Antarvedisides A-B from manglicolous lichen dirinaria consimilis (stirton) and their pharmacological profile

Article abstract of DOI:10.14233/ajchem.2019.21734

The chemical examination of acetone extract of Dirinaria consimilis resulted in isolation of six depsides of which two novel metabolites namely antarvediside A (1) and antarvediside B (2) and four known metabolites i.e. sekikaic acid (3), atranorin (4), d

Full text of DOI:10.14233/ajchem.2019.21734

Asian Journal of Chemistry; Vol. 31, No. 4 (2019), 805-812
A
SIAN
J
OURNAL OF HEMISTRY
C
https://doi.org/10.14233/ajchem.2019.21734
Antarvedisides A-B from Manglicolous Lichen Dirinaria consimilis
(Stirton) and their Pharmacological Profile
1,
1,*,
2
VINAY BHARADWAJ TATIPAMULA , GIRIJA SASTRY VEDULA
and A.V.S. SASTRY
1Pharmaceutical Chemistry Department, AU College of Pharmaceutical Sciences, Andhra University, Visakhapatnam-530003, India
2Pharmacology Department, Maharajah’s College of Pharmacy, Phoolbaugh, Vizianagram-535002, India
*Corresponding author: Tel: +91 891 2844926; E-mail: vgirijasastry@yahoo.co.in
Received: 1 October 2018;
Accepted: 24 December 2018;
Published online: 27 February 2019;
AJC-19289
The chemical examination of acetone extract of Dirinaria consimilis resulted in isolation of six depsides of which two novel metabolites
namely antarvedisideA (1) and antarvediside B (2) and four known metabolites i.e. sekikaic acid (3), atranorin (4), divaricatic acid (5) and
2’-O-methyl divaricatic acid (6). From the pharmacological screening of the isolates (1-6), it was found that 1 and 2 exhibited better
inhibition of ABTS and superoxide free radicals than that of the standard and compound 4 showed significant inhibition of protein
denaturation with IC50 value of 390 mg/mL with respect to indomethacin with 110 mg/mL. From the SRB assay results, the better IC50
values was determined by compound 2 of 10.5, 11.50 and 12.50 µg/mL on HeLa, MCF-7 and FADU cancer cell lines, respectively. Thus,
the outcomes revealed that the D. consimilis is a new source to treat free radicals, inflammation and cancer.
Keywords: Antioxidant, Protein denaturation, Inflammation, Cancer, Sulforhodamine B assay.
syringate, δ-guanidinovaleric acid, orsellinic acid, ethyl ester,
3,4-dihydroxyphenylvaleric acid, usnic acid, ethyl everninate,
2-O-methylatranorin, carbamic acid, propionic acid, glycolic
acid, 1,3-hydroxy-3-methylglutaric acid, ribonic acid, gluconic
acid, β-amyrone and various sugar and amino acid moieties
[4].
Earlier, the methanolic extract of D. applanata exhibited
good antioxidant, antimicrobial, cytotoxic and larvicidal effects
with good content of phenols [1,2], while dichloromethane
and methanolic extracts of D. confluens showed only larvicidal
and antimicrobial activities [3]. In 2016, the pharmacological
studies on D. aspera showed activity against B16-F10
melanoma and UACC-62 cells and 3T3 normal cells [4]. In
2015, the biological studies of methanolic extract from the D.
consimilis showed significant inhibitory activities against
different bacterial and fungal stains [5]. In 2016, the in vitro
anticaries activity of ethanolic extract of D. consimilis showed
moderate activity against Streptococcus mutans [5]. Earlier,
our group has reported the anti-inflammatory and cytotoxicity
studies of different extracts of D. consimilis [6,7].
INTRODUCTION
Dirinaria consimilis (Stirton) D.D. Awasthi is a foliose
lichen belongs to the Dirinaria genus and the chemical tests
of thalli of D. consimilis depicted the existence of mainly
sekikaic acid and traces of 3β-acetoxyhopane-1β,22-diol,
4-O-demethylsekikaic acid, atranorin, chloroatranorin, homo-
sekikaic acid, whereas the thallus of other species of Dirinaria
(D. applanata) contains atranorin and divaricatic acid. In 2009,
a non-reducing polyketide synthase gene have been isolated
and characterized from D. applanata [1]. The LC-HRESIMS
analysis of methanolic extract of D. applanata noticed to have
ellagic acid 3,3’-di-O-methyl ether, 5,5’-dehydrodiferulic acid,
3,3’,4-tri-O-methylellagic acid, 5’-methoxydehydrodiconiferyl
alcohol, gaigrandin, terpecurcumin Q, ergosteryl acetate,
taxuspine C and dichrostachine F [2].
In 2013, studies related to polycylic aromatic hydrocarbon
analysis of Dirinaria picta revealed the presence of naphtha-
lene, acenaphthylene, acenaphthene, anthracene, phenanthrene,
fluoranthene, pyrene, benzo anthracene and chrysene [3]. The
metabolic profile using GC-MS, LC-QTOF and MS/MS
analysis of D. picta showed existence of 5-methyluridine, ethyl
Keeping in mind of the aforementioned the chemical and
biological profile of D. consimilis as well as Dirinaria genus,
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806 Tatipamula et al.
Asian J. Chem.
we have established complete chemical and pharmacological
profile of manglicolous lichen (lichen associated particularly
on mangrove plants [8] D. consimilis.
The structures of novel and known depsides from Dirinaria
consimilis are shown in Fig. 1.
Antarvediside A (1): C38H34O14; Yield: 8 mg (0.56 %);
Rf: 0.7 (Hexane:DCM, 9:1); m.p.: 191-192 °C; IR (KBr, νmax
,
cm-1): 576.98, 702.11, 824.89, 1023.35, 1083.37, 1204.44,
1273.79, 1356.55, 1395.78, 1456.68, 1519.46, 1646.32,
2928.97, 3426.57, 3747.67, 3858.20; UV (Ethanol) λmax (log
ε) 222 nm; CHNS analysis: anal. C-63.76, H-4.82 (%), calcd.
C, 63.86, H, 4.80 (%); ESI-MS [MH]+ m/z: 715.00.
Antarvediside B (2): C46H40O18; Yield: 19 mg (1.32 %);
Rf: 0.2 (Hexane:Ethyl acetate, 7:3); m.p.: 203-204 °C; UV
(Ethanol) λmax (log ε) 219 nm; CHNS analysis: anal. C-62.76,
H-4.54 (%), calcd. C, 62.73, H, 4.58 (%); ESI-MS [MH]+ m/z:
881.21.
EXPERIMENTAL
The specimens of manglicolous lichen Dirinaria consimilis
(Stirton) was collected on the bark of mangrove plant Excoecaria
agallocha from Vainateya Island, Godavari estuary, Andhra
Pradesh, India (16°44'48"N and 81°98'19"E with 0 m elevation)
in February, 2015 [8]. This species were authenticated by Dr.
D.K. Upreti, CSIR-National Botanical Research Institute (NBRI),
Lucknow and deposited at Lichen herbarium, CSIR-NBRI,
Lucknow, India with accession numbers 15-027173.
Sekikaic acid (3): C22H26O8; Yield: 69 mg (4.79 %); Rf:
0.6 (DCM:Ethyl acetate, 1:1); m.p.: 220-221 °C; UV (Methanol)
λmax (log ε) 219 nm; IR (KBr, νmax, cm-1): 596.02, 660.91,
727.63, 756.03, 798.43, 861.89, 894.62, 953.06, 1018.59,
1039.09, 1138.46, 1203.15, 1241.04, 1283.58, 1311.71,
1353.07, 1425.20, 1458.95, 1610.97, 1644.68, 1667.87,
Extraction and isolation: The lichen specimen were gently
collected from the bark of E. agallocha and shade dried. The
dried lichen material (50 g) was powdered, suspended in acetone
for 1 week and evaporated under reduced pressure and dried
over anhydrous sodium sulphate and concentrated to obtain
dry acetone extract of D. consimilis (DC-Ac, 1.44 g). The DC-
Ac was subjected to column chromatography by using silica
gel (#230-400) resulted in four bioactive fractions. Fraction I
(100 mg) was obtained as a pale pink colour solid and re-
chromatographed over silica gel (#230-400) using hexane and
dichloromethane, which yields compound 1 (8 mg) as white
solid and compound 2 (19 mg) as a pale pink colour substance,
respectively. Fraction II (90 mg) was obtained as a white solid,
which on purification by column chromatography (#230-400)
using DCM in ethyl acetate as eluent to obtain compound 3
(69 mg) as white solid. Fraction III (281 mg) was white solid
material is subjected to column chromatography (#230-400)
using DCM in ethyl acetate as eluent to obtain compound 4
(90 mg) as colourless needles and compound 5 (162 mg) as a
white solid, respectively. Fraction IV (20 mg) was white solid
material is subjected to column chromatography (#230-400)
using hexane in ethyl acetate as eluent to obtain compound 6
(11 mg) as colourless solid.
1
2588.20, 2874.03, 2956.55, 3440.69. H NMR (CDCl3, 400
MHz) δ 0.97-1.05 (m, 6H, 2CH3), 1.69-1.72 (m, 4H, 2CH2),
2.95-3.04 (m, 4H, 2CH2), 3.85 (s, 3H, 1OCH3), 3.91 (s, 3H,
1OCH3), 6.40-6.42 (dd, J = 4, 8 Hz, 2H, 2CHar), 6.66-6.67 (d,
J = 3.2 Hz, 1H, 1CHar), 6.77-6.78 (d, J = 3.8 Hz, 1H, 1CHar),
10.39 (s, 1H, 1OH), 11.40 (s, 2H, 2OH); 13C NMR (CDCl3,
400 MHz) δ 14.18, 14.23, 24.84, 25.28, 38.53, 39.18, 55.43,
99.03, 103.60, 108.55, 111.58,116.22, 148.11, 149.53, 154.92,
164.88, 165.33, 166.60, 169.40, 174.34; CHNS analysis: anal.
C-63.16, H-6.24 (%), calcd. C, 63.15, H, 6.26 (%); ESI-MS
[MH]+ m/z: 419.33.
Atranorin (4): C19H18O8;Yield: 90 mg (6.25 %); Rf: 0.4
(DCM:Ethyl acetate, 1:1); m.p.: 196-197 °C; UV (Methanol)
1
λmax (log ε) 210 nm; H NMR (CDCl3, 400 MHz) δ 2.12 (s,
3H, 1CH3), 2.57 (s, 3H, 1CH3), 2.71 (s, 3H, 1CH3), 4.01 (s,
3H, 1OCH3), 6.43 (s, 1H, CHar), 6.54 (s, 1H, CHar), 10.39 (s,
1H, 1CHO), 11.97 (s, 1H, 1OH), 12.52 (s, 1H, 1OH), 12.58
(s, 1H, 1OH); 13C NMR (CDCl3, 400 MHz) δ 9.37, 24.02,
25.58, 52.34, 102.85, 108.56, 110.27, 112.87, 116.02, 116.08,
139.88, 152.00, 152.44, 162.89, 167.50, 169.10, 169.71,
O
37
O
O
28
33
24
46
35
32
31
35
29
O
O
28
OH
O
O
43
23
22
25
26
34
26
34 17
16
25
18
29
21
O
O
O
27
41
30
O
45
27
38
19
24
O
18
44
42
21
O
14
20
O 22
15
19
OH
12
O
14
O
39
23
20
31
12
17
36
40
OH
2
O
7
13
O
15
O
O
2
O
7
13
O
11
11
10
16
36
30
38
8
8
O
O
O
10
O
O
3
3
1
1
9
9
6
33
HO
4
37
HO
4
6
32
5
5
Antarvediside A (1)
(2)
Antarvediside B
10'
5
O
8
4
6
OH
O
9
7
O
O
18
OH
OH
14
O
18
9
9
10
16 12
13
14
8
8
18
10
O
3
OH
O
17 O
13
13
1
11
O
O
OH
OH
13
O
18
7
7
15
2
15
16
11
O
7
2
6
6
3
19
OH
14
9
8
12
O 10
9'
8'
9'
O
16
8'
1
15
5
11 O 12
1
5
11 O 12
OH
1
17
7'
14
15
17
7'
4
10
O
6
HO
4
10'
17
8'
4
2 OH
5
O
2
OH
O
9'
16
3
3
7'
Sekikaic acid (3)
Atranorin (4)
Divaricatic acid (5)
2'-O-Methyldivaricatic acid (6)
Fig. 1. Structures of novel and known depsides from Dirinaria consimilis

Vol. 31, No. 4 (2019)
Antarvedisides A-B from Manglicolous Lichen Dirinaria consimilis (Stirton) 807
172.21, 193.36; CHNS analysis: anal. C-60.76, H-4.74 (%),
calcd. C, 60.96, H, 4.85 (%); ESI-MS [MH]+ m/z: 375.11.
Divaricatic acid (5): C21H24O7;Yield: 162 mg (11.25 %);
Rf: 0.2 (DCM:Ethyl acetate, 1:1); m.p.: 138-139 °C; UV (Ethanol)
λmax (log ε) 213 nm; IR (KBr, νmax, cm-1): 590.98 659.01,
727.03, 756.05, 798.33, 861.73, 893.24, 952.35, 1018.91,
1039.69, 1077.04, 1137.74, 1202.80, 1239.87, 1283.83,
1311.80, 1352.84, 1425.59, 1458.56, 1609.55, 1645.87,
750 nm using UV-visible spectrophotometer and the experi-
ment was triplicated and the data was expressed as percentage
inhibition.
Superoxide radical scavenging assay: In this radical method
[9], the superoxide radicals generated from non-enzymatic
phenazine methosulfate/nicotinamide adenine dinucleotide
(PMS/NADH) reduces nitro blue tetrazolium (NBT) to a purple
formazan. To 1 mL of reaction mixture contained 20 mM phos-
phate buffer (pH 7.4), 73 µM NADH, 50 µM NBT, 15 µM PMS
added various concentrations of extract (50, 100, 150 and 200
µg/mL) and pure compound/standard (25, 50, 75 and 100 µg/mL)
and incubated for 10 min at room temperature and the absor-
bance was noted at 562 nm against blank using UV-visible
spectrophotometer and the experiment was triplicated and the
data was data was expressed as percentage inhibition.
1
1669.94, 2591.35, 2874.04, 2956.45, 3424.84; H NMR
(CDCl3, 400 MHz) δ 0.97-1.05 (m, 6H, 2CH3), 1.70-1.72 (m,
4H, 2CH2), 2.96-3.04 (m, 4H, 2CH2), 3.85 (s, 3H, 1OCH3),
6.40-6.42 (s, 2H, 2CHar), 6.67 (s, 1H, CHar), 6.78 (s, 1H, CHar),
11.36 (s, 2H, 2OH); 13C NMR (CDCl3, 400 MHz) δ 14.19,
14.23, 24.83, 25.28, 38.54, 39.18, 55.43, 99.03, 103.58,
108.50, 108.99, 111.59, 116.27, 148.11, 149.62, 154.98,
164.89, 165.35, 166.61, 169.39, 174.67; CHNS analysis: anal.
C-64.76, H-6.34 (%), calcd. C, 64.94, H, 6.23 (%); ESI-MS
[MH]+ m/z: 387.00
Ac − As
Ac
Percentage inhibition (%) =
×100
whereAc is the absorbance of the control, As is the absorbance
of sample.
A graph is plotted between concentrations of the sample
and their percentage inhibition to determine the IC50 values of
particular sample.
2’-O-methyldivaricatic acid (6): C22H26O7;Yield: 11 mg
(0.76 %); Rf: 0.4 (hexane:ethyl acetate, 1:1); m.p.: 142-143 °C;
1
UV (Ethanol) λmax (log ε) 212.5 nm; H NMR (CDCl3, 400
MHz) δ 0.93-1.05 (m, 6H, 2CH3), 1.66-1.80 (m, 4H, 2CH2),
2.95-3.04 (m, 4H, 2CH2), 3.85 (s, 3H, 1OCH3), 3.91 (s, 3H,
1OCH3), 6.40 (s, 2H, 2CHar), 6.46 (s, 1H, CHar), 11.18 (s, 1H,
1OH), 11.73 (s, 1H, 1OH); 13C NMR (CDCl3, 400 MHz) δ
14.35, 14.36, 24.84, 25.15, 38.87, 39.10, 55.37, 56.05, 98.89,
104.45, 104.45, 104.65, 106.48, 110.97, 124.94, 147.31, 148.73,
156.40, 156.94, 164.42, 165.55, 168.91, 175.47; CHNS analysis:
anal. C-65.66, H-6.62 (%), calcd. C, 65.66, H, 6.51 (%); ESI-
MS [MH]+ m/z: 403.05.
in vitroAnti-inflammatory activity: Protein denaturation
method [6] was employed for the evaluation of in vitro anti-
inflammatory activity for all compounds and extracts in three
sets and the mean SD values are reported. Bovine serum
albumin (BSA) protein was used in this study. The protein
was solubilized to 1 % concentration using sodium phosphate
buffer (50 mM, pH 6.4). To 0.2 mL of prepared protein, 0.1
mL of sample dissolved in DMSO (Sample size - 0.1, 0.2, 0.4,
0.6, 0.8 and 1 mg/mL for sample) was added and final volume
adjusted to 5 mL with buffer. Then the sample tubes are
incubated at 37 °C for 20 min. Thereafter, the tubes are heated
in steam bath at 95 °C for 20 min and then cooled to room tempe-
rature. Finally, the turbidity in the cooled tubes are measured
at 660 nm by UV-visible spectrophotometer (Model SL 210,
Elico India Ltd.). The percentage inhibition of serum albumin
protein denaturation was determined as follows and IC50 values
were calculated by plotting concentration vs. percentage inhibition.
General procedure for the basic hydrolysis of compounds
1 and 2: The isolate 1 or 2 (2 mg) was dissolved in methanol
and 2-3 drops of distilled water and 5 N potassium hydroxide
were added to the solution. The reaction mixture was heated
to reflux for 2-3 h. The thin layer chromatography confirmed
the completion of the reaction and the reaction mass was subjected
to gas chromatography-mass spectrometry (GC-MS) analysis.
Antioxidant activity
DPPH assay: The 1,1-diphenyl-2-picrylhydrazyl (DPPH)
assay [9] is based on the reduction of DPPH• radical in presence
of natural/synthetic antioxidant turns to non-radical DPPH
(yellow coloured). Initially, 0.004 % DPPH in methanol was
prepared and reacted with known concentrations of the extract
(50, 100, 150 and 200 µg/mL) and pure compound/standard
(25, 50, 75 and 100 µg/mL), incubated for 30 min at 37 °C.
Then the absorbance was noted at 517 nm on UV-visible spec-
trometer and the experiment was triplicated and the obtained
optical density values were converted into percentage inhibition
using the percentage inhibition formula.
ABTS radical scavenging assay: All the prepared extracts
was subjected to 2,2’-azino-bis(3-ethylbenzothiazoline-6-
sulphonic acid (ABTS) free radical scavenging assay [10]. Firstly,
the stock solution was prepared by adding 7 mM ofABTS+• to
2.45 mM potassium persulfate in water at 25 °C and stan-
dardized for 16 h. Then to each 1 mL ofABTS+• solution added
different concentrations of extract (50, 100, 150 and 200 µg/
mL) and pure compound/standard (25, 50, 75 and 100 µg/
mL), incubated for 6 min and the absorbance was measured at
C −S
C
Percentage inhibition (%) =
×100
where C is absorbance of control, S is absorbance of sample.
A graph is plotted between concentrations of the sample
and their percentage inhibition to determine the IC50 values of
particular sample.
Anticancer activity
Cancer cell lines: MCF-7 (Breast), DLD-1 (colon), HeLa
(cervical), FaDu (head & neck), A549 (lung) and normal
human mammary epithelial (NHME) cell lines were kindly
provided by National Centre for Cell Science, Pune. The cancer
cells were maintained in MEM media (containing 10 % fetal
calf serum, 5 % mixture of penicillin (100 units) and strepto-
mycin (100 µg/mL) in presence of 5 % carbon dioxide incu-
bator having 90 % humidity at 37 °C for 72 h.
Cell growth medium: All the cancer cell lines were main-
tained in minimal essential medium (MEM) (adjusted to 10
% (v/v) FBS, 1.5 g/mL NaHCO3, 0.1 mM MEM non-essential

808 Tatipamula et al.
Asian J. Chem.
amino acids and 1 mM sodium pyruvate). Three days prior to
performing assay, the cells were washed with sterilized PBS
and grown using MEM media (supplemented with 0.25 %
trypsin in versene-EDTA and 10 % FBS) and mixed to obtain
homogeneous suspension of cells. The suspension was taken
in a sterilized polypropylene tube and the cell concentration
in each well was determined by hematocytochameter chamber
under a microscope using 0.4 % trypan blue solution. The
minimal seed density must be 1 × 104 cells per well.
cm-1). The 1H and 13C NMR (Table-1) of 1 indicated the presence
of 5 methyl group protons at δH 2.09 (s, 3H), 2.17 (s, 3H), 2.55
(s, 3H), 2.62 (s, 3H) and 2.69 (s, 3H), with carbon signals at
δC 9.39 (C-34), 23.46 (C-35), 24.05 (C-33), 25.60 (C-32) and
29.73 (C-38), respectively. The 2 phenolic group protons at
δH 12.50 (s, 1H) and 12.55 (s, 1H), with carbons at δC 156.95
(C-12) and 169.71 (C-4), respectively and 2 aldehyde group
protons at δH 10.36 (s, 1H) and 11.95 (s, 1H), with carbon signals
1
at δC 182.41 (C-37) and 193.86 (C-30), respectively. The H
Sample preparation: Initially, for all the extracts (at 100
µg/mL), compounds (at 30 µg/mL) and standard (doxorubicin
at 10 µg/mL) were dissolved in DMSO and screened against
selected cancer cell lines. The active samples i.e. more than
50 % inhibition were further screened at 25, 50, 75 and 100
µg/mL concentrations for extracts; 5, 10, 20 and 30 µg/mL
concentrations for pure compounds; 2.5, 5.0, 7.5 and 10 µg/mL
concentrations for doxorubicin, hence their IC50 values were
determined by plotting percentage of inhibition vs. concen-
tration. The DMSO were used as a control.
NMR spectrum also revealed the presence of aromatic protons
at δH 6.41 (s, 2H), 6.52 (s, 2H) and 6.70-6.72 (dd, 2H), with
carbon signals at δC 104.13 (C-5), 96.78 (C-13), 152.00 (C-
10), 92.08 (C-20), 112.87 (C-23) and 139.89 (C-26). The 1H
NMR spectrum also depicted the existence of 3 methoxy at δH
3.90 (s, 3H), 3.93 (s, 3H) and 4.02 (s, 3H) with carbon signals
at δC 52.36 (C-29), 55.72 (C-31) and 55.67 (C-36), respectively,
including 6 carbonyl signals were observed at δC 165.82 (C-
21), 167.50 (C-7), 169.10 (C-14), 172.22 (C-28), 182.41 (C-
37), 193.86 (C-30).
Sulforhodamine B (SRB) colourimetric assay: The SRB
assay [7] is based on the estimation of cellular protein content.
The samples were taken in 96-well tissue-culture plate and
added 190 µL screened ideal cell suspension and mixed occa-
sionally and incubate at 37 °C with 5 % CO2 and 90 % relative
humidity for 3 h. Then add 100 µL cold trichloro acetic acid to
each well and incubate at 4 °C for 1 h. After that the plates were
gently washed using water, dried using blow dryer and air-dried
at room temperature. To each completely dried well, add 100 µL
of 0.057 % SRB solution, kept aside for 30 min and quickly
rinse with 1 % acetic acid. To the dried plate add 200 µL of 10
mM Tris base (pH 10.5) solution, shake for 5 min and measure
the OD at 510 nm. The blank contains only medium while the
control has only cancer cells with no test samples. The percentage
of growth inhibition was calculated using below formula.
The HMBC correlations (Fig. 2) revealed the relation
between methyl protons at 32-H depicted correlation with C-1
& C-6, hence the placement of methyl group was confirmed
at C-6 position as there is no possibility at C-1. Similarly, the
methyl protons at 38-Halso showed cross peaks with C-27 &
C-26, which indicates the most possibility of methyl group at
C-27 because of depisde correlation observed in C-6. More-
over, the methyl protons at 34-H showed strong cross peaks
with C-16 & C-15 which justifies the presence of methyl group
at C-16 position due to lack of possibility at C-15 and the 33-
H showed cross peaks with C-9, which proves the presence of
methyl group at C-9. In addition, the methoxy protons at 29-
H depicted correlation with C-28, which indicates the presence
of benzoate at C-25. Hence, the HMBC correlations were
consistent with a rigid structure for 1. Furthermore, the
complete confirmation is attained by GC-MS analysis of basic
hydrolyzed 1.
The basic hydrolysis of 1 (Scheme-I) was analyzed by
GC-MS showed four peaks at retention time 10.981, 16.271,
24.024 and 35.166 min, which were corresponding to m/z values
of 194, 196, 210 and 168 respectively. Hence, from the GC-MS
analysis the four components were confirmed as 1a (C10H10O5,
3-formyl-4-hydroxy-2-methoxy-6-methylbenzoic acid) with
m/z 210.19 [M]+, 1b (C8H8O4, 2,4-dihydroxy-5-methylbenzoic
acid) with m/z 168.15 [M]+, 1c (C10H12O4, 4-hydroxy-6-methoxy-
2,3-dimethylbenzoic acid)with m/z 196.20 [M]+and 1d (C10H10O4,
Absorbance of sample
Absorbance of control


Growth inhibition (%) =100 −
×100




RESULTS AND DISCUSSION
The compound 1 was isolated as a white powder with
m.p. 191-192 °C and UV absorption at λmax 222 in ethanol.
The positive mode of FT-MS-ESI provided at m/z 715.00 [MH+],
corresponding to a molecular formula of C38H34O14 (calcd. for
C38H34O14 m/z 714.68). Its FT-IR showed for the presence of
phenol group (3426.57 cm-1) and carbonyl group (1646.32
O
O
37
O
33
46
24
35
28
O
32
31
O
28
OH
O
35
29
O
43
34
23
22
25
26
26
34 17
16
25
18
29
O
O
27
21
O
41
19
30
O
45
27
38
24
O
44
42
21
O
14
20
O 22
15
19
OH
12
O
14
O
39
18
23
20
12
17
31
36
40
OH
2
O
7
13
O
O
15
O
2
O
7
13
O
11
10
11
16
36
30
38
8
O
O
O
8
O
10
O
3
1
9
3
1
9
33
6
HO 4
37
HO
4
6
1
2
32
5
5
Fig. 2. HMBC correlations of compounds 1 and 2

Vol. 31, No. 4 (2019)
Carbon No.
Antarvedisides A-B from Manglicolous Lichen Dirinaria consimilis (Stirton) 809
TABLE-1
1H, 13C NMR SPECTRAL DATA AND HMBC CORRELATIONS OF 1 AND 2
Compound 1 NMR data (400 MHz, CDCl3)
Compound 2 NMR data (400 MHz, CDCl3)
1H
13C
HMBC
1H
13C
HMBC
1
2
3
4
5
6
7
8
9
–
–
–
102.85
163.74
98.47
169.71
104.13
152.45
167.50
163.77
110.28
152.00
108.56
156.95
96.78
169.10
162.89
116.80
143.47
110.53
159.42
92.08
165.82
163.77
112.87
112.96
115.46
139.89
116.03
172.22
52.36
193.86
55.72
25.60
24.05
9.39
C-6, C-32
–
96.77
167.49
98.47
C-5
–
–
–
C-1
–
–
–
–
–
C-39
–
C-39
C-38
C-41
–
–
C-41
-
C-41
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
12.50 (s, 1H)
–
12.55 (s, 1H)
6.41 (s, 1H)
12.55 (s, 1H)
6.40 (s, 1H)
165.82
102.12
152.45
169.09
156.95
92.07
159.42
112.86
130.02
104.12
163.77
156.95
92.07
159.42
112.86
130.02
104.12
163.77
156.95
92.07
159.42
112.86
130.02
104.12
162.88
143.47
152.45
115.45
115.80
115.28
116.03
167.49
193.86
29.71
–
–
–
–
C-1, C-32
–
–
–
–
–
C-33
–
–
–
–
–
6.67-6.69 (dd, 1H)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
6.52 (s, 1H)
–
–
–
–
12.50 (s, 1H)
6.41 (s, 1H)
6.30-6.34 (dd, 1H)
–
–
–
–
–
–
–
–
C-16, C-34
C-15, C-34
6.67-6.69 (dd, 1H)
–
–
–
–
–
–
–
–
–
–
–
6.52 (s, 1H)
6.30-6.34 (dd, 1H)
–
–
–
–
6.70-72 (t, 1H)
6.67-6.69 (dd, 1H)
–
–
–
–
–
–
6.70-72 (t, 1H)
C27, C-38
C-26, C38
C-29
C-28
–
–
6.30-6.34 (dd, 1H)
–
3.90 (s, 3H)
11.95 (s, 1H)
3.93 (s, 3H)
2.62 (s, 3H)
2.55 (s, 3H)
2.09 (s, 3H)
2.17 (s, 3H)
4.02 (s, 3H)
–
–
–
–
–
–
C-1, C-6
C-9
C-15, C-16
–
C-34
C-33, C-46
6.52 (s, 1H)
–
23.46
55.67
182.41
29.73
–
–
–
–
–
–
–
–
13.39 (s, 1H)
11.95 (s, 1H)
2.55 (s, 3H)
3.87 (s, 1H)
2.85 (s, 3H)
3.90 (s, 3H)
2.69 (s, 3H)
3.90 (s, 3H)
2.85 (s, 3H)
3.99 (s, 3H)
10.36 (s, 1H)
2.09 (s, 3H)
–
–
–
C-14
10.36 (s, 1H)
2.69 (s, 3H)
C-26, C-27
52.34
23.44
55.64
25.57
55.70
24.02
60.59
182.42
9.36
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
C-11, C-13
–
C-15, C-18, C-20
–
–
–
–
C-34
–
–
–
methyl 2-formyl-4-hydroxy-5-methylbenzoate) with m/z 194.19
[M]+ in NIST library. Based on the correlations from NMR,
Mass, HMBC and GC-MS spectral analysis, the linkage of the
components of 1 was established and the structure and absolute
configuration of 1was assigned as 4-((4-((5-formyl-4-(methoxy-
carbonyl)-2-methylphenoxy)carbonyl)-5-methoxy-2,3-
dimethylphenoxy)carbonyl)-5-hydroxy-2-methylphenyl-3-
formyl-4-hydroxy-2-methoxy-6-methylbenzoate.
The compound 2 was isolated as a white powder with
m.p. 203-204 °C and UV absorption at λmax 219 in ethanol. The
positive mode of FT-MS-ESI provided at m/z 881.21 [MH+]
(calc. for C46H40O18 m/z is 880.81), corresponding to a molecular
formula of C46H40O18.
The 1H and 13C NMR of 2 (Table-1) indicated the presence
of 5 methyl group protons at δH 2.09 (s, 3H), 2.55 (s, 3H),
2.69 (s, 3H) and 2.85 (s, 6H), with carbon signals at δC 9.36
(C-46), 29.71 (C-37), 25.57 (C-41), 23.44 (C-39) and 24.02
(C-43). The 2 phenolic protons at δH 12.50 (s, 1H) and 12.55
(s, 1H), with carbon signals at δC 167.49 (C-2) and 165.82 (C-
4), respectively and 2 aldehyde protons at δH 10.36 (s, 1H)
and 11.95 (s, 1H), with carbon signals at δC 182.42 (C-45) and
193.86 (C-36), respectively. The 8 aromatic protons at 6.30-
6.34 (dd, 3H), 6.40 (s, 1H), 6.52 (s, 1H) and 6.67-6.69 (dd, 3H).
The 4 methoxy group protons at 3.87 (s, 1H), 3.90 (s, 6H) and
3.99 (s, 3H), with carbon signals at δC 52.34 (C-38), 55.64 (C-
40), 55.70 (C-42) and 60.59 (C-44), including 7 carbonyl

810 Tatipamula et al.
Asian J. Chem.
O
O
O
O
O
OH
O
O
O
O
O
O
O
O
O
O
O
O
OH
O
O
O
O
OH
O
O
O
O
O
O
2
1
HO
HO
H2O
5N KOH
OH OH
5N KOH H2O
O
O
O
O
O
O
OH
O
O
OH
O
+
+
O
OH
O
+
OH
OH
O
OH
HO
O
O
+
HO
HO
+
HO
1d
1a
1b
1c
HO
HO
O
HO
O
Scheme-I: Basic hydrolysis of compound 1
2a
2b
Scheme-II Basic hydrolysis of compound 2
2c
signals were observed at δC 162.88 (C-28), 163.77 (C-14/21),
167.49 (C-35), 169.09 (C-7), 182.42 (C-45) and 193.86 (C-36).
The HMBC (Fig. 2) confirmed the interaction of aryl
protons i.e. 5-H with C-1 (one-two correlation), 13-H with C-
39, 20-H with C-41 and 33-H with C-34, which confirm the
presence of aromatic hydrogen at positions C-5, C-13, C-20
and C-33, respectively. The methoxy protons at 38-H showed
cross peaks with C-14 i.e. one-two-three correlation, hence
justifies the presence of methoxy group at C-38. Moreover,
the methyl protons at 39-H depicted strong cross peaks with
C-11 i.e. one-two correlation, which confirms the methyl group
at C-12. Furthermore, the methyl protons showed one-three
correlation at 41-H with C-15, C-18 and C-20, hence this
support the presence of methyl group at C-19. Likewise, strong
correlation from methyl protons at 46-H to C-34 was also
noticed, which confirms the presence of methyl group at C-34
(Table-1). In addition, the complete confirmation is attained
by GC-MS analysis of basic hydrolyzed 2.
In DPPH radical scavenging assay [9], the antioxidant
capable substances are able to reduce the stable purple coloured
DPPH radical to a yellow coloured non-radical DPPH-H form.
Generally, the DPPH radical quenching activity of antioxidants
are ascribed to their hydrogen donating capacities [9]. As
shown in Fig. 3, the IC50 value for 1, 2, 3, 4, 5 and 6 on DPPH
were calculated to be 30.5, 32.0, 68.0, 53.5, 43.0 and 39.0 µg/
mL, respectively, while standard drug (ascorbic acid) with 27.0
µg/mL.
100
DPPH
ABTS
80
60
40
20
0
Superoxide
Ascorbic
acid
4
6
1
2
5
3
The basic hydrolysis of 2 (Scheme-II) was analyzed by
GC-MS showed three peaks at retention time 6.141, 21.012
and 61.941 min which were corresponding to m/z values of
182, 210 and 196 respectively. Hence, from the GC-MS analysis
the three components were confirmed as 2a (C9H8O5, 3-formyl-
2,4-dihydroxy-6-methylbenzoic acid) with m/z 196.16 [M]+,
2b (C9H10O4, 4-hydroxy-2-methoxy-6-methylbenzoic acid)
with m/z 182.18 [M]+ and 2c (C10H10O5, 2-formyl-4-hydroxy-
3-methoxy-5-methylbenzoic acid) with m/z 210.19 [M]+ in
NIST library. Based on the correlations from NMR, Mass,
HMBC and GC-MS spectral analysis, the linkage of the compo-
nents of 2was established and the structure and absolute configu-
ration of 2 was assigned as 2-formyl-4-((4-((4-((4-((3-formyl-
2,4-dihydroxy-6-methylbenzoyl)oxy)-2-methoxy-6-methyl-
benzoyl)oxy)-2-methoxy-6-methylbenzoyl)oxy)-2-methoxy-
6-methylbenzoyl)oxy)-3-methoxy-5-methyl benzoic acid.
Antioxidant activity: Generally, natural antioxidants have
great forbearance to humans and are sans of considerable side
effects [7]. The inferior IC50 values indicates superior inhibition
of free radicals. From the antioxidant results it is confirmed that
the 1, 2 and 6 depicted to have promising antiradical scaven-
ging capacities. In all the antioxidant assays, the 1, 2 and 6
showed markedly higher radical scavenging activity than other
compounds.
Sample
Fig. 3. IC50 values of isolates (1-6) against DPPH, ABTS and Superoxide
free radicals
The principal of ABTS radical assay [10] is similar to
that of DPPH assay in which decoy of the radical cationABTS+•
takes place.Among all samples, the isolates 1, 2 and 6 showed
better IC50 of 30.0, 26.0 and 37.0 µg/mL, respectively, than
that of the standard (41.0 µg/mL). The IC50 values of 3, 4 and
5 on ABTS radical were noticed to be 72.0, 57.5 and 60.0 µg/
mL, respectively (Fig. 3).
The superoxide radical generally arise from metabolic
process/ROS, which interact with other substrates in presence
of enzyme/metal catalyzed processes to generated hydroxyl
radical, H2O2 and 1O2. These radicals persuade oxidative damage
in DNA, lipids and proteins. From the superoxide free radical
assay [9], it was noticed that 1 and 2 showed better IC50 than
that of the standard. The concentration of 1, 2, 3, 4, 5 and 6
needed for 50 % inhibition of superoxide radical were found
to be 30.0, 35.0, 81.0, 48.0, 83.5 and 41.5 µg/mL, respectively,
while ascorbic acid was 35.5 µg/mL (Fig. 3). Hence, the higher
free radical quenching capabilities of the isolated metabolites
may be due to the presence of oxygenated substituents like
phenolics and carbonyls in the molecules.

Vol. 31, No. 4 (2019)
Antarvedisides A-B from Manglicolous Lichen Dirinaria consimilis (Stirton) 811
in vitro Anti-inflammatory activity: The denaturation
of biological proteins causes inflammation. The denaturation
pathways can be performed by acidic (or) alkaline reactions,
heat treatment, radiation reactions, etc. Proteins lose their
complex tertiary structure because of the externally induced
stress under the above mentioned conditions thus leading to
denaturation. Therefore, in the present work the isolates from
D. consimilis were tested for inhibition of protein denaturation
induced by heat [6]. Among the tested D. consimilis comp-
ounds, the compound 4 showed better inhibitory profile against
protein denaturation with IC50 value of 390 µg/mL, which was
far better than all other isolates of D. consimilis. The IC50 values
of compounds 1, 2, 3, 5 and 6 on protein denaturation were
found to be 878, 600, 632, 922 and 700 µg/mL, respectively,
while indomethacin with 110.0 µg/mL (Fig. 4).
During the initial screening, the isolates 2, 3, 5 and 6
depicted prominent degree of specificity against all the tested
cancer cell lines. Besides, all the samples exhibited very low
degree of specificity against NHME cell lines indicates they
are non-toxic towards normal human cells (Table-2). The
preliminary screening results revealed that the compound 2
(30 µg/mL) showed better inhibition of cell growth of MCF-7
and HeLa with 83.70 2.40 and 90.90 6.30 % cell growth
inhibition, respectively, than the doxorubicin (10 µg/mL) with
81.25 1.56 and 85.55 1.31 % cell death, respectively (Table-
2). Whereas, the 3 and 5 showed moderate degree of specificity
against tested cancer cell lines. On the other hand, the 6 showed
significant inhibitory profile against MCF-7, DLD-1, HeLa,
FaDu and A549 (Table-2).
Considering the initial screening, the active samples i.e.
compounds 2, 3, 5 and 6 were further evaluated of their IC50
values and illustrated in Fig. 5. The compound 2 depicted better
IC50 values of 11.50 µg/mL on MCF-7 with respect to doxoru-
bicin (5.50 µg/mL), whereas compounds 3 and 6 showed IC50
value of about 26.50 and 22.50 µg/mL, respectively (Fig. 5).
Compounds 2, 3, 5 and 6 revealed significant IC50 values of
22.0, 15.50, 15.0 and 17.70 µg/mL, respectively on DLD-1,
while doxorubicin with 5.4 µg/mL. Based on the SRB assay
results of HeLa, it is observed that the IC50 values of tested
compounds on HeLa were found to be in the order doxorubicin
(4.5 µg/mL) < 2 (10.5 µg/mL) < 3 (16.5 µg/mL) < 5 (17.5 µg/
mL) < 6 (18.5 µg/mL) (Fig. 5). Similarly, it is noticed that the
concentration of 2, 3, 5 and 6 needed for 50 % inhibition of
FaDu were found to be 12.5, 14.5 19.0 and 21.0 µg/mL, respec-
tively, whereas doxorubicin with 3.8 µg/mL (Fig. 5). From
the obtained data, it can be confirmed that the only compounds
1000
800
600
400
200
0
1
2
3
4
5
6
Indome-
thacin
Sample
Fig. 4. IC50 values of isolates (1-6) against protein denaturation
Anticancer activity: As chronic inflammation is crucial
in many deadly diseases like cancer. In addition, as the isolated
metabolites showed good anti-inflammatory potentialities, we
have screened the isolated secondary metabolites (2-6) for their
anticancer activity by using Sulforhodamine B (SRB) assay
[7] and calculated their IC50 values. Initially, the compounds
(2-6) at 30 µg/mL concentration were screened against breast
(MCF-7), colon (DLD-1), cervical (HeLa), head & neck (FaDu),
lung (A549) and normal human mammary epithelial (NHME)
cell lines using SRB assay and standard (doxorubicin, 10 µg/
mL) and the percentage of inhibition of cell growth was tabu-
lated in Table-2. Further, the active samples i.e. more than 50
% cell death were further screened at 5, 10, 20 and 30 µg/mL
concentrations for pure compounds; 2.5, 5.0, 7.5 and 10 µg/
mL concentrations for doxorubicin. The results are plotted to
obtain IC50 values. The lower IC50 value indicates better
inhibitory profile against cancer cell lines.
30
MCF-7
DLD-1
HeLa
FaDu
20
10
0
A549
5
2
3
6
Doxorubicin
Sample
Fig. 5. IC50 of isolates (1-6) against test panel of cell lines
TABLE-2
PERCENTAGE OF INHIBITION AND IC50 VALUES OF ISOLATES (2-6)
AGAINST SERIES OF CANCER CELL LINES AND NHME CELL LINE
Percentage inhibition (%) at 30 µg/mL concentration*
Sample
MCF-7
DLD-1
HeLa
FaDu
A549
NHME
83.70 2.40
56.21 1.09
10.91 1.24
44.95 3.42
64.69 5.26
81.25 1.56
67.60 7.90
77.19 0.57
7.80 1.15
72.23 3.20
77.21 1.06
72.67 0.21
90.90 6.30
75.89 1.77
2.55 0.79
70.17 1.19
73.97 3.44
85.55 1.31
89.70 6.80
78.89 5.82
7.01 0.93
74.83 4.01
71.04 2.38
98.50 1.21
66.50 3.20
40.75 2.20
8.68 1.53
30.78 3.15
55.82 3.20
77.92 0.41
8.07 0.60
7.19 0.45
1.82 0.46
4.51 0.73
6.66 1.33
1.20 0.81
2
3
4
5
6
Doxorubicin**
*n = 3; Mean SD; **10 µg/mL

812 Tatipamula et al.
Asian J. Chem.
2 and 6 are active against A549 with IC50 values of 22.5 and
27.5 µg/mL, respectively, while doxorubicin with 6.3 µg/mL
(Fig. 5).
Hence, form the SRB analysis it can be concluded that
the key agents responsible for the anticancer activity of D.
consimilis were 2, 3, 5 and 6. Furthermore, the low toxicity
against NHME indicates that all the compounds are less toxic
towards normal human cells. Hence, these active metabolites
can be a benchmarks for designing potent anticancer agents.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interests
regarding the publication of this article.
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The authors are thank full to the Ministry of Earth Sciences
(Grant number: MOES-2/DS/6/2017), India for the financial
support.