
peak area (y) against concentration in mg/mL of standard solutions which are responsible for many plant biological activities.21
(x). The standard equation obtained from the curve was used for The solvent efficiency on TFC, in ascending order was:
quantification of bioactive compounds in the unknown samples. hexane < methanol < ethyl acetate. The highest flavonoid content
of 208.4 mg QE/g extract was observed in the extract of ethyl ace-
2.8. Statistical analysis tate followed by methanol extract (89.9 QE/g extract). Hexane was
found to be ineffective for flavonoid extraction (Table 1). These
All the experiments were performed in triplicate and the results are in agreement with the report of Hajji et al.23 which re-
experimental data were expressed as mean ± standard deviation ported that flavonoid content in extract depend on solvent polarity.
(SD). One-way analysis of variance (ANOVA) and Duncan's multiple
range tests were carried out to determine significant differences 3.4. Antioxidant activity
(p < 0.05) between the means by SPSS (version 16.0). Correlation
coefficients were determined by SPSS (version 16.0) Pearson cor- 3.4.1. DPPH free radical scavenging activity
relation program. The DPPH free radical scavenging activity in leaf extracts of
B. ciliata is presented in Fig. 1. The methanolic leaf extract showed
3. Results and discussion excellent free radical scavenging activity (IC50 ¼ 53.5 mg/mL). In
other two leaf solvent extracts, the free radical scavenging activity
3.1. Extraction yield in ethyl acetate extract (IC50 ¼ 2593.3 mg/mL) was superior to that
of the hexane extract (IC50 ¼ 3026.7 mg/mL). Nevertheless, when
Results showed that leaf extraction yield of B. ciliata varied compared to standard, the BHT, all the tested Bergenia leaf extracts
considerably as a function of solvent nature and ranged from 8.3 to showed significantly (p < 0.05) lower DPPH radical scavenging
38.2 % with a descending order of methanol > ethyl activity. These results are indicative of the influence of the solvent
acetate > hexane (Table 1). Extraction with methanol resulted in on the antioxidant activity of the biological extracts. It has been
the highest amount of total extractable compounds whereas the reported that in plant compounds with different polarity and
extraction yield with hexane was small in comparison to other structure are present that dissolve in specific solvents having
solvents. Higher extraction yield in methanol might be due to the similar polarity.24,25 The difference in the DPPH radical scavenging
fact that it easily penetrates the cellular membrane and extracts the activity in different solvent extracts implies towards the preference
intracellular ingredients from the plant material. Moreover, results of the solvents for extraction of different types and concentrations
indicated that the plant contains more of polar substances than the of bioactive compounds.24,25 Strong DPPH radical scavenging ac-
others. Earlier reports have also suggested that methanol give tivity of B. ciliata methanolic extract can be due to higher content of
higher extraction yield than the other solvents such as acetone, total phenolic compounds.
diethyl ether, ethyl acetate and water.19,20
3.4.2. ABTS radical scavenging assay
3.2. Total phenolic content (TPC) The free radical scavenging activity of Bergenia extracts was also
determined using ABTS radical. Significant difference (p < 0.05) was
The antioxidant activity of medicinal plants, fruits and vegeta- revealed between ABTS scavenging capacities of extracts measured
bles has been reported to be positively correlated to their total as IC50 value (Fig. 2). The highest ABTS radical scavenging activity
phenolic contents due to their ability to scavenge free radicals.21,22 was found in methanol extract with an IC50 value of 5.4 mg/mL,
It becomes, therefore, mandatory to estimate total phenolic com- whereas hexane extract exerted the lowest ability to scavenge ABTS
pounds present in the Bergenia extracts. In the present study, the radical with an IC50 value of 51 mg/mL. Strong ABTS radical

Table 1
Total phenolics, flavonoid and extraction yield of different extracts of Bergenia ciliata.

Solvent Extraction yield (% w/w) Total phenolic content (mg GAE/g extract) Total flavonoid content (mg QE/g extract)

Methanol 38.2 ± 3.4 a 473.4 ± 15.1 a 89.9 ± 0.1 b

Ethyl acetate 18.5 ± 2.5 b 249.7 ± 1.3 b 208.4 ± 0.6 a
Hexane 8.3 ± 1.4 c nd nd

Values are mean ± standard deviation of triplicate experiments. Different letters in columns show significant differences at p < 0.05; nd, not detected.
 M. Singh et al. / Journal of Traditional and Complementary Medicine 7 (2017) 152e157 155

Results indicate clearly that the DPPH activities of extracts are

mainly due to the total phenolic compounds. Negative correlation
indicated that with the increase in phenolic and flavonoid content,
concentration of extract required to inhibit free radical decreases.
In comparison to DPPH assay, strong correlation existed between
ABTS assay and TFC. The correlation for ABTS IC50 value vs TPC and
ABTS IC50 value vs TFC were 0.911 and 0.782, respectively, imply
that both the total phenol and flavonoids of B. ciliata are responsible
for ABTS activities. These differences in correlation between phy-
tochemicals and antioxidant assays could be attributed to the
different mechanism of the radical antioxidant reaction. In accor-
dance with this study, Li et al.27 have also reported high negative
linear correlation between the phenolic contents and IC50 values in
Angelicae sinensis confirming that phenolics are likely to contribute
Fig. 1. Free radical-scavenging activity of B. ciliata extracts measured by DPPH method
in terms of IC50 value (mg/mL of extract). Values are mean of triplicate experiments. to the antioxidant activity of the extracts. However, presence of
Mean values with different letters differ significantly (p < 0.05) according to Duncan's other metabolites which have significantly contributed in antioxi-
multiple range test. dant activity of B. ciliata extracts cannot be ruled out.

3.5. Antimicrobial activity

The results of the disc diffusion assay (Table 2) revealed that all
the extracts tested were effective against the bacteria and actino-
mycetes studied, but showed no activity against fungi. Among the
tested extracts, methanol extract was found to be the most effective
which showed the highest effects against Bacillus megaterium strain
with inhibition zone of 9.8 mm, followed by Nocardia tenerifensis
and Bacillus subtilis with inhibition zones of 9.7 and 8.8 mm,
respectively. The ethyl acetate extract showed the similar pattern of
microbial inhibition with highest inhibition zone of 7.5 mm for B.
megaterium, followed by 6.2 and 5.5 mm inhibition zone for N.
tenerifensis and B. subtilis, respectively. In contrary to methanol and
ethyl acetate extract, the hexane extract had the greatest inhibitory
Fig. 2. Free radical-scavenging activity of B. ciliata extracts measured by ABTS method effects against Serratia marcescense (5.0 mm) and B. subtilis
in terms of IC50 value (mg/mL of extract). Values are mean of triplicate experiments. (4.7 mm).
Mean values with different letters differ significantly (p < 0.05) according to Duncan's The minimum inhibitory concentration (MIC) was determined
multiple range test.
in the methanolic extracts of B. ciliata due to its higher antimicro-
bial activity in disc diffusion assay against the tested organisms. The
scavenging ability of B. ciliata methanol and ethyl acetate extracts methanol extract gave the lowest minimal inhibitory concentration
can be attributed to the presence of flavonoids. Earlier, it has been against B. subtilis with MIC of 1250 mg/mL (Table 3). The highest
reported that ABTS radical scavenging ability of bioactive com- antimicrobial activity of methanolic extract could be due to the
pounds depends on its molecular weight, structure and presence of high contents of phenolic compounds and flavonoids present in the
number of aromatic rings.26 extract. These results are in accordance with a previous study
where similar observations were recorded in case of Bergenia lig-
3.4.3. Correlation of IC50 values of antioxidant activities with TPC ulata leaf extracts.28
and TFC
The correlation coefficients determined between the antioxi- 3.6. Quantification of bergenin, catechin and gallic acid by HPLC
dant activities and the total phenolic and flavonoid contents in all
the B. ciliata leaf extracts revealed the presence of significant high B. ciliata is reported to contain a wide array of pharmaceutically
correlation (p < 0.1). The correlation for DPPH IC50 value vs TPC and important bioactive compounds.9,29,30 Among all, three main
DPPH IC50 value vs TFC were 0.913 and 0.056, respectively. compounds viz., bergenin catechin and gallic acid are the most

Table 2
Effect of the extraction solvent on the antimicrobial activity of Bergenia ciliata.

Microorganism Zone of inhibition (mm)

Methanol extract Ethyl acetate extract n-Hexane extract

Bacillus subtilis* 8.83 ± 0.3 5.5 ± 0.5 4.66 ± 0.3

Bacillus megaterium* 9.83 ± 0.2 7.5 ± 0.5 1.83 ± 0.3
Escherichia coli* 6.00 ± 0.0 1.83 ± 0.3 na
Serratia marcescense* 7.00 ± 0.2 5.00 ± 0.3 5.00 ± 0.3
Nocardia tenerifensis** 9.66 ± 0.9 6.16 ± 0.2 na
Streptomyces sp.** 6.66 ± 0.6 1.83 ± 0.3 na
Aspergillus niger# na na na
Fusarium oxysporum# na na na

* ¼ bacteria; ** ¼ actinomycetes; # ¼ fungi; na ¼ no activity.

156 M. Singh et al. / Journal of Traditional and Complementary Medicine 7 (2017) 152e157

Table 3 bergenin showed that methanolic extract contained significantly

Minimum Inhibitory Concentration (MIC) of test organism. higher amount of these bioactive compounds.
S. no. Microorganisms MIC (mg/mL)

1 Bacillus subtillis* 1250 Conflict of interest

2 Bacillus megaterium* 2500
3 Serratia marcescense* 2500
The authors declare “no conflict of interest.”
4 Escherichia coli* 2500
5 Nocardia tenerifensis** 5000
6 Streptomyces sp.** 2500 Acknowledgment
7 Fusarium oxysporum# na
8 Aspergillus niger# na
Authors are highly grateful to the Director, GB Pant Institute of
* ¼ bacteria; ** ¼ actinomycetes; # ¼ fungi; na ¼ No activity.
Himalayan Environment and Development, Almora, for providing
necessary facilities. Ministry of Environment, Forests and Climate
Table 4 Change (MoEF & CC) is thanked for financial support.
Standard curve analysis for catechin, gallic acid and bergenin.

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