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Two new Stereoisomeric Antioxidant Triterpenes from Potentilla fulgens

Alka Choudhary, Amit Kumar Mittal, Manukonda Radhika, DebabrataTripathy, Anupam Chatterjee, Uttam Chand Banerjee, Inder Pal Singh

PII: S0367-326X(13)00243-8DOI: doi: 10.1016/j.fitote.2013.09.008Reference: FITOTE 2777

To appear in: Fitoterapia

Received date: 1 July 2013Revised date: 9 September 2013Accepted date: 14 September 2013

Please cite this article as: Choudhary Alka, Mittal Amit Kumar, Radhika Manukonda,Tripathy Debabrata, Chatterjee Anupam, Banerjee Uttam Chand, Singh Inder Pal, Twonew Stereoisomeric Antioxidant Triterpenes from Potentilla fulgens, Fitoterapia (2013),doi: 10.1016/j.fitote.2013.09.008

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Two new Stereoisomeric Antioxidant Triterpenes from Potentilla fulgens

Alka Choudhary a, Amit Kumar Mittal

b, Manukonda Radhika

c, Debabrata Tripathy

c,

Anupam Chatterjee c, Uttam Chand Banerjee

b and Inder Pal Singh

a,

aDepartment of Natural Products,

National Institute of Pharmaceutical Education and Research,

Sector-67, S.A.S. Nagar, 160062, Punjab, India.

bDepartment of Pharmaceutical Technology Biotechnology,

National Institute of Pharmaceutical

Education and Research, Sector-67, S.A.S. Nagar, 160062, Punjab, India.

cDepartment of Biotechnology and Bioinformatics, North Eastern Hill University, Shillong, 793002,

Meghalaya, India.

*Corresponding author

Dr. Inder Pal Singh

Associate Professor

Department of Natural Products

National Institute of Pharmaceutical Education and Research (NIPER)

Sector 67, SAS Nagar

Punjab-160062

India

Tel: 91-172-2292144, Fax: 91-172-2214692

Email: ipsingh@niper.ac.in; ipsingh67@yahoo.com

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Abstract

The roots of Potentilla fulgens have been used for a long time as a folk remedy for many

ailments without having information on its pharmacological action. Of the various extracts

prepared by partitioning of the methanol extract, the ethyl acetate fraction was found to

possess better antioxidant and cytotoxic activities. The degree of reduction in cloning

efficiencies of MCF-7 cell lines was more with ethyl acetate than hexane fraction of the root-

extract. Hence, this fraction was further purified and nine compounds, including two new

ursane type triterpenoids Fulgic acid A (4) and Fulgic acid B (5), were identified and

characterized. Other compounds were identified as ursolic acid, euscaphic acid, corosolic

acid, epicatechin, catechin, p-hydroxy benzaldehyde and gallic acid. Chemical structures

were elucidated by spectroscopic methods, especially HRESIMS and 2D NMR techniques.

The new compounds showed good antioxidant activity and therefore this plant can be a

source of natural antioxidants.

Keywords: Potentilla fulgens, Rosaceae, Triterpenes, Fulgic Acids, Ursane, antioxidants

1. Introduction

The genus Potentilla includes about 500 species, herbs or small shrubs with rhizomes and

belongs to family Rosaceae. The genus evolves from the Latin term potens meaning

“powerful” which is in reference to the curative properties of some species. The plants find

their natural abode in Temperate, Arctic and Alpine zones of the Northern hemisphere while

a few species have been found in high mountains of the tropics and in South America [1].

Various species of genus Potentilla are used in Ayurveda, Unani, Siddha, Chinese and

Tibetan systems of medicine [2]. Several reports in the literature have indicated the presence

of polyphenolic compounds in the plants of this genus suggesting that these plants can be

explored for their potential antioxidant properties. Polyphenols isolated from these plants are

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of immense importance both in medicine and nutrition for their potent antioxidant capacity

and protective effects on human health and diseases including neoplastic diseases [3].

Potentilla fulgens (Vajardanti) is a therapeutically and commercially important member of

this genus. The plant is generally distributed at higher altitudes in western Himalayan region,

Khasi hills and in eastern Himalaya in Meghalya, India [4]. Traditionally, the root stock is

edible and is effective against high blood pressure. In several areas of North-East India, the

root juice is taken as a remedy for stomach ailments, peptic ulcers, cough and cold and other

respiratory problems. Root powder is consumed to strengthen the teeth and as a treatment to

gingivitis and pyorrhoea. Owing to its importance in managing various tooth ailments, the

species is commercially utilized by the Vicco Laboratories, India for manufacturing Vicco

Vajardanti tooth paste and powder. The pieces of roots are also chewed along with betel,

composed of raw areca nut, locally called Kawai, and betel leaf [5].

Methanolic root extract of P. fulgens has shown activity against several tumours [6],

hypoglycemic and anti-hyperglycemic acitivity in alloxan induced and normal diabetic mice

[7], antioxidant, anti- ulcerogenic activity [8] and

anthelmintic activity [9].

Despite several uses and biological activities, there are only a few reports on the isolation of

bioactive compounds [10]. Epicatechin and a biflavanoid, Potifulgene from the butanol

soluble part of methanolic extract of the roots have been recently reported as antioxidant

molecules [11]. Primary phytochemical studies have pointed towards the presence of

triterpenoids along with polyphenols in the plant. A few triterpene compounds have been

recently reported from the ethyl acetate extract of the plant [12]. In preliminary investigations

by the authors, the methanol extract of the roots of P. fulgens was found to increase the in

vivo survival of mice bearing Ehrlich ascites cells and also showed a dose–dependent

inhibitory effect on growth of MCF-7 cells through clonogenic assay [6]. In order to

understand the chemical composition responsible for biological activities, present study was

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taken up to explore the existence of several other antioxidant and cytotoxic molecules in P.

fulgens. Methanol extract of the roots was prepared and fractionated into hexane, ethyl

acetate, butanol and aqueous soluble fractions in the same sequence. The obtained fractions

were evaluated in various antioxidant and cytotoxic assays and the fraction having both the

antioxidant and cytotoxic activity was taken up for further purification and isolation of pure

compounds. The isolated molecules were also examined in various antioxidant and cytotoxic

assays.

2. Materials and methods

2.1 General

HPLC analysis was carried out on Phenomenex C18 column (250 × 4.6 mm) connected to a

Shimadzu (USA manufacturing Inc.) HPLC system consisting of a model LC-10AT VP fitted

with SIL-20AC auto sampler and SPD-M10A VP photodiode array detector. Princeton

SPHER-C18 column (250 × 10 mm) was used for isolation of compounds. The 1H and

13C

NMR spectra were recorded on Ultrashield 400 NMR spectrometer (Bruker, Germany). The

optical rotations were measured on Autopol IV polarimeter (Rudolph, USA). Mass spectra

were recorded on a LCQ (Thermo- Quest, USA). The IR spectra were performed on a FTIR

system (Nicolet, USA). Absorbances were recorded on Multiskan (Thermo scientific) 96 well

plate reader. The extracts/compounds were dried on rotary vacuum evaporator (Buchi,

Switzerland).

2.2 Chemicals and reagents

All chemicals and solvents used for extraction and purification were of laboratory reagent

grade. All chromatographic purifications were performed with silica gel #60-120 and silica

gel G whereas all TLC analysis was performed on silica gel coated (Merck Kieselgel 60 F254,

0.2 mm thickness) plates. HPLC grade acetonitrile (JT Baker) and ultra pure water (Elga®)

were used for sample preparation and in HPLC mobile phases. For antioxidant assays, 1,1-

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diphenyl-2-picrylhydrazyl (DPPH·), Trolox, 2,2-azinobis-(3-ethylbenzthiazoline-6-sulphonic

acid) (ABTS+·

), potassium persulphate, trichloroacetic acid, potassium ferricyanide, ferrous

chloride were purchased from Loba chemie, Mumbai, India, ferric chloride from

Spectrochem, Mumbai, India, 3-(4,5-dimethylthiazole-2-yl)-2,5- diphenyltetrazolium

bromide (MTT) and quercetin dihydrate were purchased from Sigma Aldrich and L-ascorbic

acid from Himedia.

2.3 Cell lines and culture medium

MCF-7 (human breast cancer) cell line was purchased from the National Center for Cell

Science (Pune, India). Cells were grown in Dulbecco’s MEM medium (DMEM, Invitrogen-

GIBCO), supplemented with 10% Fetal Calf serum (Invitrogen-GIBCO), 100 U/ml penicillin

and 100 mg/ml streptomycin (Invitrogen-GIBCO) and 2 mM L-Glutamine (Invitrogen-

GIBCO).

2.4 Plant material

Potentilla fulgens roots were collected from Shillong peak forest area of Meghalaya state in

India. A voucher specimen was deposited in the herbarium of the Department of Botany,

North-Eastern Hill University, Shillong (accession number 11906). The roots were air-dried

in the shade and crushed to a coarse powder.

2.5 Extraction and isolation

The plant material (1 Kg root material) was dried and ground to yield a coarse powder. The

root powder was macerated with methanol at 35-37 °C for 72 h to yield 400 g methanol

extract. The concentrated extract was then suspended in water-methanol mixture (80:20) and

partitioned with hexane, chloroform, ethyl acetate and butanol, in the same order. Solvent-

solvent partitioning yielded 8 g hexane extract, traces of chloroform extract, 293 g ethyl

acetate and 30 g of butanol extract. Ethyl acetate extract (40 g) was chromatographed [silica

gel #100-200, Column H: 30 cm, i.d.: 7 cm; Eluent: hexane-ethyl acetate (0% to 90% EtOAc)

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then chloroform-methanol gradients (1% to 50% MeOH)] to yield five major fractions.

Fraction 1 (2.28 g) was column chromatographed using hexane-ethyl acetate to give

compound 1 (30 mg), compound 2 (8 mg) and compound 3 (6 mg). Fraction 2 (2.6 g) which

was eluted with 5 % EtOAc in hexane was a mixture of two compounds, which were resolved

on HPLC using Phenomenex C18 (250 × 4.6 mm, 5 μ) column and isocratic ACN/H2O, 75:25

mobile phase at flow rate 1mL/min. It furnished two compounds, compound 4 and

compound 5. Fraction 3 (15 g) on TLC analyses showed presence of compound 6 and

compound 7 which were identified as Epicatechin and catechin by co-TLC with the authentic

sample available in laboratory. Fraction 4 (5 g) yielded compound 8 and compound 9 after

chromatography on silica gel.

2.6 Antioxidant assays

2.6.1 DPPH· radical scavenging assay

Free radical scavenging activity of the test compounds/extracts was determined by the DPPH·

assay [13]. Briefly, 200μL reaction mixture contained methanolic solution of DPPH· (190 μL)

and 10 μL of different concentrations of test compounds/extracts in methanol. The reaction

mixture was incubated for 1 h at 27 °C and the absorbance was measured at 517 nm. The

change in absorbance with respect to the control (containing DPPH· only without sample,

expressed as 100% free radicals) was calculated as percentage scavenging and the IC50 value

was determined. Acorbic acid and trolox were used as positive controls. The ability to

scavenge DPPH radicals was calculated as follows:

% Inhibition = (ODblank – ODtest)/ ODblank ×100

ODblank is the absorbance of blank; ODsample is the absorbance of test (extract/compound).

The IC50 value was obtained by plotting the DPPH· scavenging percentage of each sample

against the sample concentration. The experiment was performed in triplicate for each

concentration of the individual sample.

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2.6.2 ABTS+·

radical scavenging assay

ABTS+·

radical scavenging activity of the test compounds was determined using an ABTS+·

decolorisation assay [14]. To the ABTS+·

liquid substrate system 2.45 mM potassium

persulphate was added in a stoichiometric ratio of 1:0.5 (v/v). The mixture was allowed to

stand in the dark at 27 °C for 8 h. In the ABTS+·

solution (190 μL) different concentrations of

test compounds/extracts (10 μL) were added. The reaction mixture was incubated for 6 min at

27 °C and the absorbance was measured at 734 nm. The change in absorbance with respect to

the control (containing ABTS+·

solution only without sample, expressed as 100% free

radicals) was calculated as percentage scavenging and the IC50 value was calculated. The

known antioxidants ascorbic acid and trolox were used as positive controls.

2.6.3. Reducing power assay

The reducing power assay was done by reported protocol [15-16]. Briefly each

extract/compound (0.125–1 mg) was dissolved in 1.0 mL of DMSO. To this extract solution,

phosphate buffer (0.2 M, 2.5 mL, pH 6.6) was added followed by addition of a solution of

potassium ferricyanide (1%, 2.5 mL). The reaction mixture was incubated in water bath at 50

°C for 20 min. After incubation trichloroacetic acid (10% w/v, 2.5 mL) was added to the

reaction mixture and was centrifuged at 7500 rpm (5471 g) for 10 min. Distilled water (2.5

mL) and a solution of ferric chloride (0.1%, 0.5 mL) were added to an aliquot of the upper

layer (2.5 mL). Finally the absorbance of the reaction mixture was measured at 700 nm.

Increased absorbance of the reaction mixture indicates greater reducing power. The

experiment was performed in triplicate. Quercetin dihydrate was used as a positive standard.

2.6.4. MTT antioxidant assay

MTT antioxidant assay was performed as per the reported procedure [17]. Briefly a stock

solution of test extracts/ compounds (1 mg/mL) in DMSO and an aqueous solution of MTT

(1 mg/mL) were prepared. In a capped glass vial (5 mL) mixture of aqueous solution of MTT

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(190 μL) and different concentrations of test compound/extract (10 μL) was vortexed. To it

methanol (200 μL) was added and was vortexed again. The reaction mixture was incubated at

37 °C for 6 h. After the incubation, 200 μL of the reaction mixture was taken in 96-well cell

culture plate, and the absorbance was read at 570 nm. Each sample was assayed in triplicate

and the average absorbance was noted. In control the test solution was replaced with 10 μL of

methanol. Quercetin dihydrate was used as a positive standard.

2.7 Clonogenic cell survival assay

The cell survivality was evaluated using a clonogenic assay in MCF-7 cancer cell lines. Cells

plated at two cell densities (2000 and 3000 cells) onto three 25 cm2 flasks each and for

untreated controls, four flasks were plated at one cell density (1000 cells). Five hours after

plating, the cells were exposed for 24 h with 15 to 120 µg/ml of the test samples. Cells were

washed twice with the medium and finally flasks were incubated in CO2-incubator with fresh

DMEM with 10% foetal calf serum for 8 to 10 days. The colonies were fixed and stained

with 0.2% crystal violet in 70% ethanol. Colonies with a minimum of 50 cells were counted

as the progeny of a viable cell.

2.8 Statistics

The data obtained from clonogenic survival assay are represented as a sigmoidal fit curve

with a linear X scale, the function used was Boltzmann. The results are expressed as mean

plus or minus Standard deviation (SD).

3. Results and discussion

Of the various extracts prepared by partitioning of the methanol extract viz. hexane, ethyl

acetate, butanol and aqueous extract, the ethyl acetate fraction was found to possess both the

antioxidant and cytotoxic activities. Hence, this fraction was selected for further purification

of pure compounds. Nine compounds (Fig. 1), including two new compounds, were identified

and characterized from the ethyl acetate extract of the roots of P.fulgens.

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3.1 Structure elucidation of compounds 4 and 5

Compound 4: 9 mg (0.0009% yield) was obtained as a white powder with [α]D20

-89.9 (c

0.34, CHCl3). IR spectrum revealed the presence of carboxyl group (1692 cm-1

) and hydroxyl

group (3422 cm-1

). The molecular formula was established as C30H48O5 on the basis of

ESIHRMS data at m/z: 511.3393[M+Na]+ (calcd for m/z 511.3399 [M+Na]

+) and at 493.3293

[M+Na-H2O]+. Combined analysis of the

1H,

13C and DEPT-135 NMR spectroscopic data

revealed the presence of seven CH3 (δ 16.5, 17.5, 17.7, 20.4, 21.6, 22.9 and 28.5); nine CH2

(δ 17.9, 21.5, 25.4, 27.4, 27.9, 32.8, 33.3, 34.8 and 42.3); five CH (δ 40.4, 48.1, 50.7, 66.6

and 78.9 ), six sp3 quaternary carbons (δ 38.3, 38.8, 41.7, 42.5, 43.4 and 83.1), two sp

2

quaternary carbons (δ 131.36 and 140.1) and one carboxyl carbon (δ 176.5). Absence of an

olefinic proton signal at δ 5.5-5.2 ppm in 1H NMR and the corresponding carbon signal at δ

128-126 ppm was indicative of the absence of a trisubstituted double bond at the usual

position of ursane type triterpenes. Furthermore, 13

C and DEPT-135 suggested a double bond

between two quaternary carbons. Presence of deshielded carbon signals at δ 66.5, 78.8 and

83.1 ppm was indicative of the attachment of a hydroxyl group at these positions. Exact

position of the double bond and hydroxyl groups was elucidated using detailed analyses of

the correlations observed in the 1H-

1H COSY, HSQC, HMBC and NOESY spectrum.

1H-

1H

COSY spectrum presented correlation between the proton signals resonating at δ 4.02 and

3.43, indicating their adjacent location (Fig. 2). Appearance of one doublet signal at δ 1.08

for methyl group in 1H NMR was supported by the presence of its COSY correlation with

proton at δ 2.56 (H-19). The HMBC spectrum depicted the correlations for seven methyl

groups. Methyl signal at δ 1.01 (H-23) was correlated with tertiary carbons at δ 78.8 (C-3)

and 48.2 (C-5), quaternary carbon at δ 38.8 (C-4) and a primary carbon at δ 21.7 (C-24).

Similar correlations were observed for the methyl signal at δ 0.84 (H-24). It showed

correlation with C-3, C-4, C-5 and C-23. These correlations confirmed the position of two

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germinal methyl groups C-23 and C-24 at C-4 of skeleton. The methyl signal at δ 0.91 (3H,

H-25) presented correlations with secondary carbon at δ 42.3 (C-1), a quaternary carbon at δ

38.8 (C-10) and a tertiary carbon at δ 50.7 (C-9). The common correlation by protons at δ

0.91 (H-25) and 0.81(H-26) with C-9 confirmed the position of two methyl groups, C-25 and

C-26 at the positions C-10 and C-8 of the ursane nucleus. Other correlations for H-26 with

quaternary carbon at δ 41.7 (C-8) and quaternary carbon at δ 42.5 (C-14) also supported its

location at C-8. For methyl signal at δ 1.13 (H-27) correlations were observed with

quaternary carbon at δ 41.7 (C-8), 42.5 (C-14) and 140.1 (C-13) and also with the secondary

carbon at δ 27.4 (C-15). All these correlations were indicative of the methyl group (C-27) at

C-14. Both methyl signals at δ 1.08 (H-29) and 1.38 (H-30) showed correlations with carbon

signals at δ 83.1 (C-20) and 40.3 (C-19). In addition, correlations were observed between the

H-29 and carbon signal at δ 131.4 (C-18) and between H-30 and carbon signal at δ 32.8 (C-

21). These two correlations were helpful in assignments of the C-29 and C-30 positions for

methyl groups on the nucleus. All these correlations confirmed the presence of seven methyl

groups at C-23, C-24, C-25, C-26, C-27, C-29 and C-30 along with double bond between C-

13 and C-18. Chemical shift values are presented in Table 1. A search on Scifinder database

showed only one ursane compound with C-20 hydroxyl group [18] and one with C(13,18)

double bond. However, no compound possessing both of these features has been retrieved on

the Scifinder.

Compound 5: 10 mg (0.0010% yield) was obtained as a white powder with [α]D20

-91.1 (c

0.34, CHCl3). The molecular formula was established as C30H48O5 on the basis of ESIHRMS

data at m/z: 511.3396 [M+Na]+ (calcd for m/z 511.3399 [M+Na]

+) and at 493.3293 [M+Na-

H2O]+. Comprehensive analysis of the

1H,

13C and DEPT-135 NMR spectroscopic data

revealed that there was a close agreement between the chemical shifts of both the compounds

except in the values of ring A. The differences were observed for H-1, H-2 and H-3 and the

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corresponding carbon signals. This suggested a similar planar structure of both the

compounds. Evaluation of the 2D spectra was helpful in assessment of the difference between

the two molecules. A similar pattern was observed for the COSY and HMBC correlations as

was found in compound 4. A difference was seen between coupling constants in 1H NMR and

the cross peaks in NOESY spectra of the two which confirmed the relative stereochemistry of

compound 4 and 5. In compound 4, a ddd due to protons resonating at δ 4.02 (H-2) displayed

coupling constant values of 11.8 Hz (H-1α/H-2), 4.3 Hz ( H-1β/H-2) and 3.2 Hz (H-2/H-3)

which indicated trans, cis and cis coupling of the protons respectively. The coupling constant

values for ddd at δ 3.71(H-2) were 11.9 Hz (H-1α/H-2), 9.08 Hz (H-2/H-3) and 4.4 Hz (H-

1β/H-2) suggesting the trans, trans and cis arrangements of these protons in compound 5.

Moreover the NOESY spectrum of compound 4 presented the cross-peaks between H-2 and

H-3 while the same were absent in case of compound 5. This leads to the assignment of cis

configuration of the two protons, H-2 and H-3 in compound 4 and trans arrangement in

compound 5. Furthermore, triterpenoid literature suggests that the chemical shifts associated

with the carbons C-2/C-3 are around 66/79 for α/α placement of two hydroxyl groups at these

positions, around 71/78 for β/β and around 68/83 for α/β arrangements [19-21]. The

aforementioned NOESY interpretations along with the coupling constant values and literature

comparison suggested that the compound 4 has α/α hydroxyl groups at C-2/C-3 while the

other stereoisomer 5 has α/β arrangement for them. Other key NOESY correlations were

observed between H-19/H-30 suggesting α positioning of the C-29 methyl group and β

placement of C-30 methyl group. Key NOESY correlations are presented in Fig 2.

Compounds 4 and 5 possess a rearranged ursane type carbon skeleton in which the double

bond existing between C-12/C-13 has shifted to C-13/C-18 positions.

A plausible biogenetic pathway is shown in Fig. 3. It suggests that these compounds might be

derived from germanicenyl carbocation by a sequence of hydride shifts to introduce double

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bond between C-13 and C-18. Once the nucleus is formed, the Cyt P450 monooxygenases

may act on the molecule to introduce hydroxyl and carboxyl functionalities [22, 23].

Compounds 1, 2, 3, 6, 7, 8 and 9 were characterized as Ursolic acid [24], Euscaphic acid [25],

Corosolic acid [26], Epicatechin, Catechin, p-hydroxy benzaldehyde and gallic acid by

analysis of spectral data and comparison with literature.

3.2 Antioxidant activity

Antioxidants exert their action by different mechanisms including, suppression of formation

of reactive oxygen species, scavenging the free radicals, chelation of metal ion responsible

for free radical generation and protecting the antioxidant enzymes in the body. The classical

antioxidant action is generally based upon hydrogen atom transfer and single electron transfer

by an antioxidant molecule. Secondly, the structural features like solubility, partition

coefficient, bond dissociation energy, ionization potential are a set of parameters that

determine the overall efficacy of an antioxidant. Because of the multiple reaction

mechanisms and different structural characteristics of the test compounds, no single assay can

be used to reflect the antioxidant potential of a molecule [27]. Various extracts prepared by

partitioning from the methanol extract of P. fulgens i.e. hexane extract (HE), ethyl acetate

extract (EAE), butanol extract (BE), aqueous extract (AE) and the triterpene acids were

evaluated in various antioxidant assays, DPPH·and ABTS

+· (Hydrogen atom transfer based),

Reducing power assay (single electron transfer based) and MTT assay. Antioxidant potential

of triterpene acids has been reported in the literature. Ursolic acid has been reported to

provide significant protection against UV- induced lipid peroxidation, oxidative stress and

DNA damage by preventing the oxidation of guanine bases and enhancing the levels of

antioxidant enzymes [28]. Corosolic acid on the other hand is known to reduce oxidative

stress by reducing the levels of thiobarbituric acid-reactive substances and 8-

hydroxydeoxyguanosine (oxidative stress bio-markers) [29]. Euscaphic acid is a potential

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anti-inflammatory molecule that inhibits lipopolysaccharide induced inflammatory responses

by reducing production of nitric oxide, prostaglandin E2, Cyclooxygenase-2, TNF-α and IL-

1β. Inhibition of these mediators is an important mechanism for avoiding the generation of

reactive oxygen species [30]. Owing to the involvement of different mechanisms in the

antioxidant activity of triterpene acids, the two new compounds were evaluated for their

antioxidant activity. The antioxidant activity of other phenolic compounds isolated in this

study is well documented in literature [31-33].

3.2.1 Radical scavenging activity

Lipid peroxidation involves free radical chain reaction generating peroxide radicals. The

radical scavengers react with and quench peroxide radicals to terminate the chain reactions.

DPPH·and ABTS

+· are among the most common spectrophotometric methods used to

determine antioxidant capacity. Antioxidants reduce the oxidative damage to the cellular

components caused by reactive oxygen species and hence preventing chronic diseases. The

ethyl acetate fraction obtained from the methanol extract was shown to exhibit significant

inhibition against free radicals. Comparatively less activity was observed in butanol fraction

while hexane and aqueous extracts were found to have minimal activity. Thus, the ethyl

acetate fraction was selected for isolation of molecules that could account for its antioxidant

activity.

Out of the five isolated triterpene acids, 1, 4 and 5 showed better inhibitory activity than 2

and 3 against free radicals in DPPH· and ABTS

+· assay. The results are depicted in Table 2.

Compound 1 was the most potent with IC50 of 5.3 ± 0.2 μM and 7.8 ± 0.4 μM in DPPH· and

ABTS+·

assay respectively. Compound 4 showed comparatively better antioxidant activity

than 5 (Figure 4). The IC50 for compound 4 was found to be 9.8 ± 0.5 μM and 19.9 ± 0.3 μM

in DPPH· assay and ABTS

+· assay while for compound 5, IC50 values were 11.6 ± 0.7 μM and

23.3 ± 0.3 μM respectively. The free radical scavenging activity of these compounds could be

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attributed to the presence of free hydroxyl and the carboxyl groups in these compounds.

Compounds 4 and 5 demonstrated comparatively better activity than 2. This difference can be

due to variability in the position of hydroxyl group which is present at C-20 in 4 and 5 and at

C-19 in compound 2. The free hydroxyl groups are capable of transferring hydrogen atom to

scavenge free radicals.

3.2.3. Reducing power assay

The reducing power of the compounds is due to their single electron donation capacity which

in turn is based on deprotonation and ionization potential of the reactive functional groups. In

the reducing power assay, there is reduction of the Fe3+

/ferricyanide complex to its ferrous

form in the presence of antioxidants. Tested compounds did not show activity at micromolar

concentration.

3.2.4 MTT antioxidant assay

The MTT assay is a colorimetric assay for measuring the activity of the mitochondrial

enzymes present in healthy cells. The assay involves monitoring the absorbance of purple

formazan formed as the enzymatic reduction product of MTT. The MTT antioxidant assay

utilizes the redox reaction between MTT and natural antioxidants to assess their antioxidant

potential. A dose response curve was plotted and is presented in Fig. 4A. Quercetin dihydrate

was used as a positive control. Ethyl acetate extract has showed comparable activity as

compared to the positive standard while the two compounds 4 and 5 showed good antioxidant

activity than compounds 2 and 3.

3.3 Clonogenic cell survival assay

A dose dependent reduction in the cloning efficiency was observed in ethyl acetate and

hexane treated MCF-7 cell lines (Fig. 4B). However, the degree of reduction in cloning

efficiencies was more with ethyl acetate than hexane fraction of the root extract. The

inhibition concentration (IC50) value deduced from the Sigmoidal fit graph and it was 48.38

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and 73.65 μg/ml for ethyl acetate and hexane fractions, respectively. Such reduction in

cloning efficiencies was not seen with compounds 4 and 5.

4. Conclusion

In present study, the ethyl acetate soluble fraction from the roots of P.fulgens showed good

inhibitory activity against the free radicals. Furthermore, a dose dependent reduction in the

cloning efficiency was also observed in ethyl acetate and hexane treated MCF-7 cell lines.

However, the degree of reduction in cloning efficiencies was more with ethyl acetate than

hexane fraction of the root-extract. Thus, the ethyl acetate fraction showing good results in

both the assays was selected for phytochemical investigations. From the ethyl acetate extract

nine compounds, including two new compounds were isolated. The two new compounds

exhibited good antioxidant activity but no reduction in the cloning efficiency was observed in

MCF-7 cell lines on treatment with these pure isolates. The plausible biogenetic pathway for

the two new compounds has been proposed. The antioxidant activity of the plant can

attributed to the presence of these compounds along with other phenolics and hence the plant

can be considered as a source of natural antioxidants for food and nutraceutical products.

5. Acknowledgements

This work was supported by DBT, Govt. of India (BT/59/NE/TBP/2010) to AC, UCB and

IPS; DBT-fellowship and DST-Inspire fellowship (IF 120044) to Alka Choudhary; DBT-

Fellowship to DT. Authors are thankful to Director, NIPER for support to this work.

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Figure captions

Fig. 1. Chemical structures of compounds 1-9

Fig. 2. Key COSY ( ), HMBC ( ) and NOESY ( ) correlations in compounds 4 and 5

Fig. 3. Plausible biosynthetic pathway of compound 5

Fig. 4. (A) Dose response curve for MTT assay (B) A dose dependent reduction in the cloning

efficiency observed in ethyl acetate and hexane treated MCF-7 cell lines

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Table 1:1H (400 MHz) and

13C NMR (100 MHz) data for 4 and 5 in CDCl3

4 5

δH ppm δC ppm δH ppm δC ppm

1 1.76, dd, J = 11.0 and 4.5 Hz

1.22 (overlapped with H-5, 22, 15)

42.3 2.1, dd, J = 12.1 and 4.4 Hz

0.9

46.8

2 4.02, ddd, J = 11.8, 4.3 and 3.2 Hz 66.5 3.71, ddd, J = 11.9, 9.6 and 4.4 Hz 69.0

3 3.43, d, J = 2.2 Hz 78.8 3.01, d, J = 9.5 Hz 83.9

4 - 38.3 - 39.2

5 1.22 (overlapped) 48.2 0.85, m 55.4

6 1.45 (overlapped with H-7, 16)

1.32 (m)

17.9 1.54 (overlapped with H-9, 11, 16)

1.38 (m)

18.3

7 1.45 (overlapped) 34.7 1.48 (overlapped with H-16) 34.9

8 - 41.7 - 41.5

9 1.58 (overlapped with H-11, 16) 50.7 1.54 (overlapped) 50.9

10 - 38.8 - 38.7

11 1.58 (overlapped) 21.4 1.54 (overlapped) 21.4

12 2.41, dt, J = 12.6, 2.2 and 2.0 Hz

2.0, m

27.8 2.40, dt, J = 12, 2.0 and 2.1 Hz

2.2, m

27.8

13 - 140.1 - 139.9

14 - 42.5 - 42.5

15 1.71 (overlapped with H-21)

1.22 (overlapped)

27.4 1.68 (overlapped with H-21)

1.24 (overlapped with H-22)

27.4

16 1.58 (overlapped)

1.45 (overlapped)

33.3 1.54 (overlapped)

1.48 (overlapped)

33.3

17 - 43.4 - 43.3

18 - 131.4 - 131.5

19 2.56, dq, J = 5.76 and 2.04 Hz 40.3 2.56, dq, 5.82 and 1.9 Hz 40.4

20 - 83.1 - 83.1

21 1.82

1.71 (overlapped)

32.8 1.68 (overlapped)

1.74, m

32.8

22 2.33, dt, J = 8.1, 4.0 and 3.6 Hz)

1.22 (overlapped)

25.4 2.33, dt, J = 9.4, 4.0 and 3.8 Hz)

1.24 (overlapped)

25.4

23 1.01, s 28.5 1.03, s 28.6

24 0.84, s 21.7 0.80, s 16.7

25 0.91, s 17.5 0.94, s 17.7

26 0.81, s 17.7 0.81, s 17.8

27 1.13, s 20.6 1.13, s 20.4

28 - 176.5 - 176.4

29 1.08, d, J = 6.8 Hz 16.5 1.08, d, J = 6.7 Hz 16.5

30 1.38, s 22.9 1.38, s 22.9

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Table 2: Antioxidant activity of P. fulgens extracts/ compounds/ standards

Sr. No. Test samples DPPH· ± SDa

IC50

ABTS+·

± SDa

IC50

1 Hexane extract 74.6 ± 0.2b 86.9 ± 0.3b

2 Ethyl acetate extract 5.8 ± 0.6b 12.5 ± 0.1b

3 Butanol extract 8.4 ± 0.4b 20.3 ± 0.9b

4 Aqueous extract 28.6 ± 0.1b 42.2 ± 0.7b

5 Ursolic acid 5.3 ± 0.2 7.8 ± 0.4

6 Euscaphic acid 21.0 ± 0.3 30.2 ± 0.6

7 Corosolic acid 32.8 ± 0.5 38.7 ± 0.2

8 Fulgic acid A 9.8 ± 0.5 19.9 ± 0.8

9 Fulgic acid B 11.6 ± 0.7 23.3 ± 0.3

10 Trolox 3.2 ± 0.3 5.9 ± 0.9

11 Ascorbic acid 3.6 ± 0.7 6.2 ± 0.2

a IC50 value (μM) (n = 3); b IC50 value (μg/mL) (n = 3), Values are expressed in mean ± SEM.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Graphical abstract