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Estimation of camptothecin and pharmacological evaluation of Ophiorrhiza

prostrata D. Don and Ophiorrhiza mungos L.

Article in Asian Pacific Journal of Tropical Biomedicine · February 2012

DOI: 10.1016/S2221-1691(12)60304-9

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 Asian Pacific Journal of Tropical Biomedicine(2012)S727-S731 S727

Contents lists available at ScienceDirect

Asian Pacific Journal of Tropical Biomedicine

journal homepage:www.elsevier.com/locate/apjtb

Document heading

Estimation of camptothecin and pharmacological evaluation of

Ophiorrhiza prostrata D. Don and Ophiorrhiza mungos L.
Krishnakumar G1, Rameshkumar KB2, Priya Srinivas3, Satheeshkumar K1*, Krishnan PN1
1
Biotechnology and Bioinformatics Division
2
Phytochemistry and Phytopharmacology Division Jawaharlal Nehru Tropical Botanic Garden and Research Institute Palode, Thiruvananthapuram,
Kerala, India 695 562
3
Integrated Cancer Research Programme Rajiv Gandhi Centre for Biotechnology Poojappura, Thiruvananthapuram, Kerala, India.

ARTICLE INFO ABSTRACT

Article history: Objective: To carry out the qualitative and quantitative evaluation of camptothecin, estimation
Received 4 June 2012 of total phenolic compounds and evaluation of in vitro antioxidant activity and cytotoxic activity
Received in revised from 5 July 2012 of Ophiorrhiza prostrata and Ophiorrhiza mungos. Methods: Direct Analysis in Real Time- Mass
Accepted 7 August 2012
Spectrometry (DART-MS) was employed for the detection of camptothecin in the Ophiorrhiza
Available online 28 August 2012
species, while high performance thin layer chromatography (HPTLC) was used for the estimation
of camptothecin. Total phenolic compounds were estimated by modified Folins-Ciocalteu’s
reagent method. Antioxidant activity was evaluated through DPPH radical, hydroxyl radical,
superoxide radical scavenging assays and reducing power assay. The cytotoxicity evaluation was
Keywords: performed using MTT assay on MCF-7 cell lines. Results: The presence of camptothecin was
Ophiorrhiza prostrata confirmed in both the species by the [M++H] peak at 349 by DART-MS analysis. Camptothecin
Ophiorrhiza mungos content was estimated as 1.47 毺g/gm (dry wt) in O. prostrata and 188.60 毺g/gm (dry wt) in O.
Camptothecin
mungos using HPTLC method. The moderate in vitro antioxidant activities of the methanol
HPTLC estimation
extracts corroborates with the low content of phenolic compounds in O. prostrata (9.88 GAE mg/
DART-MS analysis
Cytotoxicity g) and O. mungos (12.73 GAE mg/g). The methanol extract of O. prostrata exhibited remarkable
Antioxidant activity cytotoxicity on human breast cancer cell lines (MCF-7), with IC50 value 1.10毺g/mL compared to
O. mungos (3.48毺g/mL) and standard camptothecin (3.51毺g/mL). Conclusions: The application
of DART-MS proved to be a simple and rapid technique for the detection of camptothecin
in Ophiorrhiza species. The higher cytotoxicity for O. prostrata, despite the low content of
camptothecin suggests the presence of other potential cytotoxic compounds in O. prostrata.

camptothecin, which has drawn great attention world wide as

1. Introduction a potential inhibitor of DNA topoisomerase-1[2]. Topotecan
and irinotecan, two semisynthetic derivatives of camptothecin,
The genus Ophiorrhiza (Rubiaceae) is represented by 49 are currently being used for the treatment of colorectal and
species in India and different species of the genus have been ovarian cancer[3]. Camptothecin also possesses activity against
used in traditional medicines against snake bite, stomatitis, HIV-1[4], HSV-2[5], parasitic trypanosomas and leishmania[6].
ulcers and wound healing[1]. Ophiorrhiza species, especially The anticancer compound camptothecin has previously been
O. mungos has emerged as a source of the anticancer drug reported from Camptotheca accuminata[7], Nothapodytes
foetida [8] , M errilliodendron megacarpum [9] , E rvatamia
heyneana[10] and several Ophiorrhiza species including O.
mungos[11] and O. prostrata[12].
* C orresponding author: Satheeshkumar K, B iotechnology and B ioinformatics
Division, Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Palode, O. mungos and O. prostrata are herbaceous species,
Thiruvananthapuram, Kerala, India.
Tel: 91 9496154183
distributed in the tropical I ndo- M alaysian region [13] .
Fax: 91 04722869646
E-mail: [email protected]
Fundation Project: Supported by Kerala State SC/ST Welfare Development Fund, Grant
No.B3-2248/11 dtd. 22/03/2011(SC).
S728 Krishnakumar G et al./Asian Paicfic Journal of Tropical Biomedicine (2012)S727-S731

Though camptothecin has been estimated in Camptotheca out using TLC scanner 3, in absorbance mode at 254 nm by
accuminata [14] and N othapodytes foetida [15] , reliable comparing the peak area values of sample with that of standard
estimation of camptothecin in O. mungos and O. prostrata using the Wincats software.
are scarce[11]. Cytotoxicity of camptothecin has been tested
in different cell lines and reported as an active compound[2], 2.5. Total phenolic compounds estimation
however, the cytotoxicity of the title plants have not been
evaluated previously. Present study reports the detection of Total phenolic compounds were estimated as gallic acid
camptothecin by DART-MS and estimation of camptothecin by equivalent (GAE) mg/g dry weight using modified Folins-
HPTLC together with estimation of total phenolic compounds, Ciocalteu’s reagent colorimetric method[16].
evaluation of in vitro antioxidant and cytotoxic activities of the
methanol extracts of O. mungos and O. prostrata. 2.6. 2, 2-diphenyl- 1-picrylhydrazyl (DPPH) radical
scavenging assay

2. Materials and methods R adical scavenging activity was determined by

spectrophotometric method based on the reduction of the
2.1. Plant material stable free radical DPPH, using ascorbic acid as positive
control[17].
O. prostrata D. Don and O. mungos L. were collected from
Thiruvananthapuram, Kerala, India in August 2010 and 2.7. Hydroxyl radical scavenging assay
deposited in the herbarium of JNTBGRI (Herbarium voucher
specimens nos.TBGT 69942 and TBGT 69945 for O. prostrata and The method depends on the competition between deoxyribose
O. mungos respectively). and the plant extract for hydroxyl radical generated from Fe3+-
EDTA-ascorbate system[18]. The hydroxyl radical scavenging
2.2. Extraction activity of the extract was measured as percentage inhibition
of deoxyribose degradation and ascorbic acid was used as
Shade dried whole plant samples were powdered (5g each) positive control.
and defatted with petroleum ether using Soxhlet apparatus for
2h. The defatted samples were extracted with methanol using 2.8. Superoxide scavenging assay
Soxhlet apparatus for 4 h. and the methanol extracts were used
for further studies. The method depends on the light induced superoxide
generation by riboflavin and the corresponding reduction
2.3. Direct analysis in real time- mass spectrometry (DART- of nitroblue tetrazolium[19]. Quercetin was used as positive
MS) control.

The mass spectrometer used was a JMS-T100 LC (Accu 2.9. Reducing power assay
ToF) atmospheric pressure ionization time-of-flight mass
spectrometer (Jeol, Tokyo, Japan) fitted with a DART ion source. The presence of reductants such as antioxidants substances
The mass spectrometer was operated in positive-ion mode. in the samples causes reduction of the Fe3+/ferricyanide
The DART ion source was operated with helium gas flowing complex to the green ferrous form. The extent of reduction can
at 4.0 L/min. The gas heater was set to 300 曟. The potential be monitored by measuring the formation of Perl’s Prussian
on the discharge needle electrode of the DART source was set blue at 700 nm[20]. Ascorbic acid was used as positive control.
to 3000 V. Orifice 1 potential was set at 28V. The extracts were
positioned in the gap between the DART source and mass 2.10. Cytotoxicity assay
spectrometer for measurements. Data acquisition was from m/z
10 to 1050. Cytotoxicity was measured in vitro on human breast cancer
cell line (MCF-7), using 3-(4,5-dimethythiazol-2-yl)-2,5-
2.4. HPTLC estimation of camptothecin diphenyl tetrazolium bromide (MTT) colorimetric assay[21]. The
50% inhibitory concentration (IC50) was derived from the dose-
HPTLC was done on Camag HPTLC. 20 毺L (7.2 mg/mL) and 1 response curve.
毺L (0.63 mg/mL) each of O. prostrata and O. mungos methanol
extracts were applied on pre-coated silica gel plate 60F254 (E. 2.11. Statistical analysis
Merk, Germany) using the Linomat V applicator. Camptothecin
from Sigma Aldrich was used as the standard compound. 5.0 The analyses were done in triplicate and the results are
毺L to 10.0毺L of standard camptothecin (0.1毺g/mL) was expressed as mean 依 standard deviation. Results were
used for the estimation of O. prostrata, while 1.0毺L to 5.0毺L analyzed by one-way analysis of variance (ANOVA). P< 0.05
standard camptothecin (1.0毺g/mL) was used for the estimation was selected as statistically significant.
of O. mungos. The separation was carried out in twin trough
chamber using the solvent system toluene: acetonitrile: glacial
acetic acid (65:35:1) as mobile phase. Quantitation was carried 3. Results
 Krishnakumar G et al./Asian Paicfic Journal of Tropical Biomedicine (2012)S727-S731
S729

3.1. Direct analysis in real time- mass spectrometry (DART- scavenging models, viz., DPPH radical scavenging assay,
MS) superoxide radical scavenging assay and hydroxyl radical
scavenging assay, along with reducing power assay. The
Camptothecin has been detected in the methanol extracts of results (Table 1) are expressed as IC50 values in 毺g/mL, the
both the species by the [M++H] peak at m/z 349 (Figure1). The concentration of sample required to scavenge 50% free radical,
peak intensity was negligible for O. prostrata compared to O. except for reducing power assay. Reducing power increases
with an increase in the absorbance at 700 nm and the results
mungos. DART-MS of O. mungos also revealed the presence of are expressed as absorbance of 50 毺g/mL extract and standard
9-methoxy camptothecin at m/z 379[22]. solution at 700 nm. Both the extracts showed moderate activity,
compared to the positive controls. The content of phenolic
3.2. HPTLC estimation of camptothecin compounds were also low in both the species (Table 1).

The camptothecin content in O. prostrata was estimated as 3.4. Cytotoxicity assay

1.465 依 0.027 毺g/g dry wt (0.0001465 依 0.0000027%) (Figure 2)
and that of O. mungo was 188.6 依 12.3 毺g/g dry wt (0.01886% 依 Both the extracts reduced the viability of the cell lines in
0.0012%) (Figure 3). a dose dependent manner. IC50 was derived from the dose-
response curve (Figure 3). The methanol extract of O. prostrata
3.3. In vitro anti oxidant activity exhibited remarkable cytotoxic activity on human breast
cancer cell lines (MCF-7), with IC50 value 9.218依0.019毺g/mL,
Antioxidant activities of the methanol extracts of O. mungos
while O. mungo and standard camptothecin showed 34.81依0.03
and O. prostrata were tested in three in vitro free radical 毺g/mL and 35.15依0.03 毺g/mL respectively.

Figure 1. DART mass spectra.

A: O. mungos; B: O. prostrata.

A B
800.0

[AU]

600.0
800.0
500.0
[AU]
400.0
600.0
300.0
500.0
200.0
400.0
100.0
300.0
0.0
100.0
200.0 [mm]
80.0
70.0
100.0 60.0
50.0
40.0
0.0 30.0
20.0
1 2 3 4 5 6 7 0.00 0.10 0.20 0.30 0.40 0.50 10.0
0.60 0.70 0.80 [Rt] 1.00 0.0

Figure 2. HPTLC of camptothecin from O. prostrata (Track1plant extract and track 2 -7 standard camptothecin).
A: chromatogram; B: three dimentional densitogram.
S730 Krishnakumar G et al./Asian Paicfic Journal of Tropical Biomedicine (2012)S727-S731

B
A

600.0
600.0

[AU]
[AU]

400.0
400.0

300.0
300.0

200.0
200.0

100.0
100.0

0.0
100.0
0.0 60.0[mm]
40.050.0
0.00 0.10 0.20 0.30 0.40 20.030.0
1 2 3 4 5 6 7 0.50 0.60 0.70 [Rt] 0.0 0.010.0

Figure 3. HPTLC of camptothecin from O. mungo (Track 1-6 standard camptothecin and track 7 plant extract).
A: chromatogram; B: three dimentional densitogram.

mass spectrometry analyses[22]. DART is a simple and rapid

120 ambient ionization technique developed recently[23] and
accepted widely in natural product research as a high-
100 throughput tool for confirmation of chemical identity and
metabolomic profiling[24]. The present investigation shows
% of Cell viability

80
DART-MS as a reliable analytical technique for the detection
60 of camptothecin in Ophiorrhiza species. The ion intensities
shown by DART-MS suggests that the concentration found
40
in O. prostrata is negligible than those found in O. mungos
20 and the finding corroborates with the camptothecin content
estimated by HPTLC. DART-MS of O. mungos also revealed
0 the presence of 9-methoxy camptothecin at m/z 379[22] and the
0 10 20 30 40 50 finding corroborates with previous report of camptothecin and
9-methoxy camptothecin in O. mungos[25].
concentration of drug (mg/mL)
HPTLC has become a potential tool for identification,
authentication and quality control of phytochemicals[26]. HPTLC
Figure 3. Dose-dependent cytotoxic activity of methanol extracts of method has been successfully employed for the estimation of
O. mungos (OM) and O. prostrata (OP) standard camptothecin (CPT), on camptothecin from Nothapodytes foetida[27] and Camptotheca
MCF-7 using MTT assay. acuminata[28]. The present study showed the solvent system
toluene-acetonitrile-glacial acetic acid (6.5:3.5:0.1) (v/v) giving
the best resolution for the estimation of camptothecin in
4. Discussion Ophiorrhiza species with varying concentration.
Phenolic compounds contribute significantly to the total
The ambient ionisation techniques such as direct analysis antioxidant activity of plants[20] and the moderate in vitro
in real time (DART) and desorption electrospray ionisation antioxidant activities of O. prostrata and O. mungos were in
mass spectrometry (DESI) gives the chemical profile with corroboration with the low content of phenolic compounds in
molecular weights without sample preparation or pre- O. mungos and O. prostrata. Reactive oxygen species such as
separation as required in the case of liquid chromatography superoxide anion and hydroxyl radicals are known to exert

Table 1
Antioxidant activities of O. prostrata and O. mungos whole plant methanol extracts.
IC50 values of free radical scavenging assays (毺g/mL) Total phenolic compounds (GAE mg/g)
Ophiorrhiza species Reducing power assay
*
DPPH radical Hydroxyl radical Suproxide radical
O. mungo 64.57依2.42 80.30依6.71 55.59依5.14 0.16依0.02 12.72依0.57
O. prostrata 67.01依1.01 87.44依4.72 49.60依2.37 0.11依0.00 9.88依1.53
Standard compound# 03.30依0.15 08.73依0.70 27.32依1.06 1.11依0.02 -
*Absorbance of 50 毺g/mL extract and standard solution at 700nm. #Ascorbic acid for DPPH radical, reducing power assay and for hydroxyl radical
scavenging assay. Quercetin for superoxide radical scavenging assay. The analyses were done in triplicate and the results are expressed as mean 依
standard deviation.
 Krishnakumar G et al./Asian Paicfic Journal of Tropical Biomedicine (2012)S727-S731
S731

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