Trends in Biosciences 11(46), Print : ISSN 0974-8431, 4325-4329, 2018

Qualitative and Quantitative Analysis of Cucurbitacin B–An Anti-cancer

Compound from Trichosanthes cucumerina L. subsp. cucumerina through TLC
and HPLC
J.M. DEEPA, K.K. SABU AND C.G. SUDHA*
Biotechnology and Bioinformatics Division,
Jawaharlal Nehru Tropical Botanic Garden and Research Institute
Palode,Thiruvananthapuram
email:*cgsudha@yahoo.co.in

ABSTRACT family Cucurbitaceae (Kaushik, et al., 2015). There are mainly

The present study focuses on qualitative and quantitative 40 known species of cucurbitacins and their derivatives
(Alghasham, 2013). Among the species of cucurbitacins,
analysis of cucurbitacin B (Cu B), a promising anti-cancer
Cu B is the most abundant form (Duangmano, et al., 2012).
compound in leaf, stem and fruit rind of T. cucumerina L.
Cu B has been shown to possess strong anticancer activity
subsp. cucumerina. Leaf yielded more crude extract (4.46
in vitro and in vivo against various cancers (Garg, et al.,
± 0.52 % g/dw) followed by stem (4.12 ± 0.64 % g/dw) and
fruit rind (2.28 ± 0.31% g/dw) when they extracted in 2018). Chan et al., 2010 demonstrated that Cu B effectively
restrains liver cancer xenograft through oral administration.
ethanol. Qualitative detection of Cu B was done by TLC by
Considering the pharmacological importance of Cu B and
comparing the Rf value of the compound (0.7) in the profile
its future prospects, the present study aimed to detect Cu
which was corresponding to the reference compound Cu
B, in T. cucumerina L. subsp. cucumerina through Thin
B. Quantitative detection of Cu B done by HPLC indicated
layer chromatography (TLC) and its quantification using
highest concentration of Cu B in leaf (0.19 % g/dw)
high performance liquid chromatography (HPLC). No study
followed by stem (0.12 % g/dw) and fruit rind (0.09 % g/
is reported till date for the production of Cu B in this plant.
dw). The present observations suggest that the shoots of
Trichosanthes cucumerina L. subsp. cucumerina can be MATERIALS AND METHODS
used as a suitable source for the production of Cu B or
Plant material
production of anti-cancer drug.
Field grown 4-5 months old plants of T. cucumerina
Keywords Trichosanthes cucumerina L. subsp. L. subsp. cucumerina with mature fruits from Thirunelveli
cucumerina, Cucurbitacin B, TLC, HPLC district in Tamil Nadu state were collected during January
2016. The plant was authenticated by Dr. E.S Santhosh
Trichosanthes cucumerina L. subsp. cucumerina Kumar, JNTBGRI. The Voucher specimen (TBGT- 76856)
(Cucurbitaceae) is used extensively in the Indian traditional had been deposited in the Herbarium, Division of Plant
system of medicine for curing a variety of disorders. Earlier Systematic and Evolutionary Science of JNTBGRI.
the plant is treated as variety of Trichosanthes cucumerina
L. The plant is commonly known as “Patola” in Sanskrit
and it is a bitter perennial climber with white flowers and
small green fruits with white striations when young (Fig.1).
The plant is distributed throughout India, Bangladesh, Sri
Lanka, Burma, Malaysia and Australia (Chakravarty, 1982).
The major active constituents of the drug are triterpenoids
which include cucurbitacins, saponins, flavonoids,
carotenoids, phenolic acids which makes the plant
pharmacologically as well as therapeutically active
(Rajasree, et al., 2016). The plant is reported to have anti-
bacterial (Devendra, et al., 2009), anti-fertility (Devendra,
et al., 2009), anti-inflammatory (Devendra, et al., 2010) and
hepatoprotective activity (Kumar, et al., 2009, Rajasekaran
and Periyasamy, 2012).
The bitterness of the plant is due to cucurbitacins
which exhibit a broad range of pharmacological properties.
Cucurbitacins are highly oxidized tetracyclic triterpenoid Fig. 1. Habit of Trichosanthes cucumerina L. subsp.
compounds which are commonly found in the plants of cucumerina; inset: mature fruits
4326 Trends in Biosciences 11 (46), 2018

Table 1. Yield of crude ethanolic extracts and Cu B content of T. cucurmerina L. subsp. cucumerina
Plant parts Yield of extract (% g/dw) Concentration of Cu B (% g/dw)
Leaf 4.46 ± 0.52a 0.19 % g/dwa
Stem 4.12 ± 0.64 ab 0.12 % g/dwab
c
Fruit rind 2.28 ± 0.31 0.09 % g/dwc

Values represent mean ± SD of triplicates. Means followed by the letters in the column are significantly different as indicated by Anova
(P < 0.05).

ethanol and 10l was applied onto TLC plates (Merck, 25

Preparation of crude extracts
Leaves, stems and fruit rinds of the plant were dried
at room temperature (270C ± 20C) for 4-5 days, powdered
and 5g of each sample was extracted in 50 ml absolute
ethanol under cold condition with continuous shaking at
80-90 rpm in a gyrotary shaker (Brunswick, USA) for 24
hours and the extraction was repeated thrice. The extracts
thus obtained were combined and concentrated to dryness
using rotary evaporator (Heidolph Instruments, Germany)
under reduced pressure at 400C. The crude extract of each
sample was weighed and stored in refrigerator at 40C.
Qualitative detection of Cu B through TLC
The crude extracts of plant samples and reference
compound of Cu B (Sigma-Aldrich), were dissolved in
DC- Alufolien 20 x 20 cm Kieselgel 60 F254), using fine glass
capillaries. The plates were developed in the solvent
system, Hexane: Chloroform: Methanol (5: 4.5: 0.5). After
chromatographed to a distance of 3/4th of the plates, they
were air dried and the bands were visualised under UV at
254nm. The plates were sprayed with vanillin-sulphuric acid
reagent and placed on hot plate for 10–15 seconds for the
development of clear bands. The movement of the active
compound was expressed by its retention factor (Rf) values.
Quantitative analysis of CuB by HPLC
Quantitative analysis of Cu B was done by HPLC
which was performed on a Shimadzu system (Shimadzu
Corp. Japan), attached with two pumps (LC 6 AD Pumps),

Cu B Cu B
Cu B

A B

Fig. 2. TLC profile of leaf sample of T. cucurmerinaL. subsp. cucumerina (A) viewed under UV254 nm, (B) After
spraying with vanillin-sulphuric acid reagent.

Conc.(Ratio) MeanArea
62.5 108718
125 183636
250 338368

Fig. 3. Calibration curve of reference compound of Cu B

DEEPA et al. Qualitative and Quantitative Detection of Cucurbitacin B, A promising Anti-cancer Compound 4327

Fig. 4. HPLC chromatogram of the reference compound, cucurbitacin B

Fig. 4a. HPLC chromatogram of the leaf of T. cucumerina L. subsp. cucumerina

Fig. 4b. HPLC chromatogram of the stem of T. cucumerina L. subsp. cucumerina

Fig. 4c. HPLC chromatogram of the fruit rind of T. cucumerina L. subsp. cucumerina
4328 Trends in Biosciences 11 (46), 2018
system interface (CBM-20A), C-18 column (250 x 4.6mm,
5µm particle size) and PDA Detector (SPD M20A photo
diode array detector). The compound was detected at 254nm
using the mobile phase of 100% HPLC grade methanol with
a flow rate 1ml/min and a sample size of 10l. The
concentrated extract was sonicated for 5–10min (UP 100 H
Ultraschall Processor, Germany) and filtered through a
0.22µm nylon filter, (Spincotech Pvt. Ltd.). The column
temperature was maintained at 25°C. Cu B was detected by
comparing the retention time (Rt) of the reference compound
which was 3.9 min, with the compound having the same Rt
in the specific extract. Peak area of the crude extract was
measured at the same retention time (Rt) of reference
compound. A calibration curve was prepared using different
concentrations (62.5, 125, 250, 500, 1000 µg/ml) of reference
compound Cu B.
RESULTS AND DISCUSSION
YIELD OF EXTRACT
The yield of the total extract of various samples of T.
cucurmerina L. subsp. cucumerina after the removal of
ethanol is given in Table 1. The leaf extract showed more
biomass (4.46 ± 0.52% g/dw) which was significantly
different (Pd<0.05) from fruit rind (2.28 ± 0.31% g/dw) and
stem (4.12 ± 0.64% g/dw). Extraction is an important initial
step for the recovery and isolation of bioactive compounds
from plant samples (Singh, et al., 2016). Similarly, drying
conditions of the plant samples, solvent and its
concentration are of essential importance for the extraction.
The plant samples were dried at room temperature and
absolute ethanol was used for the extraction of Cu B in the
present system. Absolute ethanol was previously used for
the isolation of cucurbitacins from Luffa operculata
(Krepsky, et al., 2009), Cu E from Ecballium elaterium
tissue culture material (Attard, 2002) and mature plants of
T. cucumerina L. var. cucumerina (Devendra, et al., 2011).
The samples were dried at room temperature (27 0C ± 20C) in
the present study, It may be ideal as the concentration of
Cu B had reduced 47% to 86% when the fruits of wild
cucumber (Cucumis myriocarpus) and wild watermelon
(Cucumis africanus) were dried at higher temperatures
above 520C (Shadung, et al., 2016).
Qualitative detection of Cu B
The TLC profile of the samples was distinctive in
bands separated when visualized under UV at 254nm, Cu B
was identified based on the bluish pink coloured band with
Rf value (0.7) was observed in the profile of the extracts
which was exactly corresponding to the reference
compound of Cu B under UV at 254nm (Fig. 2a). Bands of
the compounds turned to black after a spray with vanillin-
sulphuric acid reagent (Fig. 2b). Pink or bluish colour of the
band in TLC chromatogram under UV at 254nm is found to
be a characteristic colour of terpenoid compounds as
observed in Cucumis callosus (Panda, et al., 2018). The
compound was further confirmed by co-spotting of both
specific extract and reference compound.
Quantitative detection of Cu B
The quantitative analysis was done through HPLC
using a calibration curve of reference compound obtained
at the concentration 62.5, 125, 250 µg/ml (Fig. 3). HPLC
profile of reference compound of Cu B showed a peak at Rt
(3.99 min) (Fig, 4). HPLC profile of ethanolic extract of leaf,
stem and fruit rind showed a peak at Rt (3.99 min) which
was same as that of reference compound of Cu B (Fig.4 a.,
b., c). The analysis showed highest Cu B in leaf (0.19 % g/
dw) followed by stem (0.12 % g/dw) and fruit rind (0.09 % g/
dw) (Table 1). Previous study in Ecballium elaterium
showed low concentration of total cucurbitacin in leaf (0.34
% w/w) and higher concentration in fruit (3.84 % w/w)
(Attard and Scicluna, 2003). Similar trend was observed in
the case of total cucurbitacin in T. cucumerina L.var.
cucumerina (Devendra, et al., 2011). Pulp of the ripened
fruits is generally eaten by birds by leaving the fruit rind,
hence the part is considered for the present analysis.
Moreover, it is not worthwhile to consider whole fruits as
seeds generally contain very low concentrations of
cucurbitacins (Jorn, et al., 2006) and seed is useful for
conventional propagation.The shoots of the plant
constitute a larger portion of biomass, which is easily
harvestable and consumed by the drug industries. Since
high content of Cu B was detected in the leaf followed by
stem in T. cucumerina L. subsp. cucumerina in the present
study, the whole shoots of the plant can be used as a
suitable source for the production of Cu B.
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Received on 15-11-2018 Accepted on 04-12-2018