Journal of Ayurveda and Integrative Medicine xxx (xxxx) xxx

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Journal of Ayurveda and Integrative Medicine

journal homepage: http://elsevier.com/locate/jaim

Original Research Article

Determination of cucurbitacin E in some selected herbs of ayurvedic

importance through RP-HPLC
Joydeb Chanda, Sayan Biswas, Amit Kar, Pulok K. Mukherjee*
School of Natural Product Studies, Department of Pharmaceutical Technology, Jadavpur University, Kolkata, 700 032, India

a r t i c l e i n f o a b s t r a c t

Article history: Background: The consumption of the fruits of cucurbitaceae plants is widely popular among Indians due
Received 1 June 2018 to their various nutritional and medicinal purposes. Some of these plants are well reported in Ayurveda
Received in revised form due to their potential therapeutic importance. In particular, the plants of this family are well-
8 September 2018
characterized by the presence of its bitter principle, Cucurbitacin E which differs within the species
Accepted 3 January 2019
Available online xxx
due to its genetic variations.
Objectives: The objective of the study was to develop a validated RP-HPLC method for standardization in
some widely consumed cucurbits with cucurbitacin E as a marker compound.
Keywords:
Ayurveda
Materials and methods: The RP-HPLC method was developed with a reverse phase C18 column, using
Cucurbitaceae acetonitrile and water (1% glacial acetic acid) as mobile phase (70:30 v/v). The flow rate and lmax were
Cucurbitacin E optimized at 1 mL/min and 230 nm respectively. The HPLC method was validated in terms of accuracy,
Standardization specificity, sensitivity, and repeatability as per ICH guideline.
RP-HPLC Results: The calibration curve was found linear in the concentration range of 1e100 mg/mL. The % RSD of
Validation precision and recovery was found to be <2%, which confirms high repeatability of the method. The re-
sults indicated that the content of cucurbitacin E was highest (0.0663% w/w) in Cucurbita pepo whereas
Lagenaria siceraria contains the lowest (0.0356% w/w).
Conclusion: The study was able to explore the variation of cucurbitacin E content in some selected food
plants of Cucurbitaceae family. The applicability of the method can be established in nutraceutical in-
dustry for the effective quality control of cucurbits for safe human consumption.
© 2019 Transdisciplinary University, Bangalore and World Ayurveda Foundation. Publishing Services by
Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/
licenses/by-nc-nd/4.0/).

1. Introduction cucurbits are a good source of glucose, fructose, essential amino

acids, vitamins, water-soluble polysaccharides, dietary fibers,
Cucurbitaceae is a large plant family, consisting of about 125 phenolic glycosides, flavonoids, terpenoids, and minerals etc. Apart
genera and 960 species. The various parts (fruit, seeds, stems, from the diverse chemical constituents, this family is very well
leaves) of the plants belonging to the cucurbitaceae family are very characterized by their presence of cucurbitacin. Cucurbitacin con-
popular for their uses in culinary purposes from the ancient time. It sists of tetracyclic cucurbitane nucleus skeleton with a variety of
is also used in Ayurvedic and folk medicine for their several ther- oxygenation functionalities at different positions with diverse
apeutic values due to the presence of a large number of metabolites chemical categories. The cucurbitacins are present as non-
(both primary and secondary). The importance of cucurbitaceae glycosylated or glycosylated triterpenoids and divided into twelve
species has been highly recognized for effective control of lifestyle categories, incorporating cucurbitacins A-T [2]. Various biochem-
diseases such as diabetes, obesity and related disorders [1]. The ical studies suggested that cucurbitacins have a potential cytotoxic
property which is responsible for making it a prominent lead for
anti-cancer drug development [3]. The hydrophobic property of the
* Corresponding author. School of Natural Product Studies, Department of cucurbitacin nucleus is a major regulating factor for their cytotoxic
Pharmaceutical Technology Jadavpur University, Kolkata, 700032, India. Telefax: effects and it increases linearly with their hydrophobicity. In
þ91 33 24146046.
particular, cucurbitacin E (Fig. 1) and their glycosides are the most
E-mail addresses: naturalproductm@gmail.com, pulok.mukherjee@
jadavpuruniversity.in (P.K. Mukherjee). widely distributed chemical constituents in food plants of Cucur-
Peer review under responsibility of Transdisciplinary University, Bangalore. bitaceae family. Cucurbitacin E has been reported to possess anti-

https://doi.org/10.1016/j.jaim.2019.01.002
0975-9476/© 2019 Transdisciplinary University, Bangalore and World Ayurveda Foundation. Publishing Services by Elsevier B.V. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article as: Chanda J et al., Determination of cucurbitacin E in some selected herbs of ayurvedic importance through RP-HPLC,
J Ayurveda Integr Med, https://doi.org/10.1016/j.jaim.2019.01.002
2 J. Chanda et al. / Journal of Ayurveda and Integrative Medicine xxx (xxxx) xxx

Raktavikara (blood disorder), Daha (burning sensation) [18].

C. grandis is used in folklore medicine as antibacterial, hep-
atoprotective, hypoglycemic, hypolipidemic, antioxidant proper-
ties. The fruits of this plant contain Cucurbitacin B, E, taraxerone,
taraxerol, b-carotene, carotenoids, b-sitosterol, Stigma-7-en-3-one
etc. as active constituents [19]. C. pepo is also mentioned as a va-
riety of Kushmandu in Ayurveda and widely used in the treatment
of mental disorder, epilepsy, urinary disorders, diabetes etc. [20]. It
contains a large number of chemical constituents including
cucurbitacin B, cucurbitacin E, dihydrocucurbitacin, acylated
phenolic glycosides (cucurbitosides), spinasterol, b-sitosterol, pal-
mitic, palmitoleic, stearic, oleic, linoleic acids etc. [21]. In Ayurveda,
Luffa acutangula is known as Kosataki, indicated in Kustha (skin
Fig. 1. General structure of Cucurbitacin E.
disorder), Pandu (jaundice), Pliharoga (Splenic disease), Sopha
(inflammation) [22]. It has also been reported to possess several
pharmacological properties like diuretic, hepatoprotective, anti-
diabetic etc. The fruits of L.acutangula contain cucurbitacin B, E as
inflammatory [4], anti-angiogenic, immunomodulatory, cytotoxic bitter principles. The plant contains a significant amount of poly-
[5], cytostatic and hepatoprotective [6] properties in both in vitro phenols (mostly phenolic acids viz. gallic acid, p-coumaric acid,
and in vivo model. It has been observed that the combination of ferulic acid, protocatechuic acid, and its glycosides, flavonoids
cucurbitacin E with other synthetic anti-cancer drugs results in (catechin, quercetin) [23,24].
synergistic action in terms of cytotoxicity with greater efficacy in With this background, the present study was aimed to develop a
tumor growth inhibition [7]. Despite the potential therapeutic ac- validated RP-HPLC method for standardization of the selected fruits
tivity of Cucurbitacin E and cucurbitacin E glycoside, their chronic of cucurbitaceae family by using cucurbitacin E as a marker com-
exposure is undesirable due to their extremely bitter and pound. The validation of RP-HPLC method was further carried out
disagreeable taste as well as their toxicological effects found in based on the ICH guidelines. This validated method can be applied
experimental animals [8]. It has been presumed that back mutated for quantitative estimation of cucurbitacin E in the cucurbitaceae
fruits produce more toxicity and bitterness whereas the suppressor food plants and their related preparations.
gene is responsible for the absence of cucurbitacins [9].
Although a large number of gourd family plants are grown and 2. Experimental
consumed, six species namely Lagenaria siceraria, Benincasa hispida,
Momordica charantia, Coccinia grandis, Cucurbita pepo, and Luffa 2.1. Instrumentation and reagents
acutangula have potential nutraceutical benefits. The therapeutic
benefits of these plants are also well documented in Ayurveda. The RP-HPLC system (Waters, Milford, MA, USA) consisted of a
Lagenaria siceraia (Bottle gourd) is known as Tumbini or Alabu in 600 controller pump, a multiple-wavelength ultraviolet-visible
Ayurveda which is indicated in Jwara (fever), Kasa (cough), Svasa (UV-Vis) detector equipped with an in-line degasser AF 2489 and a
(respiratory distress), Visa Roga (poisoning), Sopha (inflammation/ rheodyne 7725i injector having 20 mL loop volume. Membrane fil-
swelling), Vrana (Ulcers) Sula (colic pain) [10]. It is also reported as ters (0.45 mm pore size) (Millipore) were used for filtration of the
a diuretic, cardioprotective, antihyperlipidemic, anti- mobile phase. Quantitative estimation was performed with
hyperglycemic, and antioxidant. The major bioactive constituents Empower 2 software programs using the external standard cali-
in the fruit consist of cucurbitacin B, D, E, phenolic compounds viz. bration method. Acetonitrile (HPLC grade) and glacial acetic acid
phenolic glycosides, phenolic acid, flavonoids, flavon-C-glycoside (HPLC grade) were procured from Merck (Mumbai, India). All the
such as isovitexin, isoorientin, saponarin sterols like fucosterol, other solvents (AR grade) procured from Merck. Cucurbitacin E
campesterol etc [11,12]. In Ayurveda B. hispida (Wax gourd) is (purity  95% HPLC) was purchased from Chromadex Inc. USA. All
known as kusmanda, indicated in Mutraghata (Urethritis), Prameha aqueous solutions were prepared using purified water (resistivity
(Diabetes mellitus), Ashmari (kidney stone), Manasa Vikara (psy- of 18.2 MU cm at 25  C) from a Mili-Q filtration system.
chological problems) [13]. It possesses several pharmacological
properties including antioxidant, ACE inhibitory, anti-ulcer, anti- 2.2. Extraction of plant material
inflammatory, anti-obesity, anti-diarrheal activity. The presence of
a large number of chemical constituents have been reported in this The mature fruits of L. siceraria, B. hispida, M. charantia, C.
plant viz. lupeol, sitosterol, pentacyclic triterpenes, cucurbitacin B, grandis, C. pepo, and L. acutangula were collected from local market
E, triterpenoid (isomultiflorenol), trigonelline, b-sitosterol, alka- of West Bengal, India. They were authenticated and the voucher
loids such as 5-methylcytosine, triterpenoids such as cucurbitacin specimen of all of them has been retained in the School of Natural
B, sterols, glycosides [14]. M. charantia (Bitter gourd) is known as Product Studies, Jadavpur University, Kolkata, India vide voucher
karabellak in Ayurveda indicated in Kasa (cough), Svasa (Asthma), specimen numbers SNPS-1462/2016- SNPS-1467/2016 for future
Jvara (fever), Raktavikara (blood disorder), Kamala (jaundice), references. The juice was squeezed from the fruits and then filtered
Krmiroga (helminthiasis), Kustha (skin disorder) [15] (Anonymous through Whatman no. 1 filter paper. The aqueous extract was
1999). It consists of a wide variety of chemical constituents lyophilized and stored at 20  C for further use. The % yield of the
including triterpene (cucurbitane type), protein (Polypeptide P), extracts was calculated.
steroid (diosgenin), alkaloid (vicine), inorganic and phenolic acids,
phenolic glycosides, flavonoids etc. [16]. In particular, M. charantia 2.3. RP-HPLC conditions
extract possesses potential hypocholesterolemic, antidiabetic, an-
tiobesity, antimicrobial, lipid-lowering properties [1,17]. Another The chromatographic method was developed based on the
food plant, Coccinia indica (Ivy gourd) is also known as Bimbi in previous method with some modification [25]. The RP-HPLC
Ayurveda, indicated in Kasa (cough), Svasa (Asthma), Jwara (fever), method was refined by changing the mobile phase composition

Please cite this article as: Chanda J et al., Determination of cucurbitacin E in some selected herbs of ayurvedic importance through RP-HPLC,
J Ayurveda Integr Med, https://doi.org/10.1016/j.jaim.2019.01.002
 J. Chanda et al. / Journal of Ayurveda and Integrative Medicine xxx (xxxx) xxx 3

Fig. 2. Calibration curve of Cucurbitacin E.

Table 1 was set at 230 nm for maximum absorption of the compound. A

Cucurbitacin E content in cucurbits by RP-HPLC. baseline was recorded with the optimized chromatographic
Plant name Voucher specimen no. Common Cucurbitacin E method for about 15 min prior to standard and sample injection.
name content (%w/w) Each chromatographic analysis was followed by a blank run to wash
Lagenaria siceraria SNPS-1462/2016 Bottle gourd 0.0356 out any carryover from the previous analysis.
Benincasa hispida SNPS-1463/2016 Wax gourd 0.0446
Momordica SNPS-1464/2016 Bitter gourd 0.0523
2.4. Preparation of standard and sample solutions
charantia
Coccinia grandis SNPS-1465/2016 Ivy gourd 0.0511
Cucurbita pepo SNPS-1466/2016 Pumpkin 0.0663 A standard stock solution of Cucurbitacin E was prepared by
Luffa acutangula SNPS-1467/2016 Ridge gourd 0.0556 dissolving approximately 1 mg of cucurbitacin E in 1 mL methanol.
Further dilution was carried out to prepare calibration samples in
the concentration range of 1e100 mg/mL. The sample solutions
were prepared by taking 10 mg of extract in 1 mL methanol. The
Table 2 solution was filtered through 0.45 mL syringe filter prior to
Accuracy study. injection.
Excess CuE Expected CuE Average CuE Average Recovery RSD (%)
added (ng) in extract (ng) found (ng) (%) 2.5. Method validation
0 66.3 63.21 95.35 1.25
10 77.3 74.20 95.99 0.98 The RP-HPLC method validation was carried out by determining
40 107.3 103.8 96.82 1.41 linearity, specificity, accuracy and precision, limit of quantification
80 147.3 143.2 97.23 1.05 and limit of detection on the basis of International Conference on
Harmonization guidelines [26]. Method specificity was determined
by comparing the retention time of both standard and test samples.
Sensitivity was evaluated by determining the Limit of Detection
in a gradient manner and finally, isocratic method was optimized (LOD) and Limit of Quantification (LOQ) and calculated based on the
with the mobile phase of acetonitrile (solvent A) and water (solvent equation: LOD ¼ 3.3 s/S and LOQ ¼ 10 s/S, where s is the standard
B) in the ratio of 70: 30 (v/v). The pH of the solvent B was adjusted deviation and S is the slope of the calibration curve. The standard
at 3.8 by using 1% (v/v) glacial acetic acid. The mobile phase was deviation (s) was calculated by measuring the deviations of the
filtered through a 0.45 mm pore size (Millipore) membrane filter background response of an appropriate number of blank samples
followed by sonication to degas the solvent. The separation was (n ¼ 6). The accuracy of the method was determined by the stan-
carried out on a Waters Spherisorb 5 mm ODS2 column (C18, dard addition technique and expressed in terms of % RSD for the
250"  4.6", 5 mm particle size). The temperature of the column was mean recovery of the theoretical concentration. The samples were
kept at 25  C and the injection volume was 20 mL. The total run time spiked with three different amounts of standard compounds in
was set at 10 min. The flow rate was set at 1.0 mL/min and the lmax triplicate. For estimation of spike recovery, C. pepo extract was

Table 3
Intra-day and inter-day precision study.

Intra-day (n ¼ 6) Inter-day (n ¼ 6)

RT (min) Response (AU) RT (min) Response (AU)

Mean % RSD Mean % RSD Mean % RSD Mean % RSD

4.70 0.87 4,753,208 1.20 4.68 1.50 4,593,228 1.28

4.65 1.47 7,612,069 1.30 4.55 1.17 7,292,664 1.81
4.69 1.46 16,198,361 1.25 4.70 1.10 18,105,372 1.50

Please cite this article as: Chanda J et al., Determination of cucurbitacin E in some selected herbs of ayurvedic importance through RP-HPLC,
J Ayurveda Integr Med, https://doi.org/10.1016/j.jaim.2019.01.002
4 J. Chanda et al. / Journal of Ayurveda and Integrative Medicine xxx (xxxx) xxx

Fig. 3. RP-HPLC/UV chromatogram of Cucurbitacin E standard.

Fig. 4. RP-HPLC/UV chromatogram of Lagenaria Siceraria lyophilized extract.

Fig. 5. RP-HPLC/UV chromatogram of Benincasa hispida lyophilized extract.

Please cite this article as: Chanda J et al., Determination of cucurbitacin E in some selected herbs of ayurvedic importance through RP-HPLC,
J Ayurveda Integr Med, https://doi.org/10.1016/j.jaim.2019.01.002
 J. Chanda et al. / Journal of Ayurveda and Integrative Medicine xxx (xxxx) xxx 5

Fig. 6. RP-HPLC/UV chromatogram of Momordica charantia lyophilized extract.

Fig. 7. RP-HPLC/UV chromatogram of Coccinia grandis lyophilized extract.

considered as it contains highest amount of cucurbitacin E. The 3. Results

precision of the method was assessed by injecting six replicates at
three different concentrations, LQC (low-quality control), MQC 3.1. Extraction yield
(medium quality control) and HQC (high-quality control) for both
standard and extract solutions to determine the repeatability of the The extracts were weighed and the percentage yields were
method. The intra-day precision of the assay was determined by calculated. The percentage yield (%) the aqueous extracts were
analyzing three concentrations in a day whereas the inter-day found to be 5.21, 4.08, 7.25, 5.88, 3.83, 4.2% (w/w) for L. siceraria, B.
precision was carried over three successive days by analyzing the hispida, M. charantia, C. grandis, C. pepo and L. acutangula respec-
same concentrations. The robustness of the proposed method was tively. The % yield was found the maximum for M. charantia
carried out by varying different experimental conditions viz. flow whereas C. pepo was found to be lowest.
rate, mobile phase composition, detection wavelength, column
temperature and columns of the same configuration to check their 3.2. Method validation results
influences on the retention time. Values were represented as % RSD
in both cases. System suitability test was performed by using six In RP-HPLC, the linearity range of the response was found to be
replicates of test concentrations. A variation in the number of 1e100 mg/mL. The correlation coefficient was found from the cali-
theoretical plates, capacity factor, and tailing factor was also bration curve as > 0.99, which confirms that the data is closer to the
calculated. Statistical analysis was performed using the Graph Pad line of best fit. The regression equation was found to be Y ¼ 19111X-
Prism Version 5.0. The data has been represented as the mean ± % 54747 (Fig. 2). The specificity of the proposed method confirmed no
RSD. interference among the peak of standard and test samples. The

Please cite this article as: Chanda J et al., Determination of cucurbitacin E in some selected herbs of ayurvedic importance through RP-HPLC,
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6 J. Chanda et al. / Journal of Ayurveda and Integrative Medicine xxx (xxxx) xxx

Fig. 8. RP-HPLC/UV chromatogram of Cucurbita pepo lyophilized extract.

limits of detection (LOD) and limit of quantification (LOQ) were species have been shown as L. siceraria (Fig. 4), B. hispida (Fig. 5),
estimated to be 3.45 and 8.82 mg/mL respectively, which reflect the M. charantia (Fig. 6), C. grandis (Fig. 7), C. pepo (Fig. 8) and
high sensitivity of the method. The % recovery value L. acutangula (Fig. 9).
(95.35e97.23%) indicated the good accuracy of the method
(Table 2). The % RSD of intra-day and inter-day precision was re-
4. Discussion
ported to be <2% for in cases of both peak area (response) and
retention time, which confirms high repeatability of the method
The aqueous extract of Cucurbitaceae fruits is widely used by
(Table 3). The robustness of the experimental method was found to
practitioners of Ayurveda in India and also in other systems of In-
be in the range <2%. The number of theoretical plates, capacity
dian medicine. The juice and powder of the fruits are widely mar-
factor and tailing factor were found to be 4092 (desirable > 2000),
keted as a dietary supplement. In India, the fresh juice of L. siceraria
6.72 (desirable 2e10), 1.35 (desirable < 1.5), respectively, from the
and M. charantia are consumed for their anti-obesity and anti-
mean of six determinations of test concentration.
diabetic properties [27,28]. Although cucurbitacin class of com-
pounds (specifically Cucurbitacin D & E) possesses immense
3.3. Estimation of cucurbitacin E by RP-HPLC pharmacological potential viz. antitumor, hepatoprotective, anti-
inflammatory etc. [29] (Miro, 2015), their unpredictable occur-
The content of cucurbitacin E in the lyophilized extract was rence may lead to colitis with bloody diarrhea, severe abdominal
determined using the calibration curve by plotting the mean peak cramps, vomiting, and hypotension [30]. In October 2010, Indian
area (y-axis) against the concentrations (x-axis). The study Council of Medical Research (ICMR), Ministry of Health & Family
confirmed that C. pepo contains the highest amount of cucurbitacin Welfare, Government of India conducted a pilot study on the
E (0.0663% w/w) whereas the lowest amount of was reported in adverse effects of L. siceraria after consumption of its juice. The
L. siceraria as 0.0356% (w/w). The content of cucurbitacin E in the patients were reported to have suffered from diarrhea, vomiting,
other species varied within this range. The content of cucurbitacin E elevated levels of liver enzymes and excessive ulceration in distal
was presented in Table 1. The chromatogram of standard cucurbi- oesophagus [31]. There were several other cases of cucurbit toxicity
tacin E has been shown in Fig. 3. RP-HPLC chromatograms of the six which have been reported in India as well as in other countries like

Fig. 9. RP-HPLC/UV chromatogram of Luffa acutangula lyophilized extract.

Please cite this article as: Chanda J et al., Determination of cucurbitacin E in some selected herbs of ayurvedic importance through RP-HPLC,
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 J. Chanda et al. / Journal of Ayurveda and Integrative Medicine xxx (xxxx) xxx 7

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Please cite this article as: Chanda J et al., Determination of cucurbitacin E in some selected herbs of ayurvedic importance through RP-HPLC,
J Ayurveda Integr Med, https://doi.org/10.1016/j.jaim.2019.01.002