Journal of Liquid Chromatography & Related

Technologies

ISSN: 1082-6076 (Print) 1520-572X (Online) Journal homepage: http://www.tandfonline.com/loi/ljlc20

Identification of Flavonoids from Cheilanthes

albomarginata Clarke and Their Simultaneous
Determination and Quantification by UPLC/DAD
Method

Ramakanta Lamichhane, Se-Gun Kim, Amrit Poudel, Dipak Sharma, Kyung

Hee Lee, Prakash Poudel, Prakash Raj Pandeya & Hyun-Ju Jung

To cite this article: Ramakanta Lamichhane, Se-Gun Kim, Amrit Poudel, Dipak Sharma,
Kyung Hee Lee, Prakash Poudel, Prakash Raj Pandeya & Hyun-Ju Jung (2015) Identification of
Flavonoids from Cheilanthes albomarginata Clarke and Their Simultaneous Determination
and Quantification by UPLC/DAD Method, Journal of Liquid Chromatography & Related
Technologies, 38:19, 1713-1721

To link to this article: http://dx.doi.org/10.1080/10826076.2015.1091011

Published online: 23 Nov 2015.

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 Journal of Liquid Chromatography & Related Technologies, 38: 1713–1721, 2015
Copyright # Taylor & Francis Group, LLC
ISSN: 1082-6076 print/1520-572X online
DOI: 10.1080/10826076.2015.1091011

Identification of Flavonoids from Cheilanthes albomarginata

Clarke and Their Simultaneous Determination and
Quantification by UPLC/DAD Method
RAMAKANTA LAMICHHANE,1 SE-GUN KIM,2 AMRIT POUDEL,3 DIPAK SHARMA,4 KYUNG HEE LEE,1 PRAKASH
Downloaded by [Shin Seo Trading Co Ltd], [Ramakanta Lamichhane] at 04:44 24 November 2015

POUDEL,3 PRAKASH RAJ PANDEYA,1 and HYUN-JU JUNG4

1
College of Pharmacy, Deptartment of Oriental Pharmacy, Wonkwang University, Iksan, Jeonbuk, Republic of Korea
2
Department of Agricultural Biology, National Academy of Agricultural Science, Rural Development Administration, Wanju,
Republic of Korea
3
School of Health and Allied Science, Department of Pharmaceutical Science, Pokhara University, Khudi, Kaski, Nepal
4
College of Pharmacy, Department of Oriental Pharmacy & Wonkwang-Oriental Medicines Research Institute, Wonkwang University,
Iksan, Jeonbuk, Republic of Korea

Cheilanthes albomarginata Clarke, a fern from the Pteridaceae family, is traditionally used to treat peptic ulcer and stomach disorder. Six
flavonoid compounds were isolated and identified from the ethyl acetate soluble fraction of this plant. Kumatakenin 5-O-b-glucoside,
kaempferol-7-methyl ether, rhamnocitrin 3-O-b-glucoside, and kaempferol 3-O-b-glucoside were isolated for the first time from this
plant. UPLC method was developed and used for the simultaneous determination and quantification of the isolated compounds in the
methanol extract of the plant. The method was also validated using the parameters: linearity, precision, and accuracy.
Keywords: Cheilanthes albomarginata, flavonoid, identification, quantification, UPLC, validation

Introduction quantitation) according to the International Conference on Har-

Cheilanthes albomarginata (CA) is a fern from the Pteridaceae
family, which grows in rock crevices on slopes at an altitude of
1300–2700 m. It is found mainly in India, Nepal, Pakistan, and
Bhutan.[1] Traditionally, juice of the rhizome of CA is used to
treat peptic ulcer and stomach disorder in Nepal.[2] Paste of
CA rhizome is used externally to treat cuts and wounds.[3]
The previous study shows that the plant has potent antioxidant,
anti-inflammatory, and antilipid activity.[4]
Ferns are rich in flavonoids and many types of flavonoids have
been discovered from various fern species. Phytochemical inves-
tigations have revealed that some ferns contain unusualflavan-4-
ol glycosides and flavones modified in the B-ring.[5] This study
involves the isolation and identification of flavonoid compounds
from CA and their simultaneous determination and quantification
using a validated UPLC method. UPLC method was validated
with different parameters such as linearity, accuracy, specificity,
precision, and sensitivity (limit of detection and limit of
Address correspondence to: Hyun-Ju Jung, College of Pharmacy,
Department of Oriental Pharmacy & Wonkwang-Oriental Medicines
Research Institute, Wonkwang University, 344-2 Shinyong0-dong,
Iksan, Jeonbuk, 570-749, Republic of Korea. E-mail: hyun104@
wku.ac.kr
Color versions of one or more of the figures in the article can
be found online at www.tandfonline.com/ljlc.
monization ICH Q2 (R1) guideline.[6]
Experimental
Plant Materials, Extraction, Fractionation, and Isolation
Aerial parts of C. albomarginata were collected in Kaski district,
Nepal, during June/July 2011 and identified by Dr. Radhe Shyam
Kayastha, PhD., Tribhuvan University, Nepal. The voucher speci-
mens (No. 332) were deposited in the Pharmacognosy Laboratory
of Pokhara University, Lekhnath Municipality-12, Kaski, Nepal.
The collected plant samples were washed properly with water
and shade dried. Afterward, they were cut into small pieces and
extracted with methanol at 50°C. The methanol extract was fil-
tered and concentrated by a rotator evaporator using a vacuum.
The methanol extract was suspended in water and subjected to
fractionation with chloroform, ethyl acetate, and butanol. Ethyl
acetate fraction (18 g) was loaded on a normal phase Silica gel
column (650 g, Kieselgel 60, 70–230 mesh, Merk, Darmstadt,
Germany) and eluted with gradient CHCl3- MeOH (100% to
50%). The eluates were collected into test tubes (30 mL/test tube)
and divided into four fractions (F1–F4) on the basis of similatiy of
chromatogram in TLC plates.
Fraction F1 was dried and subjected to Sephadex LH-20 column
(30
2.5 cm) and eluted with 50% methanol and compound 1
(kumatakenin) was yielded. Compound 2 (rhamnocitrin or
kampferol-7-methyl ether) and compound 3 (kaempferol) were
 1714
obtained from fraction F2 after subjecting to reverse phase open
column and methanol–water (2:8–9:1) solvent system. Fraction
F3 was eluted with reverse phase (ODS-A 12 nm, S-75 µm,
YMC, Kyoto, Japan) open column (45
2.5 cm) using solvent
MeOH:H2O (1:9-1:0). Compound 4 (rhamnocitrin 3-O-
b-glucoside), compound 5 (kumatakenin 5-O-b-glucoside), and
compound 6 (kaempferol 3-O-b-glucoside) were obtained. Purifi-
cation was done by medium pressure liquid chromatography
(MPLC) using Parallelprep xy4e_050217 with a PREP UV-10 V
detector, pump 540 (Yamazen, Osaka, Japan) and Watchers SiO2
and RP18 flash cartridge columns (Isu industry Co., Seoul, Korea).
Downloaded by [Shin Seo Trading Co Ltd], [Ramakanta Lamichhane] at 04:44 24 November 2015
The fraction corresponding to single major peak were collected.
Structural elucidation of the isolated compounds was performed
by proton magnetic resonance (1H NMR), carbon magnetic reson-
ance (13C NMR), distortionless enhancement by polarization trans-
fer (DEPT NMR), HMBC NMR, and HMQC. Mass spectrum was
recorded on Finnigan LCQ spectrometer equipped with an electro
spray ion source (positive ion mode) “ESI.” The scan range was
from 100–1500 m/z. The ionization conditions were adjusted at
325°C and 3.5 kV for capillary temperature and voltage, respect-
ively. The nebulizer pressure was 35 psi and the nitrogen flow rate
was 8 L/min. The samples were dissolved in methanol.
Melting point of the compound was done in OptiMelt Auto-
mated Melting Point System (Stanford Research Systems).
Sugar moiety in the flavonoid glycosides were revealed by acid
hydrolysis and determination by means of TLC with authentic
standards. The sample and standard (glucose, sucrose, and fruc-
tose) were spotted on silica gel TLC and on the top again spotted
with acid (2 N HCl). Then, the TLC analysis was performed
using BuOH-OHAc-H2O (4:1:5) and visualized with aniline
phthalate (aniline, 9.2 mL; phthalic acid, 16 g; BuOH, 490 mL;
Diethyl ether, 490 mL; and water 20 mL) spray reagent.
Antioxidant Activity of Isolated Compounds
The antioxidant activity of the isolated compounds was ana-
lyzed by DPPH radical scavenging activity method proposed
by Hazra et al., with slight modification.[7] The results were
expressed as IC50 (quantity of antioxidant necessary to reduce
by 50% the initial free-radical DPPH concentration). The calcu-
lations were performed by linear regression.
Ultra-Performance Liquid Chromatography (UPLC) for
Quantification
An ACQUITY UPLC system (Waters Corp., Milford, MA,
USA) with a conditioned auto-sampler USA, and Halo C18
column (100 mm
4.5 mm, 1.7 µm) by Advanced Materials
Technology was used for the analytical study. After testing
several gradients, flow rates (1 mL/min–1.5 mL/min) and tem-
peratures (30°C–45°C), a suitable chromatographic condition
with solvent system of acetonitrile (A) and water (B, containing
0.1% phosphoric acid) was obtained as follow: 0–1 min
(10–15% A), 1–12 min (15–17% A), 12–14 min (17–22% A),
14–18 min (22–30% A), 18–20 min (30–36% A), 20–27 min
(36–41% A), 27–28 min (41–65% A), and 28–30 min
(65–100% A) at a flow rate, 1 mL/min and injection volume
of 4 µL. The PDA detection wavelength was set at 220 nm, on
the basis of absorption maximum of analytes. In this wavelength
R. Lamichhane et al.
there were no other (interfering) substances contributing signi-
ficantly to the absorbance. The temperature of column and
auto-sampler were maintained at 45°C and 4°C, respectively.
All solvents used were of analytical grade.
Validation of UPLC Method
Stock standard solutions equivalent to 1 mg/mL of each isolated
compound was prepared by dissolving the isolated compounds in
methanol in volumetric flask and filtered through a 0.22-µm nylon
membrane filter. The purity of the isolated compounds was deter-
mined using UPLC and all the compounds had purity in the range
of 96–98%. The standards were prepared by diluting the stock
standard solutions with methanol. Stock solution of methanol
extract equivalent to 2.5 mg/mL was prepared by dissolving in
methanol and was filtered using 0.22-µm nylon membrane filter.
For linearity the stock solution of standard compounds
were serially diluted into five concentrations: kumatakenin
(28.125–450.000 µg/mL), kampferol-7-methyl ether (180.000–
11.250 µg/mL), kaempferol (0.625–10.000 µg/mL), rhamnocitrin
3-O-b-glucoside (15.625–250.000 µg/mL), kumatakenin 5-O-b-
glucoside (3.125–50.000 µg/mL), and kaempferol 3-O-b-glucoside
(4.375–70.000 µg/mL). These solutions were analyzed individually
in triplicate for the establishment of calibration curves. Aliquots of
4.0 µL were injected and eluted with the mobile phase under the
reported chromatographic conditions. Calibration curve was con-
structed by plotting the concentration of standards versus corre-
sponding mean peak area. The correlation coefficient (r2), slope
(b), and intercept (a) of the calibration curve were calculated.[8]
Sensitivity of the method was proved by establishing the lim-
its of detection (LOD) and limits of quantitation (LOQ). The
LOD and LOQ were calculated based on the standard deviation
(SD) and the slope (S) of the calibration curve according to the
equations (1) and (2) respectively.[9] From the calibration curve
of the peak area versus concentration, the SD was the standard
deviation of the response, which was estimated by the standard
deviation of the intercepts of the regression line in the cali-
bration curves; S was the slope of the calibration curve:
LOD ¼ ð3:3
SDÞ=S ð1Þ
LOQ ¼ ð10
SDÞ=S ð2Þ
Accuracy of the proposed method was confirmed by performing
recovery experiments with the standard addition technique. First
an unspiked sample of methanol extract of CA was analyzed and
the concentrations of compounds in the sample were recorded.
Next, three known concentrations of each standard were added
to a known volume of the fresh sample (methanol extract of
CA) and analyzed. The recovery of the additional amount of
standard was checked by using following formula:[9]
Recoveryð%Þ ¼fðamount found  original amount)

100g=amount spikedg
Where, “original amount” is the amount of individual marker
compounds in the methanol extract of CA and “amount spiked”
 Quantification of Flavonoids from C. albomarginata
is the additional amount of marker compounds added to the
methanol extract sample solution. The experiments were per-
formed in triplicate.
Intra-day and inter-day variations and the repeatability of six
samples were chosen to determine the precision of the
developed assay. Intra-day precision was validated with three
concentrations of all standard solutions under the optimized
conditions within a day. Inter-day precision was validated with
the standard solutions for three consecutive days. Repeatability
was conducted by analyzing the extractive solution six times in
the same day. All investigated components were expressed as
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relative standard deviation (RSD).[10]
The newly established analytical method was subsequently
applied for the simultaneous determination of six isolated com-
pounds in the methanol extract of CA. The isolated compounds
were quantified by linear regression of the standards. Each com-
pound was analyzed in triplicate to determine the mean content.
Results and Discussion
Extraction, Isolation, and Identification
The freeze dried methanol extract and other fractions were
weighed and the yield was calculated (Table 1). The weight of
dry plant material taken for extraction was 1.5 kg. The water
extract showed considerable yield value that indicated that the
plant CA might contain large amounts of polar compounds.
The ethyl acetate fraction yielded six compounds. A previous
study has shown the presence of compound 1 and compound 2
in this plant but they did not include sufficient NMR and mass
spectrum data.[11] In this study, we isolated and identified these
compounds using their UV, MS, 1H, and 13C NMR data. Com-
pound 5 was identified as a new compound, whereas compound
3, 4, and 6 were known compounds but identified for the first
time from this plant.
Compound 1 was obtained as yellow colored needle shaped
crystals, melting point 248–249°C and the HRESIMS spectrum
showed molecular ion peak [M þ H]þ at 315.07 corresponding
to molecular formula C17H14O6. The UV spectra (kmax) were
at 220, 264.7, and 364.5 nm. The 1H-NMR spectrum (Table 2)
showed signals for two meta-coupled aromatic protons at dH
6.36 (d, J ¼ 2 Hz, H-6) and 6.74 (d, J ¼ 1.85 Hz, H-8) of ring
A and ortho-coupled aromatic protons dH 6.93 (d, J ¼ 8.7 Hz,
H-30 , 5’) and 7.96 (d, J ¼ 9.2 Hz, H-20 , 6’) of B-ring, suggesting
a flavonol skeletal pattern. In addition, the two singlet proton
peaks at dH 3.85 and 3.80 support the presence of two methoxy
group which were confirmed from the 13C NMR spectral signals
(Table 3). A very low field broad singlet at dH 10.30 clearly
Table 1. Yield of different extract/fraction of C. albomarginata
Dry Yield (gm/gm of
Extract/Fraction Weight (g) dried plant)
Methanol 325 0.22
Chloroform 94 0.06
Ethyl acetate 16 0.01
Butanol 31 0.02
Water 157 0.10
1715
Table 2 1H-NMR (500 DMSO-d6) data of compound 1–6
1
Compound H-NMR (500 MHz, DMSO-d6) values
Compound 1 d3.80 (3H, s, 3-OMe), 3.85 (3H, s, 7-OMe), 6.36
(1H, d, J = 2 Hz, H-6), 6.74 (1H, d, J = 1.85 Hz,
H-8) 6.93 (2H, d, J = 8.7 Hz, H-30 , H-50 ) 7.96 (2H,
d, J = 9.2 Hz, H-20 , H-60 )
Compound 2 d 6.35 (1H, d, J = 1.84 Hz, H-6), 6.73 (1H, d,
J = 2.3 Hz, H-8), 6.94 (2H, d, J = 8.8 Hz, H-30 ,
H-50 ), 8.09 (2H, d, J = 9.16 Hz, H-20 , H-60 )
Compound 3 d 6.16 (1H, s, H-6), 6.38 (1H, d, J = 1.8 Hz, H-8),
6.80 (2H, d, and J = 8.7 Hz, H-30 , H-50 ), 8.07 (2H,
d, J = 8.7 Hz, H-20 , H-60 )
Compound 4 3.22 (1H, m, J = 2.3 , 7.8, H-500 ), 3.30 (1H, m, J
9.6, 8.44 H-400 ), 3.44 (2H, m, J 9.15, 7.8 H-200 ,
H-300 ), 3.53(1H, m, H-6a00 ), 3.71 (1H, J 2.3 Hz,
11.95 m, H-6b00 ), 3.86 (4H, s, 7-OCH3), d 5.30
(1H, d, J = 7.35 Hz, H-100 ), 6.31 (1H, d,
J = 2.3 Hz, H-6), 6.67 (1H, d, J = 1.85 Hz, H-8),
6.88 (2H, d, J = 8.75 Hz, H-30 , H-50 ), 6.88 (2H, d,
J = 8.7 Hz, H-20 , H-60 )
Compound 5 d 4.8 (1H, d, J = 7.75 Hz, H-100 ), 3.16 (1H,
J = 9.96 Hz, 4.4 Hz, m, H-400 ), 3.52 (1H, J = 11,
J = 3.45 Hz , H-600 ), 3.75 (1H, H-600 ), 3.41–3.30
(3H, m, H-200 , H-300 , H-500 ), 3.75 (3H, s, 3-OCH3)
3.86 (3H, s, 7-OCH3), 6.86 (1H, d, J = 2.3 Hz,
H-6), 7.00 (1H, d, J = 2.3 Hz, H-8), 6.90 (2H, dd,
J = 9.15 Hz, 2.3 Hz, H-30 , H-50 ), 7.90 (2H, dd, J
6.9, 2.3 Hz, H-20 , H-60 )
Compound 6 d 5.43 (1H, d, J = 7.35 Hz, H-100 ), 3.57 (2H,
J = 8.8 Hz, d, H-400 ), 3.14–3.21 (H), 3.08 (2H, d,
J = 4.52 d,), 6.09 (1H, d, J = 0 Hz, H-6), 6.30 (1H,
d, J = 0 Hz, H-8), 6.91 (2H, dd, J = 9.15 Hz,
2.3 Hz, H-30 , H-50 ), 8.05 (2H, dd, J 6.9, 2.3 Hz,
H-20 , H-60 )
indicated the presence of a chelated hydroxyl group. The 13C
NMR and DEPT spectra showed 17 signals including six meth-
ine (-CH-), two methyl (-CH3), and nine tetra-substituted C-
atoms. In the HMBC spectrum, the C-7 (d165.6) of ring A
has correlated peaks with H-6, H-8 and also with O – methyl
group, unequivocally showing that the methyl group is attached
to the oxygen bound to C-7 (Fig. 1a). Similarly, in the HMBC,
the C-3 (d138.3) of ring B has correlated peaks with O–methyl
group confirming that another methoxy group is attached to
C3 (Fig. 1b). The structure was further supported by the COSY
and HMQC correlations. Thus, compound 1 was identified as
kumatakenin (3,7-dimethoxyl kaempferol) (Fig. 2).
Compound 2 was obtained as light yellow powder and the
HRESIMS spectrum showed molecular ion peak [M þ H]þ at
301.06 corresponding to molecular formula C16H12O6. The 1H
and 13C NMR spectral data (Table 2 and 3) were similar to those
of compound 1 except lacking one methoxy proton peak. The
methoxy group was found to attach at C-7 from the HMBC
(Fig. 1a). The UV spectrua (kmax) were at 264.7 and
365.5 nm. Based on the evidences from COSY, HMQC, and
HMBC correlations, compound 2 was determined to be Rham-
nocitrin (Kampferol-7-methyl ether) (Fig. 2).
 1716 R. Lamichhane et al.
13
Table 3. C NMR (500 DMSO-d6) data of compound 1–6 attached at C-3. The C-3 signal was seen to shift upfield by
about d2.0 compared to compound 3, which also confirmed
Compound the glycosylation at C-3.[13] The sugar moiety was established
Position
to be b-glucopyranose from the COSY and coupling constant
Aglycon 1 2 3 4 5 6
of the glucose protons. In a glucose sugar, the Karplus angle
2 156.5 148.00 156.6 158.0 160.9 159.9 between H-3 and H-4, are nearly 180° and thus 3J3,4 and 3J4,5
3 138.3 136.50 135.8 134.5 140.3 133.0 to be significant (8-14 Hz).[14,15] In contrast, for galactose sugars
4 178.5 176.60 176.0 178.2 173.7 176.9 the Karplus angle between H-3 and H-4 as well as H-4 and H-5
5 161.2 156.60 161.1 161.4 158.7 155.6 are nearly 60° and thus the observed Js are much smaller, if
6 98.3 98.01 97.9 97.7 103.1 94.0 present at all (0-7 Hz). The coupling constant of sugar moiety
7 165.6 165.40 165.2 165.9 164.0 161.0 confirms the presence of glucose ring. The UV spectra (kmax)
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8 92.9 92.58 93.1 91.7 96.4 99.3 were at 220, 264.7, and 346.5 nm. Based on the evidences from
9 156.8 160.90 159.2 157.0 158.0 156.6 COSY, HMQC, and HMBC correlations, compound 4 was
10 105.7 104.60 103.1 105.2 109.9 102.9 determined to be rhamnocitrin 3-O-b-glucoside (Fig. 2). This
10 120.7 122.10 122.3 121.3 121.0 121.0 compound is reported to be present in CA for the first time from
20 130.7 130.10 129.3 131.1 130.4 130.4 this study.
30 116.3 116.00 119.9 114.7 116.1 116.1 Compound 5 was white needle shaped crystal and the HRE-
40 161.4 159.80 161.1 160.3 153.8 155.6 SIMS spectrum of compound 5 showed molecular ion peak [M þ
50 116.3 116.00 119.9 114.7 116.1 116.1 H]þ at 477.10 corresponding to molecular formula C23H24O11.
60 130.7 130.10 129.3 131.1 130.4 130.4 1
H-NMR and 13C-NMR spectra (Table 2 and 3) of compound
3-OCH3 56.6 61.4 5 were almost similar to compound 1 except the presence of
7-OCH3 60.2 56.50 55.7 55.1 additional sugar peak. From the 13C-NMR and DEPT, the sugar
Glucose constitutes six carbons: five methine (-CH-) and one methylene
1″ 102.6 104.19 101.0 (-CH2-) groups. The anomeric proton signal at about dH 4.8
2″ 74.4 74.13 74.1 occurred as a doublet with a coupling constant J ¼ 7.35, indicat-
3″ 76.7 76.43 76.4 ing that it was in axial position. This confirmed that the sugar
4″ 70.0 70.45 69.8 confirmation was b position. Proton-carbon correlation (HMBC)
5″ 77.1 78.18 78.1 of anomeric proton at d 4.8 (1 H0 , J ¼ 7.35) with the C-5 (dC
6″ 61.2 61.44 60.8
158.78) indicated the sugar attached with the ring A at C-5
position (Fig. 1c). In the 13C NMR spectrum, the C-4 signal
Compound 3 was obtained as yellow crystal and the HRE- was shifted dH 4.75 upfield compared to kumatakenin (compound
SIMS spectrum showed molecular ion peak [M þ H]þ at 1), which occurs when there is a glucoside at the C-5 hydroxyl
287.05 corresponding to molecular formula C15H10O6. 1H- group.[13] Sugar was established to be glucose from the COSY
NMR and 13C NMR, data (Table 2 and 3) combined with DEPT and coupling constant values. We know that the J values for H-
spectrum, confirmed the compound 3 to be kaempferol.[12] 3 and H-4 for glucose (8–14 Hz) are higher than that of galactose
Compound 4 was obtained as yellow powder and its HRE- (0–7 Hz).[14,15] The UV spectra (kmax) were at 220, 260, and
SIMS spectrum showed molecular ion peak [M þ H]þ at 339.6 nm. The 13C signals for the sugar moiety was compatible
463.11 corresponding to molecular formula C22H22O11. 1H- with literature on 5-O-glucosides.[13] Hence, compound 5 was
NMR and 13C NMR spectra (Table 2 and 3) were almost similar identified as kumatakenin 5-O-b-glucoside (Fig. 2). This kind
to that of compound 3 except additional peaks for sugar moiety. of flavonoid, having O-glycosylation in position 5, is less fre-
The sugar was hexose sugar with five methine (-CH-), one quent in the case of plant flavonoids.[5] Kumatakenin 5-O-b-
methylene (-CH2-) group. In HMBC spectrum the anomeric pro- glucoside is a new flavonoid isolated from CA and, to the best
ton dH 5.3 (1 H00 , J ¼ 7.35, b position) showed correlation with of our knowledge, this is first report of kumatakenin 5-O-b-
C-3 (Fig. 1d). This indicated the presence of sugar moiety being glucoside. Its melting point was found to be 215°C.

Fig. 1. Showing Key HMBC correlation in different isolated compounds 1–6.

 Quantification of Flavonoids from C. albomarginata 1717
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Fig. 2. Structure of compounds 1–6 isolated from C. albomarginata.

Table 4. Antioxidant activity of isolated compounds
Compound 6 was yellow amorphous powder, and the HRE-
Compounds IC50 (µg/mL) SIMS spectrum showed molecular ion peak [M þ H]þ at
449.09 corresponding to the molecular formula C21H20O11.
Kaempferol 3-O-b-glucoside 54.53 The 1H-NMR and 13C NMR spectra (Table 2 and 3) are similar
Kumatakenin 5-O-b-glucoside 131.63 to kaempferol except for the presence of additional peaks for
Rhamnocitrin3-O-b-glucoside 200.23 sugar moiety. From the DEPT, the sugar constitutes of six car-
Kampferol-7-methyl ether 16.54 bons; five methine (-CH-) and one methylene (-CH2-) groups.
Kaempferol 4.15 The anomeric proton signal at dH 5.43 occurred as a doublet
Kumatakenin 178.57 with a coupling constant J ¼ 7.35, indicating that it was in axial
Vitamin C 1.29 position.[13] In HMBC spectrum (Fig. 1d), the anomeric proton

Fig. 3. UPLC chromatograms: standard mixture (A); C. albomarginata methanol extract (B). Peaks: Kaempferol 3-O-b-glucoside (6),
Kumatakenin 5-O-b-glucoside (5), Rhamnocitrin 3-O-b-glucoside (4), Kaempferol (3), Kampferol-7-methyl ether (2), and Kumatakenin (1).
 1718 R. Lamichhane et al.

Table 5. Regression data, linearity, LOD, and LOQ for the isolated compounds analyzed by UPLC

Isolated compounds Regression equation R2 Linearity range (µg/mL) LOD (µg/mL) LOQ (µg/mL)

Kaempferol 3-O-b-glucoside y ¼ 7893.3x þ 6708.9 0.999 4.375–70 1.41 4.27

Kumatakenin 5-O-b-glucoside y ¼ 3897.2x þ 479.04 0.999 3.125–50 0.37 1.13
Rhamnocitrin 3-O-b-glucoside y ¼ 9241.7x þ 50693 0.998 15.625–250 1.42 4.32
Kampferol-7-methyl ether y ¼ 26617x þ 1387 0.999 11.25–180 0.05 0.18
Kaempferol y ¼ 7559.2x þ 23392 0.999 0.625–10 1.42 4.32
Kumatakenin y ¼ 9510.8x þ 10057 0.999 28.125–450 3.24 9.83
Y ¼ peak area; x ¼ concentration (µg/mL). Tests were conducted in triplicate. R2 ¼ Correlation coefficient.
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dH 5 showed correlation with C-3. This indicated that sugar
moiety was attached at C-3. This confirmed that the sugar
confirmation was b. The sugar moiety was identified to be
glucopyranoside from 1H-NMR and TLC analysis. The UV
spectra (kmax) were at 220, 264.7, and 346.5 nm. Compound 6
was identified to be kaempferol 3-O-b-glucoside (Fig. 2). This
compound was isolated for the first time from this plant.
The spectroscopic data for the six purified compounds are
shown in Table 2 and Table 3.
Antioxidant Activity
Compounds such as kaempferol (3), kampferol-7-methyl ether
(2), and kaempferol 3-O-b-glucoside (6) showed good

Fig. 4. Comparison of UV spectra of the compound peaks in the chromatograms of C. albomarginata methanol extract (A) and standard
mixture (B).
 Quantification of Flavonoids from C. albomarginata 1719

Table 6. Analytical results of intra-day and inter-day variability (n ¼ 3)

Concentration Intra-day mean  SD RSD Inter-day mean  SD RSD

Isolated compounds (µg/mL) (µg/mL) (%) (µg/mL) (%)

Kaempferol 3-O-b-glucoside 17.500 17.38  0.20 1.13 17.94  0.59 3.16

8.750 8.47  0.13 1.42 8.76  0.41 4.27
4.370 3.53  0.08 1.83 3.58  0.12 2.87
Kumatakenin 5-O-b-glucoside 12.500 12.60  0.26 2.09 12.66  0.27 2.14
6.250 5.99  0.14 2.44 6.29  0.12 2.03
3.125 2.70  0.06 2.43 2.84  0.06 2.31
Rhamnocitrin 3-O-b-glucoside 62.500 62.26  0.53 0.85 64.76  0.94 1.45
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31.250 28.74  0.46 1.61 29.35  0.83 2.83

15.620 12.50  0.05 0.43 12.76  0.50 3.90
Kampferol-7-methyl ether 45.000 41.99  0.72 1.64 45.49  2.19 4.81
22.500 21.27  0.10 0.46 21.83  0.58 2.66
11.250 9.21  0.11 1.17 9.85  0.46 4.64
Kaempferol 2.500 2.61  0.02 0.88 2.53  0.07 3.14
1.250 1.22  0.01 1.01 1.23  0.02 1.82
0.620 0.56  0.01 2.22 0.57  0.01 3.39
Kumataenin 225.000 227.62  1.15 0.61 232.74  2.46 1.29
112.500 111.47  0.32 0.34 113.29  1.36 1.46
56.250 52.94  0.13 0.31 54.13  0.92 2.04

antioxidant activity compared to Vitamin C (Table 4). The methanol extract and the corresponding standard (isolated)
remaining compounds did not show efficient antioxidant activity. compounds.

Optimization of Chromatographic Condition Method Validation

From the suitable chromatographic condition that was obtained,
a complete chromatographic separation of the isolated com-
pounds was reached within 32 min, with good chromatographic
resolution for the target compounds. The standards and
methanol extract of CA were analyzed in 220-nm wavelength.
Figure 3 shows a typical UPLC chromatogram of the CA
UPLC analyses of linearity, specificity, precision, accuracy, and
repeatability were performed to demonstrate that the present
method is selective, precise, and reproducible. To determine lin-
earity, five concentrations of each standard solution were
injected individually. Calibration curve equations were obtained
by plotting the peak areas versus concentrations. As all of the R2

Table 7. Recovery data of the analytical method for the determination of six marker compounds (n ¼ 3)

Standard solution Standard solution Recovery RSD

Isolated compounds original concentration (µg/mL) observed concentration (µg/mL) (%) (%)

Kaempferol 3-O-b-glucoside 40.00 40.37  0.69 100.92 1.72

20.00 20.37  0.91 101.87 4.51
10.00 10.46  0.40 104.64 3.90
Kumatakenin 5-O-b-glucoside 40.00 42.03  2.00 105.04 4.76
20.00 20.96  0.38 104.78 1.85
10.00 9.77  0.40 97.72 4.17
Rhamnocitrin 3-O-b-glucoside 100.00 100.61  0.78 104.61 0.75
50.00 52.31  1.00 104.62 1.91
25.00 25.23  0.89 100.91 3.53
Kampferol-7-methyl ether 100.00 103.52  0.12 103.52 0.12
50.00 51.76  0.43 103.51 0.85
25.00 25.76  0.43 102.44 3.18
Kaempferol 10.00 10.03  0.18 100.32 1.85
5.00 5.22  0.12 104.38 2.32
2.50 2.53  0.03 101.34 1.18
Kumatakenin 100.00 101.57  3.63 101.57 3.58
50.00 49.57  2.39 99.14 4.83
25.00 25.12  1.15 100.47 4.61
 1720
Table 8. Repeatability data and content of isolated compounds in CA (n ¼ 3)
Isolated compounds RSD (%) Amount (mg/g of methanol extract)
Kaempferol 3-O-b-glucoside 2.68
Kumatakenin 5-O-b-glucoside 4.92
Rhamnocitrin 3-O-glucoside 0.45
Kampferol-7-methyl ether 1.44
Kaempferol 1.94
Kumatakenin 0.87
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values were >0.9996, linearity was verified. Detail information
regarding the calibration curve, linear range, LOD, and LOQ is
listed in Table 5.
Specificity was determined by comparing the peak purity of
six marker (isolated) compounds of the extract with standards
(Fig. 4). The results showed that the peaks were pure and lacked
interference by impurities.
The precision of the developed method was determined by
intra-day and inter-day variation, along with repeatability of
the six compounds. The RSDs of intra-day and inter-day analy-
sis were in the ranges of 0.31–2.44% and 1.29–4.81%, respect-
ively (Table 6). These data show that the described method has
an acceptable degree of precision. The accuracy was measured
by determining the recovery of standard addition. Three stan-
dard solutions were added to the known concentration of meth-
anol extract of the CA and the percentage of recovery and RSD
were calculated. The measured data showed a recovery of
97.72–105.04% and RSD of 4.83–0.12 (Table 7).
Repeatability was determined by evaluating the RSD for each
compound after injecting a known concentration of methanol
extract (2.5 mg/mL) for six times. The RSD values ranged from
0.45 to 4.92 (Table 8). Total concentration of each compound in
the methanol extract was found using the regression equation.
Quantification of Isolated Compounds
Using the newly established validated method, the quantifi-
cation of the six compounds in the methanol extract of CA
was determined. Then, the content of compounds in dry plant
was determined using the methanol extract yield value (Table
1). The results were expressed as content of the isolated
compounds per gram of the methanol extract of CA (Table 8).
Table 8 indicates that kumatakenin occurs in huge amount in
CA. Kumatakenin is an ethyl ether flavonol and has been
reported to have antiviral activity against HIV.[16] Kaempferol
and rhamnocitrin 3-O-b-glucoside were found to be available
in considerable amounts in CA. These two flavonoids have
shown good evidence of prevention of atherosclerosis, a main
cause for cardio-vascular problems.[17] Kamepferol commonly
found in plant- derived foods and in traditional medicines has
been known to have different biological properties (antioxidant,
antimicrobial, anticancer, anti-inflammation, etc.[18] Kaempferol
was found to improve insulin-stimulated glucose uptake in
mature 3T3-L1 adipocytes and also inhibited the differentiation
of 3T3-L1.[19] The presence of biologically active different fla-
vonoids indicates that CA might be a useful remedy for different
diseases and that is a part of further research.
R. Lamichhane et al.
Amount (mg/g of dried plant)
3.48  0.21 0.76  0.95
5.69  0.15 1.25  0.68
16.34  0.18 3.59  0.81
17.39  0.84 3.82  3.82
0.40  0.02 0.08  0.09
161.35  1.34 35.49  6.09
UPLC Fingerprint of CA
A fingerprint of a plant is a chromatogram that helps to identify
and characterize that plant.[20] The obtained UPLC chromato-
gram of CA (Fig. 3B) can also be a fingerprint of the plant
CA. The detectable chemical components present in the extract
are nicely separated in the chromatogram. For this reason,
the elution time was made up to 30 min, which might seem a
longer time for UPLC method. The developed UPLC chromato-
gram of CA will be highly helpful to identify and characterize
the CA.
Conclusions
A new glucoside (kaempferol-3-O-b-glucoside) and five known
flavonoids were isolated and identified. This article cites, for the
first time, the isolation of compound 3, 4, 5, and 6 from C. albo-
marginat. A reliable and acceptable UPLC method was
developed and validated and was used for the quantification
of isolated compounds. The UPLC fingerprint of CA indicated
that further research can be done on this fern to isolate other
more new and potent compounds.
Acknowledgments
We are very thankful to Dr. Hari Prasad Devkota, Kumamoto
University, Japan and Prof. Dr. Oh Hyuncheol, Wonkwang
University, Republic of Korea for their motivation and valuable
information regarding the research work. We also want to
acknowledge Mr. Lal Bahadur Thapa, Tribhuvan University,
Nepal for the valuable information regarding the plant
Cheilanthes albomarginata.
Funding
This research was supported through the Basic Science
Research Program by the National Research Foundation of
Korea (NRF-2010-0024284), under Science and Technology,
Ministry of Education.
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