Journal of Chromatographic Science 2014;52:334– 338
doi:10.1093/chromsci/bmt035 Advance Access publication April 9, 2013
Simultaneous Determination by HPLC of Quercetin and Kaempferol in Three Sedum
Medicinal Plants Harvested in Different Seasons
Luyao Wang, Qing Mei and Dingrong Wan*
Pharmaceutical College, South-Central University for Nationalities, Wuhan 430074, China
*Author to whom correspondence should be addressed. Email: wandr666@163.com
Received 9 July 2012; revised 14 January 2013
A high-performance liquid chromatography method was estab-
lished for the fast quantification of quercetin and kaempferol in
three Sedum crude medicines: Sedi Herba (Sedum sarmentosum
Bunge.), Sedi Linearis Herba (Sedum lineare Thunb.) and Sedi
Emarginati Herba (Sedum emarginatum Migo.). The column used
was a YMC-pack ODS-A (250 3 4.6 mm, 5 mm), the mobile phase
was a solution of methanol– 0.4% phosphoric acid (47:53) with a
flow rate of 1.0 mL/min at 3588 C and the detection wavelength
was 360 nm. The calibration curves for quercetin and kaempferol
were linear over the range of 0.01–0.62 mg for quercetin and
0.02– 0.78 mg for kaempferol, and the average recoveries were
99.72% [relative standard deviation (RSD): 1.63% and 99.50%
(RSD: 1.16%), respectively].
In conclusion, the method established in this paper is accurate
and repeatable. It can be used for the determination of quercetin
and kaempferol, controlling the quality of the three crude drugs.
Furthermore, the experimental data showed that the best harvest
season for the three Sedum medicinal species should be
the full-bloom period between the end of April and the beginning
of May.
Introduction
Sedi Herba (Sedum sarmentosum Bunge.), Sedi Linearis
Herba (Sedum lineare Thunb.) and Sedi Emarginati Herba
(Sedum emarginatum Migo.) are three traditional Chinese
crude drugs that have been used in folk medicine to treat
hepatitis, dysentery, herpes zoster and swelling. Sedi Herba,
which possesses activities against fever and has the function of
detoxifying, has been recorded in the Chinese Pharmacopoeia
(2010) (1) because of its activities against acute or chronic
hepatitis and its effect of reducing glutamic-pyruvic transamin-
ase. In addition, Sedi Linearis Herba and Sedi Emarginati Herba
are both used in folk medicine to detoxify, treat fever and
stanch bleeding (2, 3).
According to the current state of research, the active
pharmaceutical ingredients of Sedi Herba that possess liver-
protecting activities and reduce glutamic-pyruvic transamin-
ase primarily exist in aqueous and n-butanol extracts (in
which portions the glycosides and the flavonoids principally
exist). This suggests that its medicinal properties may result
from pharmacologically active bioflavonoids (4). There are a
few reports on the determination of quercetin in granules
and capsules of Sedi Herba (5, 6), but none on the simultan-
eous determination of quercetin and kaempferol in the three
Sedum crude drugs. The more active compounds detected,
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the better the quality of the drugs can be controlled. Thus,
these two compounds of Sedum medicinal plants harvested in
different seasons were simultaneously determined in this re-
search. Moreover, a credible analytical method was developed
to control their quality, the best harvest season for the three
Sedum medicinal plants was confirmed and their relationship
was also inferred.
Experimental
Materials and methods
Materials and reagents
The standards of quercetin and kaempferol were obtained from
the National Institute for the Control of Pharmaceutical and
Biological Products in China (batch number: 100081-200406,
110861-200808). The experimental samples were three Sedum
medicinal species that were harvested in different seasons and
from different places in Hubei province: the school campus of
South-Central University for Nationalities (SCUEC, Wuhan,
Hubei province), Sheshan of Wuhan and Huangmei, as presented
in Table I. The three Sedum medicinal species were identified as
Sedum sarmentosum Bunge., S. lineare Thunb. and S. emargi-
natum Migo. by professor Dingrong Wan (Pharmaceutical
College, SCUEC). Chromatographic grade methanol was pur-
chased from Tedia (Fairfield, OH; batch number: MS1922-001)
and water was purified by an Aquapro Hi-End Water Treatment
Solution Provider (ASWO-0005-U, Ever Young Enterprises).
Phosphoric acid, concentrated hydrochloric acid and all other
chemicals were of analytical grade.
Apparatus
The high-performance liquid chromatography (HPLC) analysis
was performed on an Agilent 1200 HPLC system composed of
a quaternary pump (DE62958975), an autosampler, a column
thermostat, temperature-controlled sample trays, an online
degasser and an ultraviolet (UV) detector. The analytical column
was a YMC-Pack ODS-A column (5 mm, 250  4.6 mm).
The full wave scanning of the three Sedum medicinal plants
was performed on a UV-visible (Vis) spectrophotometer (UV-
1800PC, Mapada Co., Shanghai, China).
An Aquapro Hi-End Water Treatment Solution Provider (ASWO-
0005-U, Ever Young Enterprises) and an AP-01P Vacuum Pump
(Auto Science Company, Tianjin, China) were used to produce
and filter the ultrapure water. A rotary evaporator (R-1001N) and
a vacuum pump were used for sample preparation.
Table I
Samples and their Sources

Species Habitat Date of collection Specimen number

Sedum sarmentosum Campus of SCUEC, Wuhan May 2, 2008 080502
Jun. 13, 2009 090613
Sept. 27, 2011 110927
Sedum lineare Huangmei, Hubei May 2, 2011 110502
Oct. 7, 2009 091007
Sedum emarginatum Sheshan, Wuhan May 3, 2008 080503

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Oct. 4, 2008 081004
Dec. 1, 2008 081201 Figure 2. Content of quercetin and kaempferol as a function of refluxing time.

Pharmacopoeia, but similar to that of 2.5 h. Therefore, 2 h was

chosen as the best extraction time.
The crude drugs were crushed, passed through a 20-mesh
sieve, extracted only by methanol and analyzed. The chromato-
grams showed hardly any quercetin or kaempferol. This suggests
that both quercetin and kaempferol primarily exist as glycosides
in the three drugs and acidic hydrolysis is needed before analyz-
ing. Thus, methanol with different acidity levels was used as the
extraction solvent. An increased concentration of HCl signifi-
cantly improved the extraction efficiency, and methanol –25%
HCl (4:1) was found to be the best solvent.

Sample preparation
The dried experimental samples (approximately 2.0 g, dried at
608C) were crushed and passed through a 20-mesh sieve, accur-
ately weighed and refluxed for 2 h in 50 mL of methanol –25%
HCl (4:1). The extract was cooled and filtered and the extraction
flask was rinsed with methanol. The filtrate was combined with
the methanol rinse and diluted to a final volume of 50 mL with
methanol. Each sample was filtered through a 0.45 mm
membrane into an amber glass HPLC vial before analysis.

Optimization of chromatographic conditions

Figure 1. Chemical structures of quercetin and kaempferol A. Quercetin;
B. Kaempferol.
Methods
Preparation of standard solution
The stock standard solution of quercetin (31 mg/mL)
and kaempferol (39 mg/mL) was prepared by dissolving the
dried powders of quercetin and kaempferol (Figure 1) in metha-
nol (1, 7).
Optimization of the sample extraction
All samples were extracted via both heated reflux and ultrasonic
methods (7, 8). The results showed that refluxing was the better
method.
To determine the ideal extraction time, samples were refluxed
for 1, 1.5, 2 or 2.5 h (Figure 2). The results showed that the
extraction efficiency improved as the reflux time increased.
In addition, the extraction efficiency of 2 h was much better
than that of the regulation time (1 h) in the 2010 Chinese
Based on the results of the full wave scanning of the three
Sedum medicinal species and the absorbing wavelength of the
standard solution of quercetin and kaempferol, 360 nm was
chosen as the detection wavelength. To choose the mobile
phase, a series of solutions was studied with different ratios
of methanol– 0.4% phosphoric acid. Ultimately, the ratio of
47:53 was selected for the mobile phase with a flow rate of
1.0 mL/min. The temperature of the column was maintained at
358C and the injection volume was 20 mL.
The chromatographic conditions above were optimized to
separate the primary marker peaks of each Sedum sample
with good resolution (R . 1.5) and theoretical plate numbers
(quercetin and kaempferol: n . 8,000). The chromatograms of
the standard solution and the three Sedum medicinal plants are
shown in Figure 3.
Results and Discussion
Results
Method validation
The reliability of the HPLC method established in this paper was
proven by checking its linearity, precision, repeatability, stability
and recovery.

Simultaneous Determination by HPLC of Quercetin and Kaempferol in Three Sedum Medicinal Plants Harvested in Different Seasons 335
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Figure 3. Chromatograms of reference substances and samples A. Standard solution B. Sedi Herba (Sedum sarmentosum Bunge.) C. Sedi Linearis Herba (S. lineare Thunb.) D. Sedi
Emarginati Herba (S. emarginatum Migo.) 1. quercetin 2. kaempferol.

Table II calibration curves of each marker component indicate good

Chromatographic Peak Area Values of Different Contents for the Standard Curve of Quercetin linearity over the range under investigation.
Content (mg) 0.0155 0.0310 0.1550 0.3100 0.4650 0.6200
Peak area (s) 48.7116 126.8467 568.5184 1,125.5443 1,707.6754 2,283.0361
Precision
To measure precision, the six standard solution samples were
Table III each analyzed thrice to determine the mean. The relative stand-
Chromatographic Peak Area Values of Different Contents for the Standard Curve of Kaempferol ard deviations (RSDs) of the peak area values of the two stan-
Content (mg) 0.0195 0.0390 0.1950 0.3900 0.5850 0.7800 dards were 0.02 and 0.08% for quercetin and kaempferol,
Peak area (s) 71.6298 154.9874 807.6650 1,619.0909 2,446.2890 3,269.3787 respectively. The results showed that the method is highly
precise.

Table IV
Regression Equations and Related Parameters
Repeatability
Compound Equation Range (mg) R2 Six Sedum sarmentosum Bunge. (May) samples were taken,
Quercetin Y ¼ 3674.7x – 1.0590 0.01 –0.62 0.9999 treated according to the previously described method and
Kaempferol Y ¼ 4201.6x – 11.647 0.02 –0.78 1.0000 analyzed thrice to determine the mean. The average contents of
quercetin and kaempferol in S. sarmentosum were 1.1644 mg/g
(RSD: 0.61%) and 0.1742 mg/g (RSD: 2.79%), respectively,
Linearity indicating good repeatability.
To calculate the regression equation, six different concentra-
tions of the standard mixture solution were used in this re-
search; the results are shown in Tables II and III. The regression Stability
equations took the form Y ¼ aX þ b [the Y-axis was the value of The solution of one Sedum sarmentosum Bunge. (May) sample
the chromatographic peak area (mAU) and the X-axis was the was analyzed at 0, 2, 4, 8, 12 and 24 h after sample preparation
weight of the marker component]. The regression equations of (Table V). The RSDs of quercetin and kaempferol peak area
quercetin and kaempferol and the parameters for linearity are values were 0.83 and 0.55%, respectively. This showed that the
presented in Table IV. The high correlation coefficients of the two components in these samples were stable for at least 24 h.

336 Wang et al.

Table V
Chromatographic Peak Area Values of Quercetin and Kaempferol in Sedum sarmentosum Bunge. (May) in 24 h

Time (h) Area (s) 0 2 4 8 12 24 RSD (%)

Quercetin 1,726.1261 1,719.8047 1,706.8201 1,694.9199 1,698.3202 1,690.6211 0.83
Kaempferol 289.6402 288.3961 287.6873 286.8962 285.8619 285.4063 0.55

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Table VI Table VIII
Recovery of Quercetin in Sedum sarmentosum Bunge. (May) (n ¼ 6) Amounts of Quercetin and Kaempferol in Three Sedum Medicinal Species Harvested in Different
Seasons
Sample Quercetin in Spiked Determined Recovery Mean RSD (%)
amount the samples quercetin quercetin (%) recovery Sample/harvested season Content of quercetin Content of
(g) (mg) amount (mg) amount (mg) (%) (mg.g21) kaempferol (mg.g21)
0.5000 0.59 0.62 1.19 98.35 99.72 1.63 S. sarmentosum Bunge. (May 2) 1.1754 0.1793
0.5005 0.59 0.62 1.23 101.65 S. sarmentosum Bunge. (Jun. 13) 1.0634 0.1414
0.5004 0.59 0.62 1.22 100.83 S. sarmentosum Bunge. (Sept. 27) 0.4323 0.0553
0.5000 0.59 0.62 1.18 97.52 S. lineare Thunb. (May 2) 0.5595 0.8077
0.5003 0.59 0.62 1.20 99.17 S. lineare Thunb. (Oct. 7) 0.1547 0.0542
0.5004 0.59 0.62 1.22 100.83 S. emarginatum Migo. (May 3) 0.0275 1.4226
S. emarginatum Migo. (Oct. 4) 0.0582 0.3027
S. emarginatum Migo. (Dec. 1) 0.0733 0.3623

Table VII
Recovery of Kaempferol in Sedum sarmentosum Bunge. (May) (n ¼ 6)

Sample Kaempferol in Spiked Determined Recovery Mean RSD

amount the samples kaempferol kaempferol (%) recovery (%)
(g) (mg) amount (mg) amount (mg) (%)
0.5000 0.090 0.078 0.165 98.21 99.50 1.16
0.5005 0.090 0.078 0.170 101.19
0.5004 0.090 0.078 0.169 100.60
0.5000 0.090 0.078 0.166 98.81
0.5003 0.090 0.078 0.166 98.81
0.5004 0.090 0.078 0.167 99.40

Recovery and accuracy

The accuracy was determined by calculating the recovery after a
standard addition procedure. In this study, six precisely weighed
Sedum sarmentosum Bunge. (May) samples were refluxed
according to previously described method and analyzed by HPLC
after adding 2 mL of quercetin and 2 mL of kaempferol (concen-
trations: 0.31 and 0.039 mg/mL) to each sample. Each sample so-
lution was injected thrice and the mean and RSD were calculated.
The results for quercetin and kaempferol are presented in
Tables VI and VII and show the accuracy of the method.

Sample analysis
All Sedum samples harvested in different seasons were weighed
accurately, prepared according to the previously described
method and analyzed by injecting thrice into the HPLC. The
amounts of quercetin and kaempferol in these Sedum samples are
listed in Table VIII, and the variation with respect to the harvest
Figure 4. Amount variation with respect to season of harvest of three Sedum species
season of the three Sedum medicinal plants is shown in Figure 4. A. Sedum sarmentosum Bunge. B. S. lineare Thunb. C. S. emarginatum Migo.

Discussion
The results showed that the amounts of quercetin and kaemp-
ferol in both S. sarmentosum and S. lineare harvested at the
beginning of May were higher than in those plants harvested
in September and October, whereas only the content of kaemp-
ferol in S. emarginatum Migo. harvested at the beginning of May
was higher than that in October and December. The content of
quercetin in S. emarginatum Migo. increased in the later
harvest months. In addition, comparing the amount of quercetin
and kaempferol showed that the quercetin content was much
higher than kaempferol in all S. sarmentosum samples harvested
in different seasons, whereas the content of quercetin was lower

Simultaneous Determination by HPLC of Quercetin and Kaempferol in Three Sedum Medicinal Plants Harvested in Different Seasons 337
than kaempferol in S. lineare harvested at the beginning of May,
but higher in October. Regarding S. emarginatum in different
seasons, the quercetin content was always so much lower than
kaempferol that quercetin almost needs not be taken into con-
sideration when selecting the best harvest season. In conclusion,
the contents of quercetin and kaempferol varied in all Sedum
medicinal samples harvested in different seasons. Considering
the results published earlier on the determination of their total
flavonoids (4), it is recommended that the full-bloom period
from the end of April to the beginning of May is the best harvest
season for the three Sedum medicinal plants.
In addition, it was found that the varying amounts of quercetin
and kaempferol in S. sarmentosum and S. lineare were similar
according to harvest season, whereas both species differed
markedly from S. emarginatum. According to the preceding
analysis, and considering their morphological and anatomic
characteristics (9), it can be concluded that the genetic relation-
ship between S. sarmentosum and S. lineare is closer than that
between either species and S. emarginatum.
Acknowledgments
The author gratefully acknowledges the financial support by
Natural Science Foundation of Hubei Provence (Grant No.
2009CDB419), and Projects in the National Science and Technology
Ministry Pillar Program of China (Grant No. 2012BAI27B06).
338 Wang et al.
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