Journal of Chromatographic Science, Vol.

46, February 2008

Determination and Quantitation of Five Cucurbitane

Triterpenoids in Momordica charantia by Reversed-Phase
High-Performance Liquid Chromatography with
Evaporative Light Scattering Detection

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Yan-Hong Wang1, Bharathi Avula1, Yi Liu1, and Ikhlas A. Khan1,2,*
1NationalCenter for Natural Products Research, Research Institute of Pharmaceutical Sciences; 2Department of Pharmacognosy, School of
Pharmacy, The University of Mississippi, MS 38677

Abstract 5β, 19-epoxy-3β, 25-dihydroxycurubita-6, 23 (E)-dienen had sig-

nificant antidiabetic activity in vivo (3) and lipid abnormalities in
A simple and specific analytical method for the quantitative HepG2 cells (7). Cucurbitane-type triterpenoids and related gly-
determination of five cucurbitane-type triterpenoids isolated cosides are the major chemical constituents in this plant and
from the fruit of Momordica charantia is developed. The exist in the fruits, seeds, leaves, and vines of M. charantia (8–11).
triterpenoids present in the fruits of Momordica charantia are The antidiabetic and antitumor activity of M. charantia has
separated with an acetonitrile (0.1% acetic acid)–water (0.1%
aroused interests in its chemical analysis and in the development
acetic acid)–methanol (0.1% acetic acid) gradient at a flow rate
of 0.5 mL/min. The high-performance liquid chromatography
of dietary supplement products. Therefore, it is necessary to
separation was performed on a Phenomenex C18 reversed-phase develop analytical methods for the identification of M. charantia
column. By using an evaporative light scattering detector, plant material, for the quality assurance of dietary products, as
the main triterpenoids of Momordica charantia could be detected well as the chemical fingerprinting of M. charantia. To date,
at levels as low as 10 µg/mL. The method was validated for there is no such method reported for the analysis of cucurbitane
precision, repeatability, and accuracy. The relative standard triterpenoids in this plant by LC–ELSD, except for the analysis of
deviation was between 0.6–4.4%. The method was sensitive, quick, a single compound, momordicoside A by Shui Wang (12). It is
and accurate for the determination of main triterpenes and important to note that momordicoside A occurs widely in plants
saponins in Momordica charantia, and can be of Cucurbitaceae family. In our current investigation, we have
used for quality control of Momordica charantia and its related developed and present a reliable and sensitive method for the
dietary supplements.
identification and quantification of five major cucurbitane-type
compounds (Momordicoside A, 1; Momordicoside L, 2;
Introduction Momordicoside F2, 3; Momordicoside, K., 4; and 3β,7β,25-trihy-
droxy cucurbita-5, (25E)-dien-19al, 5) (Figure 1) from the fruits
of M. charantia utilizing liquid chromatography–evaporative
Bitter melon, Momordica charantia L. (Cucurbitaceae), is a light scattering detector (LC–ELSD).
widely cultivated plant and grows in tropical areas, including
Asia, East Africa, the Caribbean, and South America. The fruits of
this plant have been used not only as a vegetable, but also as tra-
ditional medicine for the treatment of bitter stomachic, as a lax- Experimental
ative, an anti-diabetic, an anthelmintic in children, treatment of
feverish conditions, and an antiviral for both measles and hep- Plant material and chemicals
atitis (1). Various studies have shown that the saponin fraction of The standard compounds 1–5 were isolated at the National
Momordica charantia inhibits the increase of blood glucose and Center for Natural Products Research (NCNPR), the identity and
serum neutral fat (2–6). One recently published investigation purity was confirmed by chromatographic (TLC, HPLC)
indicated that the major cucurbitane triterpenoids from its dried methods, by the analysis of the spectral data (IR, 1D- and 2D-
fruits, 3β, 7β-25-trihydroxycucurbita-5-23 (E)-dien-19-al and NMR, HR-ESI-MS) and comparison with published spectral data
* Author to whom correspondence should be addressed.
(8,9). The percent purity of compounds 1–5 are 98.2%, 92.5%,

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 Journal of Chromatographic Science, Vol. 46, February 2008

87.2%, 96.4%, and 99.1%, respectively. Acetonitrile, methanol, 25% B, 65% C for 15 min. Total run time was 43 min. The ELS
water, and glacial acetic acid were HPLC grade purchased from detector was adjusted to 40°C, at a gain of 11 and with a nitrogen
Fisher Scientific (Fair Lawn, NJ). Plant materials studied were pressure of 2.1 bar.
from 3 different populations of M. charantia (MC-1 to MC-3)
(Voucher numbers: 2774, 2945, 3190) procured from India. Standard Preparation
Voucher specimens of all samples are deposited at the National One milligram of each standard compound was dissolved in
Center for Natural Products Research (NCNPR), The University 1.0 mL methanol (stock solution). Five additional calibration
of Mississippi, Mississippi. levels were prepared by diluting the stock solution with
methanol. Within the range of concentrations injected
Chromatographic instrument and conditions (800.0–50.0 µg/mL for compounds 1, 3, and 4, 1000–125 µg/mL
HPLC experiments were performed on a Waters 2695 Alliance for compound 2 and 500.0–30.0 µg/mL for compound 5, respec-
Separations Module (Waters, Milford, MA) connected with a tively) the detector response is a function of the mass and follows
Sedex 55 ELS detector (Sedex, Alfortville, France) using a an exponential relationship (the log of response versus log of
Gemini column (150 × 4.6 mm; 5 µm particle size) from

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concentration is linear).
Phenomenex (Torrance, CA), and maintained at 25°C. The
mobile phase consists of methanol (A), acetonitrile (B), and Sample preparation
water (C), all containing 0.1% acetic acid, which were applied in Finely powdered dried plant materials (1.0 g) of M. charantia
the following gradient elution: 0–5 min, 10% A, 25% B, 65% C; samples were sonicated in 5.0 mL of methanol–water (90:10, v/v)
5–36 min, 10% A, 25% B, 65% C to 4% A, 70% B, 26% C; 36–38 at 35°C for 25 min, followed by centrifugation for 15 min at 9000
min, 4% A, 70% B, 26% C to 100% B, then held for 5 min. Each rpm. The supernatant was transferred into a 50-mL flask. The
run was followed by a 5 min washing procedure with 100% ace- procedure was repeated four times. The combined supernatants
tonitrile. The flow rate was adjusted to 0.5 mL/min, and the were evaporated under nitrogen gas utill the volume was less
injection volume was 10 µL. Re-equilibration was with 10% A, than 6 mL. The concentrated extracts were moved into a 10.0-
mL volumetric flask, and the previous flask was
rinsed with 3 mL methanol–water (90:10, v/v).
The rinsing solvents were combined with the con-
centrated extracts. The final volume was adjusted
to 10.0 mL with methanol–water (90:10, v/v) and
mixed thoroughly.
Prior to injection, an adequate volume (ca. 2
mL) was passed through a 0.45-µm nylon mem-
brane filter. The first 1.0 mL was discarded, and
the remaining volume was collected in an HPLC
sample vial. Each sample solution was injected in
triplicate.

Precision
Precision (intra- and inter-day assay) of the
method was determined by analyzing five indi-
vidual samples of M. charantia on three consecu-
tive days. The samples were extracted and assayed
Figure 1. Structure of standard compounds (1–5). under optimized conditions (Table I).

Table I. Calibration Data, Range, LOD, LOQ for Compounds 1–5 and Content of Triterpenoids by Intra- and Inter-Day
Precision of One Sample (MC-1, M. charantia) Assayed Under Optimized Conditions*

Intra-Day (n = 5)
Regression Concentration LOD LOQ Inter-Day
Analyte Equation Range(µg/mL) r2 (µg/mL) (µg/mL) Day 1 Day 2 Day 3 (n = 15)

1 Y = 1.75 e + 000X – 5.02 e-001 50–800 0.999 30 50 0.234 (2.31) 0.235 (0.63) 0.234 (2.19) 0.234 (0.93)
2 Y = 1.61 e + 000X – 5.11 e-001 125–1000 0.998 60 110 0.152 (3.73) 0.154 (3.74) 0.152 (4.40) 0.152 (0.38)
3 Y = 1.73 e + 000X – 3.14 e-001 50–800 0.999 30 50 0.085 (2.42) 0.088 (3.64) 0.086 (3.31) 0.086 (0.63)
4 Y = 1.61 e + 000X – 1.57 e-001 50–800 0.996 30 50 0.110 (2.98) 0.110 (3.96) 0.111 (4.28) 0.110 (0.68)
5 Y = 1.84 e + 000X + 1.84 e-001 30–500 0.998 10 30 0.036 (0.76) 0.033 (2.08) 0.036 (2.09) 0.035 (0.77)

* Calibration data = regression equation and correlation coefficient (r 2); Content = Values in mg/100 mg of dry plant material weight; % relative standard deviations are given in parentheses.

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Journal of Chromatographic Science, Vol. 46, February 2008

Recovery M. charantia preparations are available throughout the United

The accuracy of the method was determined by analyzing the States as dietary supplements. According to USP, test procedures
percentage recovery of the main constituents in extract of M. cha- for the assessment of the quality of pharmaceutical products
rantia. The sample (MC-1) was spiked with 0.1 mL of standard require the determination of certain analytical parameters like
stock solution (1.0 mg/mL) containing compounds 1–5. The accuracy, precision, peak purity, linearity, and limit of detection.
spiked sample was extracted and assayed under optimized condi- The accuracy of our method was confirmed by determining the
tions. recovery. One sample (MC-1) was spiked with known amounts of
the standard compounds 1–5. Compared to the theoretical
amounts, the recovery rates were found to be 98.2% for 1,
Results 103.2% for 2, 98.3% for 3, 97.3% for 4, and 102.9% for 5. An indi-
cator for precision is the relative standard deviation (RSD). All
samples were injected in triplicate, and the standard deviation of
To demonstrate the feasibility of our method, we analyzed
compounds 1–5 was below 1.5% for all samples. Intra- and inter-
three populations of M. charantia. The fruit extracts (MC-1 to

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day variation of the assay was determined and showed to be lower
MC-3) were analyzed using LC–ELSD, and the relative concen-
than 4.5%, with a maximum RSD of 4.39% (Table I). Peak purity
trations of triterpenoids are shown in Table II. Of the three pop-
and identity were verified by studying PDA and ELS data, as well
ulations of plant materials, only the MC-1 sample provided a
by spiking samples with reference compound and by comparing
clear assignment of all five triterpenoids. Compound 1 was not
their retention times. No indications of impurities were found.
detected in MC-2 and MC-3 fruit extracts. Furthermore,
Calibration data indicated the linearity of the detector response
Compound 5 was not detected in the MC-3 plant sample.
for standard compounds 1, 3, 4 from 800.0 to 50.0 µg/mL, for
compound 2 from 1000–125 µg/mL, and for compound 5 from
500.0 to 30.0 µg/mL. Table I shows the calibration data, calcu-
Discussion
Table II. Content of Triterpenoids Found in Three
Optimal chromatographic conditions were obtained after run- Populations of M. charantia
ning different mobile phases with a reversed phase C18 column.
The different columns tried were: Gemini C18, Lichrosphere 5 Three Populations of M. charantia*
RP 18, Luna C18, Luna Phenyl-Hexyl, and Synergi POLAR-RP.
Analyte MC-1 MC-2 MC-3
The best results were observed with the Gemini C18 column
using water, acetonitrile, and methanol, all containing 0.1% 1 0.234 ND† ND
acetic acid as mobile phase. The optimal separation temperature 2 0.152 0.437 DUL
for this method was determined to be 25°C with a flow rate of 0.5 3 0.086 0.194 DUL
mL/min. Increasing the column temperature to 30°C or more 4 0.110 DUL DUL
reduced the sensitivity of peaks remarkably. By LC–ELSD, the 5 0.035 DUL ND
gradient was changed to a slightly concave one that enhanced
* Values in mg/100 mg of dry plant material weight.
the resolution. † ND = not detected; DUL = detected under limits of quantitation.

The separation of a standard mixture containing five cucurbi-

tane triterpenoids is shown in Figure 2. The com-
pounds were isolated from M. charantia (for peak
assignments see Figure 2, for corresponding
structures see Figure 1) and represent the major
triterpenoids of this species. By using a
methanol– acetonitrile–water gradient as eluent
and reversed phase C-18 material as stationary
phase, a runtime of less than 43 min was required
to separate all five triterpenoids because
momordicoside F2, momordicoside K, and 3β,
7β, 25-trihydroxycucurbita-5,(23E)-dien-19-al
held the similar structures and retention times.
For a successful separation of triterpenoids iso-
lated from M. charantia, not only did the separa-
tion conditions need to be carefully investigated,
but also the method of detection. As these com-
pounds are not sensitive to UV absorption, an
ELS detector is considered a better alternative
because this method of detection is based on Figure 2. A typical HPLC–ELS chromatogram of standard compounds (1–5) (A), three different popu-
mass and not UV absorbance, making these com- lations of Momordica charantia (B–D).
pounds easily detectable.

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 Journal of Chromatographic Science, Vol. 46, February 2008

lated limit of detection and limit of quantitation (the limit of 3. H. Liva, M. Tanaka, S. Takaoka, M. Oda, O. Mogami, M. Uchida,
detection and the limit of quantitation were determined by serial and Y. Asakawa. Momordica charantia constituents and antidiabetic
screening of the isolated major compounds. Chem. Pharm. Bull.
dilution based on a signal to noise ratio of 5:1 and of 10:1, respec- 54(7): 1017–1021 (2006).
tively). 4. I. Ahmed, E. Adeghate, E. Cummings, A. K. Sharma, and J. Singh.
Beneficial effects and mechanism of action of Momordica charantia
juice in the treatment of streptozotocin-induced diabetes mellitus in
rat. Mol. Cell. Biochem. 261(1&2): 63–70 (2004).
Conclusions 5. A. Tongia, S. K. Tongia, and M. Dave. Phytochemical determination
and extraction of Momordica charantia fruit and its hypoglycemic
potentiation of oral hypoglycemic drugs in diabetes mellitus
The HPLC–ELSD method described in this paper is the first (NIDDM). Indian Journal of Physiology and Pharmacology 48(2):
detailed report of an analytical method capable of determining 241–244 (2004).
five main triterpene and triterpenoid glycosides of M. charantia. 6. T. Miura, Y. Itoh, N. Iwamoto, M. Kato, and T. Ishida. Suppressive
activity of the fruit of Momordica charantia with exercise on blood
The developed method allows a reliable and accurate qualitative glucose in type 2 diabetic mice. Biol. Pharm. Bull. 27(2): 248-250

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and quantitative analysis of different populations of M. cha- (2004).
rantia. It fulfills the requirements of a validated method. The 7. P. V. Nerurkar, Y.K. Lee, E. H. Linden, S. Lim, L. Pearson, J. Frank, and
method described in this paper can be applied as an analytical V. R. Nerurkar. Lipid lowering effects of Momordica charantia (Bitter
tool for determining the authenticity of M. charantia plant mate- Melon) in HIV-1-protease inhibitor-treated human hepatoma cells,
HepG2. Br. J. Pharmacol. 148(8): 1156–1164 (2006).
rial and will assist in the quality and safety assessment of com- 8. H. Okabe, Y. Miyahara and T. Yamauchi. Structures of momordico-
mercial botanical products. sides F1, F2, G, I, K, and L, novel cucurbitacins in the fruits of
Momordica charantia L. Tetrahedron Lett., 23(1): 77–80 (1982).
9. H. Okabe, Y. Miyahara, T. Yamauchi, K. Miyahara and T. Kawasaki.
Studies on the constituents of Momordica charantia L. I. Isolation
Acknowledgments and characterization of momordicosides A and B, glycosides of a
pentahydroxy-cucurbitane triterpene. Chem. Pharm. Bull., 28(9):
2753–2762 (1980).
This research was funded in part by “Botanical Dietary 10. Y. Miyahara, H. Okabe and T. Yamauchi. Studies on the constituents
Supplements: Science-Base for Authentication” funded by the of Momordica charantia L. II. Isolation and characterization of
Food and Drug Administration grant number FD-U-002071-01. minor seed glycosides, momordicosides C, D and E. Chem. Pharm.
Bull., 29(6): 1561–1566 (1981).
The authors would like to thank Dr. Troy Smillie for his contin- 11. C.I. Chang, C.R. Chen, Y.W. Liao, H.L. Cheng, Y.C. Chen and C.H.
uous guidance and support. Chou. Cucurbitane-type triterpenoids from Momordica charantia.
J. Nat. Prod., 69: 1168–1171 (2006).
12. S. Wang, L. Tang, Y. Guo, F. Yan and F. Chen. Determination of
momordicoside A in bitter melon by high-performance liquid chro-
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