Phytochemical investigation and characterization of isolated chemical constituents from Kyllinga triceps Rottb.

Article abstract of DOI:10.14233/ajchem.2017.20574

Four compounds have been isolated by column chromatography from Kyllinga triceps namely quercetin dihydrate (1), rutin (2), β-sitosterol (3) and stigmasterol (4). Their structures have been elucidated by, FTIR, HR-EIMS, ¹H NMR and ¹³C NMR spectroscopic studies.

Full text of DOI:10.14233/ajchem.2017.20574

Asian Journal of Chemistry; Vol. 29, No. 6 (2017), 1393-1400
ASIAN JOURNAL OF CHEMISTRY
https://doi.org/10.14233/ajchem.2017.20574
Phytochemical Investigation and Characterization of Isolated
Chemical Constituents from Kyllinga triceps Rottb.
1,*
1
2
3
4
NISHANT VERMA , K.K. JHA , SHAMIM AHMAD , SUDHIR CHAUDHARY and MOHAMMAD ALI
1T.M. College of Pharmacy, Teerthanker Mahaveer University, Moradabad-244 001, India
2Translam Institute of Pharmaceutical Education & Research, Meerut-250 001, India
3B.B.S. Institute of Pharmaceutical & Allies Sciences, Knowledge Park-III, Greater Noida-201 310, India
4Faculty of Pharmacy, Jamia Hamdard University, New Delhi-110 062, India
*Corresponding author: E-mail: nishantvermamiet@gmail.com
Received: 6 February 2017;
Accepted: 1 March 2017;
Published online: 10 April 2017;
AJC-18360
Four compounds have been isolated by column chromatography from Kyllinga triceps namely quercetin dihydrate (1), rutin (2), β-
1
sitosterol (3) and stigmasterol (4). Their structures have been elucidated by, FTIR, HR-EIMS, H NMR and 13C NMR spectroscopic
studies.
Keywords: Column chromatography.
In present we wish to report some of the phyto-constituents
from whole plant of Kyllinga triceps which could be regarded
as the ever identified compound.
INTRODUCTION
Plants make a significant contribution to health care due
to their recognition in traditional medicinal systems [1]. Herbal
medicine are supposed to be safer than synthetic counterparts
because the phytochemicals, in the plant extract, target the
biochemical pathway [2].Kyllinga triceps is a herb belonging
to family Cyperaceae. It is a small genus of annual or perennial
herbs, distributed in the tropical and sub tropical region of the
world. About eight species occur in india. Kyllinga triceps
Rottb. Is a small tufted herb, up to 12 inches high with a short
rhizome and linear leaves, one half or nearly as long as the
stem, found in Kumaon at altitudes of 5000-6000 ft. and from
the upper gangetic plain to West Bengal, Sundarbans and
Deccan Peninsula. The plant has the same vernacular name as
Kyllinga monocephala [3].
EXPERIMENTAL
Fresh plant of Kyllinga triceps was collected in the month
of April, 2011 from Venkateswara University, Tirupati,
India. The plant was identified and authenticated by Dr. K.
Madhava Chetty, Asst. Professor, Department of Botany, Sri
Venkateswara University, Tirupati, Andhra Pradesh, India.
A voucher specimen has been deposited at the College of
Pharmacy, TMU Moradabad (TMU/Consult/2011-12/206) for
future reference.
Preparation of the extract and isolation: The air-dried
whole plant of Kyllinga triceps (1 kg) was defatted with n-hexane
and petroleum ether followed by extraction with chloroform,
ethyl acetate and n-butanol using Soxhlet extractor [7]. The
resulting extracts were tested for the presence of flavonoids
by Shinoda test [8]. Ethyl acetate fraction was found to have
the highest amount of flavonoids [9]. Systematic fractionation
of ethyl acetate fraction resulted in the isolation of two consti-
tuents designated as compound 1, compound 2 and petroleum
ether extract was obtained as dark green oily mass [10]. The
defatted material was extracted further with solvents of increa-
sing polarity. Systematic fractionation of petroleum ether
extract resulted in isolation of two constituents designated as
compounds 3 and 4 [11].
Traditionally the decoction of roots is used as refrigerant,
demulcent and tonic. It is given to relieve thirst in fevers and
in diabetes. It is also used as antidote to poisons. Oil distilled
from the root is used to relieve pruritus of the skin, internally,
oil is given in torpor of the liver [4]. Synonym of Kyllinga
triceps is Kyllinga tenuifolia and Cyperus triceps (Rottb.) [5].
Fresh juice of the plant is used externally to wash the wounds.
It is used in the treatment of indigestion. The oil of roots is
used to promote the action of liver and relieve pruritus [6].
Decoction of roots is used in diabetes and to relieve thirst in
fever. The literature reveals that no isolation work by column
chromatography has been done on Kyllinga triceps.

1394 Verma et al.
Asian J. Chem.
Structural determination of compound 1: Compound
(1H, d, J = 1.8, C8-H), 7.55 (1H, d, J = 2.1, C2’-H), 6.75-
6.78 (1H, d, J = 8.4, C5’-H), 7.40-7.44 (1H, dd, J = 8.7, 2.1,
C6’-H), 9.2 (1H, s, C-4’-OH), 9.1 (1H, s, C3’-OH), 9.4 (1H,
s, C3-OH), 12.36 (1H, s, C-5, OH), .10.69 (1H, s, C7-OH).
13C NMR (Chemical shift in ppm) represents 15 carbons.
The 13C NMR of compound 1 represents 10 quarternary atoms
and 5-CH carbons. The multiplicity and chemical shift of diffe-
rent carbons of compound 1 matches with the existing literature
for quercetin dihydrate is shown in Table-3.
1 was isolated from ethylacetate extract of Kyllinga triceps. It
was crystallized in methanol as a yellow crystalline powder
having melting point 313-316 °C. It is slightly soluble in water
and diethyl ether and soluble in methanol, ethanol and acetone
[12]. Chromatography was performed on precoated silica gel
G plates and spots were detected by spraying it with FeCl3
(2 %) in ethanol. The Rf value recorded in two solvent systems
is given below.
Solvent system
Chloroform:ethyl acetate:formic acid (5:4:1)
n-Butanol:acetic acid:water (4:1:5 upper layer) 0.82
Rf value
0.76
TABLE-3
13C NMR SPECTRA VALUES OF COMPOUND 1
Position Signal Multiplicity Position Signal Multiplicity
The spots appeared blue on spraying it with FeCl3 and in
iodine chamber the spots appeared yellowish brown giving
a clue that compound is containing phenolic group. The
compound gave a positive Shinoda test thus strengthening the
assumption that it is a flavonoid compound.
2
3
4
5
6
7
8
9
146.0
135.5
175.0
156.0
92.8
-C-
-C-
10
1’
2’
3’
4’
5’
6’
–
103.2
122.2
115.3
144.9
147.4
114.8
120.5
–
-C-
-C-
-C-
-CH-
-C-
-C-
-CH-
-C-
-C-
164.8
93.5
-CH-
-CH-
–
The UV spectrum of the compound 1 showed two major
absorption band at 374.8 and 257.5 which gives an idea of
its flavonol structure. The bathochromic shift in the UV was
observed with sodium acetate at 391.6 and 258.4 (were related
of 7 OH group of the flavonoid quercetin) and with aluminium
chloride was found to be 435.4 and 270.4 (related to 5 OH
group), hypsochromic shift was observed with boric acid at
342.6 and 236.8 nm, confirms the presence of 3,3’,4’OH group
depicted in Table-1.
-CH-
-C-
161.4
Mass spectrum of isolated compound 1 (molecular weight
302.2) recorded prominent peak at 301 (M-H+), which on
further fragmentation yields prominent fragments weighing
273, 178.8, 150 and 107. Which matches with the existing
literature for quercetin dihydrate and thus the compound is
quercetin dehydrate (Table-4).
TABLE-1
EFFECT OF SHIFT’S REAGENT ON
THE ABSORPTION MAXIMA OF COMPOUND 1
TABLE-4
MASS SPECTRUM DATA OF COMPOUND 1
m/z
Name of the fragment
M-H+ Base peak
(M-H+ -CO)
Shift in
band-I
Shift in
band-II
Shift’s reagent
Band-I
Band-II
301.1
273.1
178.8
150.0
107.1
Quercetin (Q)
Q + MeONa
Q + AlCl3
374.8
426.6
435.4
365.0
342.6
391.6
332.6
257.6
285.8
270.4
255.2
236.8
258.4
233.8
(301-C7H6O2)
51.8
60.6
-9.8
2.2
-5.4
-2.4
-20.8
0.8
(301- C7H4O4)
C7H6O2-CO-CO2
Q + HCl
Q + Boric acid
Q + AcONa
Q + NaOH
-32.0
16.8
-42.2
OH
-23.6
3'
OH
2'
1'
The IR spectrum of the isolated compound 1 (KBr), pre-
sented various bands mentioned in the Table-2.
4'
2H O
2
·
8
5'
HO
O
2
TABLE-2
IR SPECTRA OF COMPOUND 1
7
6
6'
Wavenumber
3
Functional groups and Bonds
(cm-1)
OH
4
3582
O-H str. free non bonded
5
3400, 3321
1650
O-H str. vibrational bond in phenol
C=O Aryl ketonic structure
OH
O
1611, 1522, 1462 C=C aromatic ring streatching (1650-1450)
1327 C-H in-plane bending of aromatic ring
1254, 1209, 1002 C-O str. of phenol, strong band (1260-1000 cm-1)
Structure of quercetin dihydrate
HPLC analysis of the isolated compound 1 was performed
1125
C-O-C str. of aryl ether (1085-1150)
by Shimadzu high performance liquid chromatographic
system, LC 2010CHT with UV & PDA detector in combination
with Class LC solution software. Kromasil C18, 5µ (250 × 4.6
mm) column was used [13]. Gradient elution was performed
using anhydrous sodium dihydrogen orthophosphate buffer
and acetonitrile [14]. Flow rate was maintained at 1.5 mL/min
839, 791, 721
1170
Out of plane C-H bending (900-675)
C-(C=O)-C str. and bend vibrations (1300-1100)
The 1H NMR (Chemical shift δ in ppm, coupling constant
J is in Hz) spectrum (300 MHz, DMSO-d6) recorded for Cc#1
depicted following δ values 6.07 (1H, d, J = 2.1, C6-H), 6.29

Vol. 29, No. 6 (2017)
Phytochemical of Isolated Chemical Constituents from Kyllinga triceps Rottb. 1395
TABLE-6
EFFECT OF SHIFT’S REAGENT ON THE
ABSORPTION MAXIMA OF COMPOUND 2
and injection volume was 20.0 µL and detection wavelength
was 370 nm [15]. The gradient flow was maintained as given
below in the Table-5.
Shift in
band-I
(nm)
Shift in
band-II
(nm)
Band-I
(nm)
Band-II
(nm)
Shift’s reagent
TABLE-5
GRADIENT ELUTION OF COMPOUND 1 WITH
POTASSIUM DIHYDROGEN ORTHOPHOSPHATE
BUFFER AND ACETONITIRILE
Rutin (R)
357.6
410.6
428.0
359.6
363.6
385.4
411.2
–
257.6
273.4
275.0
257.2
258.0
274.2
274.4
–
R + MeONa
R + AlCl3
53.0
70.4
2.0
15.8
17.4
-0.4
0.4
Time
0
Volume of buffer
Volume of acetonitrile
R + HCl
95
55
20
20
55
95
95
5
45
80
80
45
5
R + Boric acid
R + AcONa
R + NaOH
6.0
18
25
28
35
40
45
1.8
16.6
16.8
53.6
TABLE-7
IR SPECTRA OF COMPOUND 2 isolated from Kyllinga triceps
5
Wavenumber (cm-1)
3592
Functional groups and bonds
O-H str. (non-bonded, free) vibrational band
(3700-3584)
Retention time of compound 1 was observed at 16.568
min. with a peak area of 5546254 which occupies 99.065 %.
Table-5 and chromatogram proving it a pure entity which
matches with the retention time of quercetin.
3408, 3321
1663
O-H str. bonded (3550-3200)
C=O aryl ketonic structure (1685-1666),
frequency reduced due to conjugation and
intramolecular H bonding
Structural determination of compound 2: Compound
2 was isolated from ethyl acetate extract of Kyllinga triceps. It
was crystallized in methanol as a yellow powder, soluble in
water and methanol (under hot conditions) and insoluble in
chloroform, acetone and ether. The melting point was found
to be 242-245 °C. Chromatography was performed on pre-
coated silica gel 60 F254 plates and spots were detected by
spraying it with 1 % ethanolic aluminium chloride. Yellow
fluorescence was observed under UV 365 nm [16]. The Rf value
recorded in two solvent systems is given below.
1611, 1561, 1521
1321
C=C aromatic ring stretching (1650-1450)
C-H in-plane bending of aromatic ring
C-O str. of phenol, strong band (1260-1000
cm-1)
1199, 1013
1150
C-O-C str. of aryl ether (1085-1150)
Out of plane C-H bending (900-675)
C-(C=O)-C str. and bend vibrations (1300-
1100)
865, 823, 723, 685
1264
7.41-7.43 (1H, d, J = 1.8, C2’-H), 6.71-6.73 (1H, d, J = 8.4,
C5’-H), 7.41-7.44 (1H, dd, J = 8.8, 2.2, C6’-H), 9.56 (1H, s,
4’-OH), 9.06 (1H, s, 3’-OH)12.47 (1H, s, C-5, OH), 10.74
(1H, s, C-7 OH), 5.23 (1H, d, J = 3.9, H1-G), 5.12 (1H, d, J =
1.6, H1-R), 0.8 (3H, J = 5.8, CH3-R).
Solvent system
Methanol:water:acetic acid (50:50:6)
Ethyl acetate:water:formic acid (6:1:1)
Rf value
0.63
0.69
13C NMR (chemical shift in ppm) displayed the signals
for various carbons of the compound 2 (DMSO-d6) where there
are One CH3, one CH2, 14 CH and 11 quarternary carbons,
The assignment of chemical shift was facilitated on comparison
with 13C NMR data of rutin available in the literature. The
chemical shift of various carbons is given in Table-8.
The glycoside was first eluted with two dimensional TLC
with ethyl acetate:water:formic acid (6:1:1) in first direction.
The layer was dried at 100 °C for 15 min, the separation zone
was sprayed with 5 M sulphuric acid in water:methanol (1:1)
and the plate is kept at 110 °C for 40 min to hydrolyze the
glycoside. After cooling elution in second direction was done
with ethyl acetate:2-butanol:water (60:25:5). Plates were dried
at 80 °C for 15 min and then sprayed with saturated solution
of ethylenediammoniumsulphate in acetone:water (1:1). The
sugar under these conditions give rise to fluorescent blue spots
which may be observed under UV (365 nm) [17].
TABLE-8
13C NMR SPECTRAL DATA OF COMPOUND 2
ISOLATED FROM Kyllinga triceps
Chemical
shift (δ
scale)
Chemical
shift
Carbon
No.
Multi-
plicity
Carbon
No.
Multi-
plicity
The compound in methanol gave two significant absorp-
tion band at 357.6 nm (band-I) and 257.6 (band-II) which gives
an idea of flavonol structure. Significant bathochromic shift
in the absorption maxima is observed with sodium methoxide,
aluminium chloride and and sodium hydroxide hypsochromic
shift is observed only with HCl [18]. The shift in the absorption
maxima was observed on treating it with shift reagents which
is given in Table-6.
(δ scale)
157.06
133.70
177.80
156.80
99.10
C-2
C-3
-C-
-C-
121.60
101.60
74.50
76.90
72.30
76.30
67.40
101.60
70.80
71.03
70.48
68.69
18.18
–
C-6’
C1-G
C2-G
C3-G
C4-G
C5-G
C6-G
C1-R
C2-R
C3-R
C4-R
C5-R
C6-R
–
-CH-
-C-
C-4
-C-
-CH-
-CH-
-CH-
-CH-
-CH2-
-CH-
-CH-
-CH-
-CH-
-CH-
-CH3
–
C-5
-C-
C-6
-CH-
-C-
164.50
94.03
C-7
C-8
-CH-
-C-
161.60
104.40
122.04
115.60
145.20
148.70
116.70
C-9
The IR spectra of the isolated compound was performed
with KBr disc method and various bands observed are
mentioned in the Table-7.
1H NMR (chemical shift δ in ppm, coupling constant J
is in Hz) spectrum (300 MHz, DMSO-d6) recorded as 6.0-
6.08 (1H, d, J = 1.8, C6-H), 6.26-6.27 (1H, d, J = 2.1, C8-H),
C-10
C-1’
C-2’
C-3’
C-4’
C-5’
-C-
-C-
-CH-
-C-
-C-
-CH-

1396 Verma et al.
Asian J. Chem.
TABLE-10
The mass spectrum of the compound 2 on fragmentation
gave the following fragments shown in Table-9.
GRADIENT ELUTION OF RUTIN WITH
POTASSIUM DIHYDROGEN ORTHOPHOSPHATE
BUFFER AND ACETONITIRILE
TABLE-9
MASS FRAGMENTATION DATA OF COMPOUND 2
Time (min)
Volume of buffer
Volume of acetonitrile
0
5
80
75
20
25
30
30
20
20
m/z
Name of the fragment
M-H+ Base peak
609.15
596.00
463.00
325.00
300.00
8
70
M + H+ - (CH3)
10
12
15
15
70
M+ - (C6H10O4)
M+ - (C15H9O6)
M+ - (C12H21O9)glycone part)
80
80
Stop
M-H+ and M+H+ are the most abundant and characteristic
peaks in flavonols, compound 2 seems to be rutin as the mass
fragmentation pattern and presence of base peak at 609.15
(M-H+) on matching with the existing literature increases the
probability of the compound to be rutin.
petroleum ether to purify the remaining extract. This extract
is further subjected to column chromatography using gradient
elution with toluene and methanol, homogeneity was estab-
lished by TLC studies over precoated silica gel G plates and
the spots were developed by either exposing the plates to iodine
vapours or spraying them with 5 % ethanolic sulphuric acid
followed by heating the plates at 110 °C for 10 min. The Rf values
recorded in two different solvent systems are given below.
OH
O
HO
Solvent system
Benzene:chloroform (2:1)
Toluene:chloroform:methanol (1:1:0.1)
Rf value
0.64
0.57
OH
Compound 3 was found to be soluble in organic solvents
like petroleum ether, benzene, dichloromethane, chloroform
and methanol but insoluble in water. The melting point was
found to be 160-166 °C [19].
O
C
O
OH
O
The IR spectrum of the compound 3 gave the following
peaks mentioned in Table-11.
O
O
OH
OH
CH3
OH
OH
TABLE-11
IR SPECTRA OF COMPOUND 3
ISOALTED FROM Kyllinga triceps
OH
OH
Structure of rutin
Wavenumber (cm-1)
3337.9
Functional groups and bonds
O-H str. vibrational band of bonded –OH
CHunsymmetrical str., of steroidal ring and
tertiary CH group
The identity of the isolated compound 2, which may be
2962
rutin was further confirmed by HPLC using Shimadzu high
performance liquid chromatographic system LC 2010CHT
with UV detector. Kromasil C18 column was used. Gradient
elution was conducted using potassium dihydrogen ortho-
phosphate buffer (A) and acetonitrile (B). The buffer (A) was
prepared by taking 0.140 g of anhydrous potassium dihydrogen
orthophosphate (KH2PO4) dissolving it in 900 mL of HPLC
grade water and adding 0.5 mL of orthophosphoric acid and
making volume up to 1000 mL with water. The solution was
filtered through 0.45 µ membrane filter and degassed in a
sonicator for 3 min. HPLC grade acetonitrile was used. Flow
rate was kept at 1.6 mL/min and detection wavelength was
maintained at 258nm and injection volume was 20 µL. About
5 mg of the sample was weighed to a in a 25 mL volumetric
flask. About 10 mL of methanol was added and sonicated
cooled and made up to 25 mL with methanol. The gradient
flow was maintained as given in Table-10.
2872
1453
C-H symmetrical str.
-CH2 cyclic bending vibration
OH in plane bending vibration
C-H in plane bending of cycloalkenes
C-H out of the plane bending vibration
1374
1054, 965
836
It is observed that with acetic anhydride and triethylamine
at room temperature isolated compound afforded an acetate
derivative, m.p., 143-145 °C. Thus compound 3 appeared to
be stigmasterol and this assumption was confirmed by recor-
ding and analyzing 1H NMR, 13C NMR and mass spectrum of
compound 3 [19].
The 1H NMR (chemical shift δ in ppm, coupling constant
J is in Hz) spectrum (300 MHz, CdCl3) recorded as 1.12 (3H,
t, J = 7.05, C-29), 0.83 (3H, d J = 5.1, C-27), 0.78, (3H, d J =
6.3, C-26), 0.96 (3H, d, J = 6.3, C-21), 2.21 (1H, m, C-20),
0.62 (3H, s, C-18)1.18 (3H, s, C-19), 5.12 (1H, dd, J = 8.4,
8.4, C-23), 4.98 (1H, dd, J = 8.4, 8.4, C-22), 5.28 (1H, t, J =
7.5, C-6), 3.45 (1H, m, C-3), for methine and methylene
protons of steroidal ring and side chain.
The retention time obtained was 3.257 min with a peak
area of 4113012 and peak area % of 98.347.
Structural determination of compound 3: Compound
3 was isolated from petroleum ether extract of the entire plant
of Kyllinga triceps. Petroleum ether extract is first treated with
alcoholic KOH to saponify the fats and washed again with
13C NMR (300 MHz, CDCl3, ) apart from other signals
showed the presence of one oxy carbon (δ c, 72.1) four olefinic

Vol. 29, No. 6 (2017)
Phytochemical of Isolated Chemical Constituents from Kyllinga triceps Rottb. 1397
carbons (δC, 129.2, 138.3, 121.69, 140.76) and six methyl carbon
(δC, 12.24, 18.9, 19.39, 19.39, 12.05, 21.2) the multiplicity
and chemical shift of different carbons of compound 3 are
shown in Table-12.
wavelength was maintained at 205 nm and injection volume
was 20 µL.About 10 mg of the sample was weighed to a 10 mL
volumetric flask.5 mL of methanol was added and sonicated
cooled and volume was made up to 10 mL. Retention time of
Compound 3 was found to be 6.089 min with peak area of
2581295 and peak % of 99.6 [20].
TABLE-12
13C NMR SPECTRAL DATA OF COMPOUND 3
Structural determination of compound 4: Compound
4, was isolated from petroleum ether extract of Kyllinga triceps.
Petroleum ether extract was first treated with alcoholic
potassium hydroxide to saponify the fatty acids which removed
the fatty acids present in the extract. After removal of fatty
acids, petroleum ether extract is subjected to gradient elution
column chromatography using, toluene and methanol (95:5),
68 fractions were obtained. The fractions were concentrated
and purified and subjected to identification through TLC by
using various solvents. The spots of isolated compound was
detected by exposing the plates to iodine vapours or observing
the plates in UV light and also by spraying the plates with
(5 %) sulphuric acid reagent in methanol followed by heating
the plates at 110 °C for 10 min. The Rf value recorded in two
different solvent system are given below.
Chemical
shift (δ
scale)
Chemical
shift (δ
scale)
Carbon
No.
Multi-
plicity
Carbon
No.
Multi-
plicity
12.05
12.24
18.98
19.39
21.07
21.07
21.2
18
29
27
19
11
21
26
15
28
16
2
CH3
CH3
CH3
CH3
CH2
CH3
CH2
CH2
CH2
CH2
CH2
CH2
CH
37.2
39.2
1
12
20
13
4
CH2
CH2
-CH
-C-
40.48
42.32
42.32
50.18
51.24
55.97
56.8
-CH2
-CH
-CH
-CH
CH
CH
CH
CH
CH
-C-
9
24
17
14
3
24.3
25.4
28.9
71.8
31.6
121.69
129.29
138.30
140.76
–
6
31.6
7
23
22
5
31.9
8
31.9
25
10
CH
Solvent system
Chloroform: ethanol (9.6:0.4)
Chloroform: ethyl acetate (8.5:1.5)
Rf value
0.55
0.63
36.5
-C–
–
–
Mass spectroscopic fragmentation of the isolated compound
3 yielded M+ at m/z 412, with other prominent fractions at m/z
395 (C29H47), 255 (C19H29O), 311 (C23H35), 297 (C22H33O) and
233 (C17H26O). Fragmentation pattern is shown in Table-13.
Compound 4 was found to be soluble in organic solvents
like petroleum ether, benzene, dichloromethane, chloroform
and methanol but insoluble in water. The melting point was
found to be 172-174 °C [21].
The IR spectrum of the compound 4 gave the following
peaks mentioned in Table-14.
TABLE-13
MASS FRAGMENTATION DATA OF ISOLATED COMPOUND 3
m/z
412
395
311
297
255
233
Name of the fragment
M+ molecular ion peak
(M+ -OH)
(M+ -OH-C6H13)
(M+ -H)- C7H14
M+ - (C10H19)
(M+ -H)- C7H14-C5H7
TABLE-14
IR SPECTRA OF COMPOUND 4
Wavenumber
Functional groups and bonds
(cm-1)
3476
2962
2831
1653
O-H str. vibrational band of bonded –OH
CH unsymmetrical str. of geminal dimethyl group
CH str. of steroidal ring and tertiary CH group
C=C str. of unconjugated alkene showing weak
absorption band
29
28
1453
-CH bending vibration due to –CH2 scissoring
vibration
22
21
18
27
1379
1077
O-H bending vibration
24
-C-O stretching (1260-1000 cm-1)
C-H in plane bending of cyclo alkenes (1000-650
cm-1)
20
17
23
25
26
953, 837
12
16
11
13
14
19
1
4
It is observed that with acetic anhydride and triethylamine
at room temperature isolated compound afforded an acetate
derivative. Thus compound 4 appeared to be a steroidal com-
pound and this assumption was confirmed by recording and
analyzing 1H NMR, 13C NMR and mass spectrum of compound
4.
15
2
9
10
8
7
3
5
HO
6
Structure of stigmasterol
The 1H NMR (chemical shift δ in ppm, coupling constant
J is in Hz) spectrum (300 MHz, CdCl3 recorded as 0.80 (3H, t,
J = 7.05, C-29), 0.84 (3H, d J = 6.3, C-27), 0.61 (3H, d J =
9.3, C-26), 1.10 (3H, d J = 9.3, C-21), 2.24 (1H, m, C-20),
1.18 (3H, s, C-19), 0.61 (3H, s, C-18), 5.27 (1H, t J = 5.4, C-
6), 3.41 (1H, m, C-3), for methine and methylene protons of
steroidal ring and side chain.
The identity of the isolated compound 3 which may be
stigmasterol was further confirmed by HPLC using Shimadzu
high performance liquid chromatographic system LC
2010CHT with UV detector, Kromasil C18 column was used,
gradient elution was conducted using toluene (A) and methanol
(B). Flow rate was maintained at 2 mL/min and detection

1398 Verma et al.
Asian J. Chem.
The 13C NMR of compound 4 was recorded in CDCl3 and
the chemical shift of different carbon is mentioned in Table-15.
at 1.0 SLM. For sample preparation 10 mg of sample was
dissolved in 5 mL of acetone, sonicated, slightly warmed at 40
°C for 0.5 min and made up to 10 mL and injected immediately.
Retention time of compound 4 was found to be 3.025
min with peak area of 946261 with peak % of 99.62.
TABLE-15
13C NMR SPECTRAL DATA OF COMPOUND 4
Chemical
shift (δ
scale)
Chemical
shift (δ
scale)
Carbon
No.
Multi-
plicity
Carbon
No.
Multi-
plicity
RESULTS AND DISCUSSION
The four compounds were isolated (two from ethyl acetate
fraction and two from petroleum ether extract) of Kyllinga
triceps. Both the compounds isolated from ethyl acetate
fraction gave positive Shinoda test for flavonoids and in TLC
also. The compound gave blue spots on spraying it with ferric
chloride thus confirming the phenolic nature of both the com-
pound. Compound 1 was crystallized in methanol as a yellow
crystalline powder having melting point 313-316 °C. It is slightly
soluble in water and diethyl ether and soluble in methanol,
ethanol and acetone. Compound 2 was crystallized in methanol
as a yellow powder, soluble in water and methanol (under hot
conditions) and insoluble in chloroform, acetone and ether. It
was found to have melting point 242-245 °C.
11.84
11.97
18.77
19.04
19.37
19.78
21.09
23.11
24.29
26.19
28.22
29.23
31.69
31.93
31.93
18
29
21
27
19
26
11
28
15
23
16
25
2
CH3
CH3
CH3
CH3
CH2
CH3
CH2
CH2
CH2
CH2
CH2
CH2
CH
33.99
36.13
36.52
37.28
39.81
42.32
42.30
45.90
50.19
56.11
56.80
71.80
121.68
140.78
–
22
20
10
1
CH
CH2
CH2
-CH
-C-
12
4
-CH2
-C-
13
24
9
-CH
-CH
-CH
CH
CH
CH
CH
–
17
14
3
6
7
CH
5
8
-CH
–
Both the compounds when treated with shift reagent
depicted bathochromic and hypsochromic shift which is the
characteristic feature of flavonol glycosides.
Mass spectroscopic fragmentation of the isolated com-
pound 4 yielded M+ at m/z 412, with other prominent fractions
at m/z 395 (C29H47), 255 (C19H29O), 311 (C23H35), 297 (C22H33O),
233 (C17H26O). Fragmentation pattern is shown in Table-16.
IR spectroscopic analysis of compound 1 showed
absorption band at 3582 cm1 that is characteristic of free
OH stretching, absorption bands at 3400, 3321 cm1 are due to
bonded OH group, absorption band at 1650 cm-1 is due to aryl
ketone and the decrease in the absorption frequency is due to
intramolecular hydrogen bonding between OH group of 3C,
C=O group of 4C and OH group of 5 carbon.Absorption at 1611,
1522, 1462 is due to C=C aromatic stretching. Absorption at
839, 791, 721 cm-1 is quiet prominent due to out of plane (oops)
C-H bending in aromatic ring.
TABLE-16
MASS FRAGMENTATION DATA OF ISOLATED COMPOUND 4
m/z
414
397
383
257
Name of the fragment
M+ molecular ion peak merged/C29H50O
(M+ -OH)/C29H49 base peak
(M+ -OH-CH3)/C28H47
(M+ -OH)-C10H21/C19H29
Compound 2 gave various absorption bands which are
3592 cm1 (free OH groups), 3408, 3321 cm1 (due to bonded
OH groups), 1663 cm1 (aryl ketonic group representing intra
molecular hydrogen bond), 1611, 1561, 1521 cm1 (C=C aro-
matic ring stretching), 865, 823, 723, 685, cm1 due to out of
plane (oops) C-H bending in aromatic ring. The absorption
bands were found in the same range so possibilities are that in
both the compounds some of the bonds and functional groups
are common. For further confirmation both the compounds
were subjected to 1H NMR, 13C NMR and Mass spectroscopic
analysis.
29
28
22
21
27
24
20
17
23
25
26
18
12
16
11
13
14
19
1
15
2
9
10
8
1
The H NMR and data of compound 1 resembles with
7
that of quercetin with singlets at δ value 9.2, 9.1, 9.4, 12.36,
10.69 for OH at C4’, C3’, C3, C5 and C7 respectively. Doublet
were observed at δ value 6.07, 6.29, 7.55, 6.78 for protons at
C6, C8, C2’ and C5’ and double doublet at 7.40-7.44 for, C-
6’H, which matches with the existing literature for quercetin.
3
5
HO
4
6
Structure of β-sitosterol
The identity of the isolated compound 4 which was found
1
to be β-sitosterol was further confirmed by HPLC using Shimadzu
high performance liquid chromatographic system with LC 8A
pump and UV detector and polymer ELS detector in combi-
nation with class LC10A software. Kromasil RP C-8, 250 ×
4.6 mm × 5 µ column was used. Isocratic elution was conducted
using methanol:water (99:1). ELSD (evaporative light scatte-
ring detection) conditions includes nebulized temperature of 75
°C, evaporation temperature 90 °C and gas flow was maintained
The H NMR and data of compound 2 resembles with
that of quercetin but some extra peaks were observed at at
δ value 5.23 (for H1-G, glucose moiety), 5.12 (for H1-R,
rhamnose moiety) and 0.8 for CH3-R, which confirms that
compound 2 is a glycoside of quercetin with glucose and
rhamnose moiety attached with it. Other peaks of aglycone
moiety resembling quercetin were observed as singlets at δ
value 9.56, 9.06, 12.47, 10.74 for OH at C4’, C3’, C5 and C7

Vol. 29, No. 6 (2017)
Phytochemical of Isolated Chemical Constituents from Kyllinga triceps Rottb. 1399
respectively. Doublet were observed at δ value 6.08, 6.27, 7.44
and 6.73 for protons at C6, C8, C2’, C5’.and double doublet
was also observed at 7.41-7.44 for, C-6’H, some more peak
doublet which matches with the existing literature for rutin.
of compound 3 the proton NMR showed the proton at C-3
appeared as a multiplet at δ 3.41 and revealed the existence of
signals for olefinic proton at C-6 (δ 5.28), C-22 (δ 4.98), C-23
(δ 5.12), angular methyl proton at C-18 (δ 0.62), C-19 (δ 1.18),
were observed. In case of compound 4 the proton NMR showed
the proton at C-3 appeared as a multiplet at δ 3.41 and revealed
the existence of signals for olefinic proton at C-6 (δ 5.27),
C=C is not present in C-22 and C-23, so it can be easily distin-
guished from another compound 3 which have prominent peaks
downfield due to olefinic protons in this position. Angular
methyl proton at C-18 (δ 0.61), C-19 (δ 1.18), were observed.
The 13C NMR signals of compound 3 has shown recogni-
zable signals 140.76 and 121.69 ppm, which are assigned C5
and C6 double bonds respectively as in ∆5 spirostene. The
value at 19.39 ppm corresponds to angular carbon atom (C19).
Spectra show twenty nine carbon signal including six methyls,
nine methylenes, eleven methane and three quaternary carbons.
The other alkene carbons appeared at C-22 (δ 138.3), C-23
13
The value of C NMR of compound 1 and 2 resembles
closely with each other and with that of existing literature.
The δ value of compound 2, differs from that of compound 1
due the presence of carbons in the glucose and rhamnose
moiety (Tables 3 and 8).
The retention time of isolated compound 1 was found to
be 16.568 min and that of compound 2 was 3.257 min.
Mass spectrum of isolated compound 1 (molecular weight
302.2) recorded prominent peak at 301 (M-H+), which on
further fragmentation yields prominent fragments weighing
273, 178.8, 150 and 107. Which matches with the existing
literature for quercetin dihydrate and thus the compound is
quercetin dihydrate. Mass spectrum of isolated compound 2
(M-H+ and M+H+) are most abundant and characteristic peaks
in flavonols. Compound 2 seems to be rutin as the mass frag-
mentation pattern and presence of base peak at 609.15 (M-H+)
on matching with the existing literature increases the prob-
ability of the compound to be rutin.
13
(δ 129.29). The C NMR signal of compound 4 has shown
recognizable signals 140.78 and 121.68 ppm, which are assig-
ned C5 and C6 double bonds respectively as in ∆5 spirostene
which closely resembles compound 1. The value at 19.37 ppm
corresponds to angular carbon atom (C19). The values of C-22
(δ 33.99), C-23 (δ 26.19) differed from the compound 3 and
thus proves that there is no double bond in the C-22 and C-23
position this is further strengthened by the 1H NMR values at
these carbons.
Compounds 3 and 4 were isolated from the petroleum
ether fraction of Kyllinga triceps from which the fatty acids
were saponified using alcoholic KOH and the remaining extract
was further purified with petroleum ether and subjected to
column chromatography using gradient elution technique. Both
compounds 3 and 4 gave positive tests for steroids and alcohols
so it was assumed that compound contains steroid nucleus
along with a alcoholic functional group attached to it. Both
the compounds were found soluble in organic solvents like
petroleum ether, benzene, dichloromethane, chloroform and
methanol but insoluble in water.
The weak molecular ions of compound 3 were given at
m/z 412 and the characteristic peaks were given at m/z 395
that corresponds to (M+-OH). Other ion peaks are m/z 311 due
to (M+-OH-C6H13), peaks at 297, 255 due to the formation of
carbocation by β bond cleavage of side chain leading to the loss
ofC7H14 and C10H19 from molecular ion M+, that corresponds
to the M+115 and M+157. The molecular weight and fragmen-
tation pattern indicate that the compound presenting this
information is stigmasterol. The compound 4 on mass fragmen-
tation gives a weak molecular ion peak M+ at 414 which appear
merged with 411, other prominent peaks appears at m/z 297
which is the base peak, most abundant fragment and is due to
(M+OH), other peak at m/z 383 and 257 are due to (M+-OH-
CH3) and (M+-OH)-C10H21. The data of this compound sindicates
that the compound is β-sitosterol.
Compound 3 is a white crystalline substance with melting
point 160-166 °C. The IR spectroscopic analysis showed the
absorption band at 3337.9 cm-1, which is characteristic of
OH stretching. Absorptions at 2962 cm1 and 2872 cm1 is due
aliphatic CH stretching. Other absorption frequencies include
1453 cm1 as a result of bending frequency for cyclic (CH2)n
and 1374 cm1 for OH in plane bending vibration. The absorp-
tion frequency at 1054, 965 cm1 signifies –CH out of the plane
bending of cycloalkene. The out of plane CH vibration of
unsaturated part was observed at 881 cm1. These absorption
frequencies resemble the absorption frequencies observed for
stigmasterol.
Further confirmation of both the compounds 3 and 4
was achieved by conducting the HPLC. For the compounds 3
gradient elution was done with toluene and methanol using
UV detector and the retention time of 6.08 min was obtained.
For compound 4 isocratic elution was done using methanol:
water (99:1), ELS detector was used due to weak chromophoric
group in this compound ELSD (evaporative light scattering
detection) conditions includes nebulized temp of 75 °C, evapo-
ration temperature 90 °C and gas flow was maintained at 1.0
SLM (standard liters per min).The retention time of 3.02 min
was obtained which was found to be reproducible.
Compound 4 is a white crystalline substance with melting
point 172-174 °C. On subjection to IR spectroscopic analysis,
the observed absorption bands are 3476 cm1 that is charac-
teristic of OH stretching. Absorption at 2962 cm1 and 2831
cm1 is due aliphatic unsymmetrical and symmetrical CH stret-
ching. Other absorption frequencies include 1653 cm1 as a
result of CH=CH stretching of unconjugated alkene showing
a weak absorption band, 1453 cm1 bending vibration due to
–CH2 scissoring vibrations, 1379 cm1 is due to OH in plane
bending vibration. The absorption frequency at 1077 is due to
–C-O vibration of –C-OH group, 953, 837 cm1 signifies –CH
in plane cyclo alkenes. These absorption frequencies resemble
the absorption frequencies observed for β-sitosterol. In case
1
From the findings of IR, H NMR, 13C NMR and MS
spectral data and their comparison with those described in the
literatures showed that, compound 3 was stigmasterol and
compound 4 was β-sitosterol both of them were isolated from
petroleum ether extract of the Kyllinga triceps and chemical

1400 Verma et al.
Asian J. Chem.
5. C.P. Khare, Indian Medicinal Plant, 356 (2007).
structures elucidated respectively. It was carried out by means
of various physical (solvent extraction, TLC, Column chro-
matography) and spectral techniques. The only difference
found between the two compounds is the presence of C22=C23
double bond in Stigmasterol and C22-C23 single bond in β-
sitosterol.
6. S. Aneela, A. Dey and S. De, Int. J. Pharm. Life Sci., 5, 3385 (2014).
7. S. Sasidharan, Y. Chen, D. Saravanan, K.M. Sundram and L.Y. Latha, Afr.
J. Tradit. Complement. Altern. Med., 8, 1 (2011).
8. B. Sambandam, D. Thiyagarajan, A. Ayyaswamy and P. Raman, Int. J.
Pharm. Pharm. Sci., 8, 120 (2016);
https://doi.org/10.22159/ijpps.2016.v8i9.12288.
9. Y.M. Musa, Int. J. Sci. Technol. Res., 4, 282 (2015).
10. Y. Zhu,Y. Liu,Y. Zhan, L. Liu, Y. Xu, T. Xu and T. Liu, Molecules, 18,
15648 (2013);
Conclusion
The study concluded the traditional use of the plant drug
in the treatment of diabetes probably due to the isolated flavo-
noids i.e. quercetin, rutin and such other compounds.
https://doi.org/10.3390/molecules181215648.
11. V.S.P. Chaturvedula and I. Prakash, Int. Curr. Pharm. J., 1, 239 (2012);
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ACKNOWLEDGEMENTS
13. N. Sanghavi, S.D. Bhosale andY. Malode, J. Sci. Innov. Res., 3, 594 (2014).
14. T. Seal, J. Appl. Pharm. Sci., 6, 157 (2016);
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The authors are thankful to Hon’ble Chancellor, Teerthanker
Mahaveer University, Moradabad, India for providing the
facility to carry out the research work.
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