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Isolation of a new triterpene derivative

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and in vitro and in vivo anticancer
activity of ethanolic extract from root
bark of Zizyphus nummularia Aubrev
ab a
Sarbani Dey Ray & Saikat Dewanjee
a Department of Pharmaceutical Technology, Jadavpur
University, Kolkata700 032, India
b Dr B.C. Roy College of Pharmacy & Allied Health
Sciences, Bidhannagar, Durgapur713 206, India
Published online: 25 Nov 2014.

To cite this article: Sarbani Dey Ray & Saikat Dewanjee (2014): Isolation of a new triterpene
derivative and in vitro and in vivo anticancer activity of ethanolic extract from root bark of
Zizyphus nummularia Aubrev, Natural Product Research: Formerly Natural Product Letters,
DOI: 10.1080/14786419.2014.983921
To link to this article: http://dx.doi.org/10.1080/14786419.2014.983921

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 Natural Product Research, 2014
http://dx.doi.org/10.1080/14786419.2014.983921

Isolation of a new triterpene derivative and in vitro and in vivo anticancer

activity of ethanolic extract from root bark of Zizyphus nummularia Aubrev
ab a
Sarbani Dey Ray and Saikat Dewanjee *
a
Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700 032, India; bDr B.C. Roy
College of Pharmacy & Allied Health Sciences, Bidhannagar, Durgapur 713 206, India
(Received 12 September 2014; final version received 27 October 2014)
Downloaded by [Selcuk Universitesi] at 18:25 30 January 2015

The work is designed to isolate, identify and screen for possible anticancer activity in
vitro and in vivo of the ethanolic extract and isolated identified compound from
Zizyphus nummularia root bark.

The various parts of Zizyphus nummularia has been used traditionally in several disease
conditions. However, its anticancer activity and mechanism of action remain to be
elucidated. Considering this, the objective of this study was to isolate, identify and screen
for possible anticancer activity in vitro and in vivo of the ethanolic extract (EE) and
isolated identified compound (IC) from Z. nummularia root bark. The in vitro activity
against human breast cancer, leukaemia, ovarian cancer, colon adenocarcinoma and human
kidney carcinoma cell lines was determined. The in vivo activity in female Swiss albino
mice against Ehrlich ascites carcinoma (EAC) was determined. The isolated compound is a
new triterpene derivative. EE/IC showed cytotoxicity against different cell lines. The
administration of EE/IC decreased tumour parameters such as tumour volume, viable
tumour cell count and increased body weight, haematological parameters and life span in
comparison to the EAC control mice.
Keywords: anticancer; Zizyphus nummularia; triterpene derivatives; Ehrlich ascites
carcinoma; cell lines

1. Introduction
The majority of the world’s population in developing countries still relies on herbal medicines to
meet their health needs in cases when synthetic medicine could not relieve patients who suffer from
painful illnesses such as cancer (Nadkarni 2005). In the modern system of medicine, several
chemotherapeutic agents have been developed as a result of screening of the medicinal

*Corresponding author. Email: s.dewanjee@yahoo.com

q 2014 Taylor & Francis

 2 S. Dey Ray and S. Dewanjee

plants in various parts of the world (Radha et al. 2008). However, one of the major limitations
in the currently available treatment modalities for cancer is their side effects (Joensuu 2008).
Hence, alternate treatment for cancer is being tested. Plant-derived natural products such as
flavonoids, terpenes, alkaloids and so on have received considerable attention in recent years
due to their diverse pharmacological properties including cytotoxic and cancer chemo-
preventive properties (Doughari et al. 2009; Mann et al. 2009). Therefore, there is a growing
interest in the pharmacological screening of medicinal plants.
Zizyphus nummularia Aubrev (Rhamnaceae) commonly known as Jharber in Hindi is a
most commonly occurring branched thorny shrub species in the Indian desert with a height of 1
and 2 m and light coloured bark (Rathore 2009). The leaves are antipyretic and have the
property to reduce obesity. The fruit is cooling, tonic, digestible, laxative aphrodisiac and
removes biliousness, thirst, vomiting and burning sensations (Chopra et al. 1986). The dried
fruits contain alkaloids, triterpenoids and saponins. They are used to purify the blood and aid
digestion. The fruits are also used internally in the treatment of a range of conditions including
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loss of appetite, chronic fatigue, diarrhoea, pharyngitis, bronchitis, burns, anaemia, irritability
and hysteria (Singh et al. 2002). The plant has been screened for certain pharmacological
effects, namely anthelmintic (Bachaya et al. 2009), antibacterial (De Boer et al. 2005; Nair &
Chanda 2006), and antifertility effects (Shah et al. 2009). Phytochemical reports on Z.
nummularia have revealed the presence of polysaccharides, a pectin composed of L-rhamnose,
D-galacturonic acid, D-galactose, L-arabinose, peptide alkaloids, cyclopeptides, flavonoids,
saponins, triterpenoids, fatty acids, ziziphin N, O, P and Q, and dodecaacetylprodelphinidin B3
(Bachaya et al. 2009). The compounds nummularogenin (Srivastava 1984) and zizynummin
(Sharma & Kumar 1983) have been isolated from the plant. Very few works had been carried
out on the screening of anticancer activity only on Zizyphus jujuba. One study reported that the
chloroform fraction of Z. jujuba extract induced a concentration-dependent effect on apoptosis
and a differential cell cycle arrest in HepG2 cells (Huang et al. 2007).
Considering the above-mentioned findings, this study was aimed to provide information on
the quantitative compositions of the isolated phytoconstituents and in vitro as well as in vivo
anticancer activities of the ethanol extract and the isolated identified compound (IC) from root
bark of Z. nummularia.

2. Results and discussion

The fractions 34 – 48 showed one major spot with slight impurity over TLC silica plates after
employing vanillin sulphuric acid reagent as a spraying reagent. The fractions were combined,
concentrated and washed with hexane – methanol (9:1) and the yield of the compound was 0.133 g.
The bioactive eluents were subjected to HPTLC separation employing solvent systems of ethyl
acetate – hexane (4:6) at wavelength of 450 nm, and the R f value, height, area were 0.52, 15.5 and
1
693.3, respectively. The melting point of the isolated compound was 325 – 3288C. H NMR and
13
C NMR study indicates the presence of various types of protons and carbons present in the
isolated compound. The presence of carbonyl groups is also confirmed from IR analysis. MS (FAB;
m/z): 357 [M þ 1]. From NMR study of keto – enol tautomerism of the isolated compound, it is
observed that the average chemical shift value for the enol vinyl portion was 4.563 and keto
methylene proton was 2.995. Among the keto and enol form of the compounds, the enol form is the
least polar tautomer due to the presence of intra-molecular hydrogen bonding which helps in the
reduction of dipole – dipole repulsion of carbonyl groups. But in the keto form, there is an increase
in dipole – dipole repulsion. It was also noticed that using non-polar solvents the enol content of the
system was increased (82.5% in C6D12). By using polar solvents the enol content was decreased
(6.42% in D2O). Using AM1 semi-empirical method, the dipole moment and energy after
minimisation for the enol form is 2.955 debye and 2159.85 kcal/mol, whereas for the keto form it
 Natural Product Research 3

is 4.715 debye and 2180.89 kcal/mol. On the basis of the above-mentioned findings, the isolated
compound is a new triterpene derivative having polycyclic structures with two diketone groups
which is reported for the first time. The structure is given in Figure 1. The cytotoxicity of the
ethanolic extract (EE)/IC was studied using the MTT assay in five human cancer cell lines as
compared with standard drug tamoxifen. From Table 1, it is observed that the IC showed high
cytotoxicity in all cell lines in comparison to EE. But the activity of IC is lower than tamoxifen. The
LD50 value of EE and IC was evaluated in female Swiss albino mice and found to be 3025 and 2345
mg/kg b.w., respectively. Anticancer activity of EE and IC against Ehrlich ascites carcinoma (EAC)
cell-bearing mice was assessed by the parameters such as viable EAC cell (% inhibition in cell
growth), mean survival time (MST), percentage (%) increase of life span (% ILS) and tumour
volume. The average number of viable tumour cells per mouse of tumour control group was found
to be 8.74 ^ 0.41 cells/mL. Treatment with EE (100 and 200 mg/kg) and IC (50 and 100 mg/kg)
decreased the viable cells significantly (P , 0.05) (Table 2). It was also observed that the MST for
the control group was 17 days, whereas it was 22, 30, 35, 40 and 43 (P , 0.05) days for the groups
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treated with EE (100 and 200 mg/kg), IC (50 and 100 mg/kg) and mitomycin C (1 mg/kg),
respectively. Among the test samples, the highest increase in the life span of tumour-bearing mice
treated with IC (100 mg/kg) was found to be 77.63% as compared with the control group whereas it
was 96.97% for mitomycin C (1 mg/kg).
Ascitic fluid is the direct nutritional source for cancer cells and a rapid increase in ascitic
fluid with cancer growth would be a means to meet the nutritional requirement of cancer cells
(Shimizu et al. 2004; Ding et al. 2011; Jin et al. 2011; Mathan et al. 2011; Osman et al. 2011;
Wang et al. 2011; Yuan et al. 2011). EE and IC by decreasing the nutritional fluid volume and
arresting the cancer growth increase the life span of EAC-bearing mice. In this investigation,
treatment with EE and IC decreased the viable cancer cell count and increased the total cell
count of the cancer-bearing mice. The reliable criteria for judging the value of any anticancer
drug are the prolongation of the life span of animals (Bala et al. 2010). Thus, EE and IC have
potent anticancer activity against EAC-bearing mice. It can be observed from Figure 2 that
haemoglobin content, white blood cells (WBC) and red blood cells (RBC) count of cancer-
bearing mice on day 10 showed significant changes when compared with the normal mice.

Figure 1. Structure of identified new triterpene derivative.

 4 S. Dey Ray and S. Dewanjee

Table 1. In vitro anticancer activity (IC 50) (mg/ml for EE and mM for IC and tamoxifen) of EE/IC against
human breast cancer (MCF-7), leukaemia (K-562), ovarian cancer (OVCAR-3), human colon
adenocarcinoma (HT-29) and human kidney carcinoma (A-498).
Human colon Human kidney
Human breast Leukaemia Ovarian cancer adenocarcinoma carcinoma
Compound cancer (MCF-7) (K-562) (OVCAR-3) (HT-29) (A-498)
EE 20.71 ^ 0.012* 19.21 ^ 0.027* 15.35 ^ 0.044* 26.42 ^ 0.011* 24.45 ^ 0.006*
IC 11.24 ^ 0.001* 11.17 ^ 0.062* 11.51 ^ 0.04* 14.65 ^ 0.08* 15.27 ^ 0.05*
Tamoxifen 8.21 ^ 0.004* 10.44 ^ 0.01* 9.30 ^ 0.021* 12.25 ^ 0.012* 11.22 ^ 0.02*
(standard)

Note: Both standard and test (EE/IC) are maintained in triplicate. *p , 0.05 compared with tamoxifen (standard).

The total WBC count was found to decrease with an increase in the haemoglobin content and
RBC count. The serum biochemical parameters were restored to normal levels comparable to
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saline control indicating protection from the cancer cell-induced hepatotoxicity by EE and IC
(Saha et al. 2011). The effects of EE and IC on different serum biochemical parameters are
shown in Figure 3. Mice of EAC control group showed significant (P , 0.05) decrease in the
activities of serum glutamic pyruvic transaminase (SGPT), serum glutamic oxaloacetic
transaminase (SGOT) and alkaline phosphatase (ALP) when compared with the respective
normal values. In our study, 9 days of inoculation with EAC brought the significant elevation
in the levels of SGPT, SGOT and ALP. Treatment with EE and IC restored the elevated
biochemical parameters more or less to normal range thereby indicating the protective effect on
the cancer-induced complications.
It was reported that the reduction in free radicals reduces the cancer risk using free radical
reaction inhibitors (antioxidants) (Black 2004). In our case, the isolated compound contains
two diketone moieties present in the triterpene derivative skeleton. The hydrogen atom attached
to the vicinal carbon atom (positions 1, 4, 13 and 16) relative to carbonyl groups are active
hydrogen as they are alpha to an electronegative group (here carbonyl group). In general, free
radical-scavenging reactions need the antioxidant to donate an electron or an active hydrogen
atom such as one in reactive hydroxyl group. But it was reported earlier that ecdysteroids,
which do not have active hydroxyl groups, are also active antioxidants and free radical
scavengers (Cai et al. 2002). They exert the activity by donating the active hydrogen. Since the
extract contains octadecahydro-picene-2,3-14-15-tetranone as one of the molecule possessing
eight active hydrogen at positions 1, 4, 13 and 16, the EE and IC may exhibit the anticancer
activity by inhibiting free radicals due to donation of the active hydrogen. The presence of
diketone moiety and the triterpene skeleton may be responsible for the activity.

Table 2. Effect of EE and IC on survival time, increased life span, tumour volume, viable cell count and
nonviable cell count of EAC-bearing mice (n ¼ 10).
EAC EE EE IC IC Mitomycin
Parameters control (100 mg/kg) (200 mg/kg) (50 mg/kg) (100 mg/kg) C (1 mg/kg)
Tumour volume (mL) 3.9 ^ 0.27 2.86 ^ 0.43 2.11 ^ 0.22 1.58 ^ 0.23 1.20 ^ 0.11 0.66 ^ 0.17
MST (days) 17 22 30 35 40 43
ILS (%) 0 39.62 53.60 59.14 77.63 96.97
Viable cell ( £ 107) 7 8.74 ^ 0.41 2.71 ^ 0.16 1.73 ^ 0.06 1.41 ^ 0.16 1.22 ^ 0.51 0.95 ^ 0.06
Nonviable cell ( £ 10 ) 0.23 ^ 0.04 1.7 ^ 0.05 1.9 ^ 0.04 2.33 ^ 0.05 2.64 ^ 0.18 2.93 ^ 0.06
7
Total cell ( £ 10 ) 8.73 ^ 0.03 3.22 ^ 0.04 3.41 ^ 0.18 3.55 ^ 0.03 3.65 ^ 0.06 3.79 ^ 0.03
Viable (%) 96.98 61.67 55.78 41.92 33.72 25.84
Nonviable (%) 4.92 39.43 44.47 62.73 68.37 78.25

Note: Experimental groups are compared with EAC control groups ( p , 0.05).
 Natural Product Research 5
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Figure 2. Effect of EE and IC on haematological parameters of EAC cell-bearing mice (n ¼ 10).

Experimental groups are compared with EAC control group (P , 0.05).

3. Experimental
Animals: All the experiments were carried out in accordance with the institutional animal
ethical committee of Dr B.C. Roy College of Pharmacy and A.H.S. (BCRCP/IAEC/8/2012).
Female Swiss albino mice (weighing 20 ^ 2 g, 8 weeks old) were housed in cages at an ambient
temperature of 21 ^ 18C with 50 – 60% relative humidity in a 12 h light/dark cycle. They had
free access to the standard pellet diet and drinking water during the experiments.
Plant material: Root bark of Z. nummularia were collected in September 2010 from
Durgapur, India and authenticated by the Taxonomists of Botanical Survey of India [Ref. CNH/
I-I/20/2010/Tech.II/171]. A voucher specimen (BCRCP/DP/PT/02/06) has been deposited at
our laboratory for future reference.

Figure 3. Effect of EE and IC on different biochemical parameters in EAC-treated mice (n ¼ 10).

Experimental groups are compared with EAC control group (P , 0.05).
 6 S. Dey Ray and S. Dewanjee

3.1. Extraction, isolation and identification

The root barks were shade dried at room temperature, pulverised in an electrical grinder and
macerated with 70% ethanol and occasional shaking for 1 week. The extract was filtered to remove
particulate matter and concentrated at low heat. Finally, the concentrated crude extract was
lyophilised to obtain the lyophilised powder. For isolation, 50.10 g of crude extract was again
dissolved in 200 mL methanol then stored for 1 day at room temperature. The methanol was
removed to obtain a dirty white colour solid which on further wash with petroleum ether yields a
white powder. The yield of the final extract was 3.79 g. Initially, an admixture was prepared with the
final extract with silica gel (60 – 120 mesh) and packed in silica gel (60 – 120 mesh) column and
eluted in hexane initially. Finally, the column was eluted with an increasing solvent polarity from
hexane to ethyl acetate. Several fractions were then subjected to TLC analysis and sprayed with
various reagents for initial identification of phytoconstituents. The HPTLC analysis was carried out
using CAMAG HPTLC. Melting point of the isolated compound was measured on a capillary
melting point apparatus and was uncorrected. The proton and carbon magnetic resonance spectra
1 13
( H NMR, C NMR) were recorded on a Bruker 400 MHz instrument (Bruker, Karlsruhe,
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Germany). The infrared spectra of compounds were recorded in KBr on Fourier Transform (FTIR-
8400S, Shimadzu, Kyoto, Japan) infrared spectrophotometer. Mass spectra were recorded on LC –
MS/MS (API-4000 TM, Applied BioSystems, MDS SCIEX, Ontario, Canada). Elemental analyses
were performed on a Perkin-Elmer model 2400 Series II analyser (Perkin Elmer, Waltham, MA,
USA). For keto – enol tautomerism, four different solvents such as CDCl 3, C6D12, DMSO-d6 and
D2O were used. The dipole moment and energy at AM1 semi-empirical level were calculated by CS
MOPAC pro under CS Chemoffice software package (Chemoffice 1999). The elemental analysis
(%) of the isolated compound (C 22H28O4) is as follows (calculated: C, 74.13; H, 7.92; O, 17.95;
found: C, 74.20; H, 7.90; O, 17.89). The isolated compound showed the following spectral
1
characteristics. H NMR (CDCl3): d 2.195 (d, 2H, ZCH2Z attached to 1-a CvO and 1 b CvO,
position no. 1, 4, 13 and 16), 1.629 (s, H, ZCHZ attached to 1 b CvO, position no. 5, 22, 12 and 17),
1.275 (s, 1H, ZCHZ, position no. 8, 21, 9 and 18), 1.418 (d, 2H, ZCH2Z, position no. 6, 7, 10, 11, 19
13
and 20). C NMR (CDCl3): d 207.012 (1-carbonyl relative to
1 ZCZCZand another ZCvO, position no. 2, 3, 14 and 15), 30.932 (CH of cyclohexane relative
to 1-b C (vO)ZCZ, position no. 5, 22, 12 and 17), 29.701 (CH of cyclohexane relative to
21
ZCHZCH2Z, position no. 8, 21, 9 and 18). IR (KBr, cm ), 1642, 1687 (CvO str), 1453 (CZH
str of OvCZCH2), 2869 and 2942 (CZH str of ZCH2ZCH2Z).

3.2. In vitro anticancer activity

Five different cell lines, namely human breast cancer (MCF-7), leukaemia (K-562), ovarian
cancer (OVCAR-3), human colon adenocarcinoma (HT-29) and human kidney carcinoma (A-
498) cancer cells, were obtained from the National Centre for Cell Sciences (Pune, India) and
used for the assay. The cultures were maintained in Dulbecco’s modified eagle’s medium
containing 10% heat inactivated foetal bovine serum, penicillin (100 units/mL) and streptomycin
2
(100 mg/mL) at 378C in 5% CO2. Cells were grown in 25 cm tissue culture flasks until confluent
and used for cytotoxicity assays. The cytotoxicity of the EE/IC was determined by MTT assay
(Mosmann 1983; Holbeck 2004). Tamoxifen is used as standard drug. It was purchased from Fisher
Scientific, Waltham, MA, USA, possessing a purity of 99.24%.

3.3 In vivo anticancer activity

EAC cells were obtained by the courtesy of Indian Institute for Chemical Biology, Kolkata,
6
India and were maintained by peritoneal transplantation of 2 £ 10 cells per mouse every 10
days. Ascitic fluid was drawn from cancer-bearing mice at the log phase (days 7 and 8 of
 Natural Product Research 7

cancer bearing) of the cancer cells. Mitomycin C is used as standard drug. It was purchased
from Fisher Scientific, possessing purity greater than 98.0%.

3.4. Acute toxicity study

For the determination of LD 50 of the EE and IC, initially pilot study was conducted on two
groups (10 animals in each group) of mice to select the dose ranges for subsequent study. For
LD50 estimation, there were five groups and in each group 10 female Swiss albino mice were
taken. Five different doses (mg/kg body weight) of EE/IC were administered intraperitoneally
to animals. All 10 animals in a particular group received same and one dose of EE/IC. Lethality
of EE/IC was examined by injecting EE/IC intraperitoneally into female albino mice. The
percentage of mortality at different doses after 24 h was calculated and the values were
transformed into probit scale and LD50 for EE/IC was calculated (Lorke 1983).
Treatment schedule: Seventy female Swiss albino mice were divided into seven groups (n
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6
¼ 10). Each animal of all groups except Group I received EAC cells (2 £ 10 cells/mouse i.p.)
and this was taken as the zeroth day. Group I served as saline control (5 mL/kg 0.9% NaCl w/v
i. p.). Group II served as EAC control. Twenty-four hours after EAC transplantation, Groups III
and IV received EE (200 and 400 mg/kg bw, i.p.), Groups V and VI received IC (50 and 100
mg/ kg bw, i.p.) and Group VII received reference drug mitomycin C (1 mg/kg, bw, i.p.) daily
for nine consecutive days (Samanta et al. 2004). Twenty-four hours of last dose and 18 h of
fasting, six animals of each group were sacrificed by carbon dioxide asphyxiation followed by
cervical dislocation for the measurement of cancer and biochemical parameters, and the rest
were maintained with food and water ad libitum to check the percentage increase in life span.
Blood was collected by cardiac puncture for haematological and biochemical estimations. The
anticancer activity of the EE and IC was measured in EAC animals with respect to the
following parameters such as tumour volume, %ILS, cancer cell count, viable/nonviable
tumour cell count (Bala et al. 2010). The details are given in the Supplementary section.

3.5. Statistical analysis

All data are expressed as mean ^ SEM. The significance of the values is checked by Student’s t-test.

4. Conclusion
A new compound was isolated and its structure was identified by chromatographic and spectral
techniques. The new IC is a new triterpene derivative having polycyclic structures with two
diketone groups. According to the results of this study, it was clearly indicated that ethanol
extract and the new IC of Z. nummularia had significant anticancer activity against five
different cell lines in vitro as well as against EAC in vivo. Administration of EE and IC could
significantly increase the life span, haemoglobin content of RBC and total cell count of the
infected mice. In addition to this, EE and IC of Z. nummularia show decrease in viable and
nonviable cell count and SGOT, SGPT and ALP values. The pronounced anticancer activity of
both EE and IC of Z. nummularia was possibly due to the compound having triterpene nucleus
with active hydrogen atom attached to vicinal carbonyl group which neutralises free radicals.
These results suggested that ethanol extract and IC of Z. nummularia had potent anticancer
activity and could be utilised as a new natural anticancer agent in various areas of therapy.

Acknowledgements
One of the authors (S.D.R.) wishes to acknowledge the authority of Dr B.C. Roy College of Pharmacy &
A.H.S. for providing the necessary facility for carrying out the animal experiment.
 8 S. Dey Ray and S. Dewanjee

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