Phytochemical Screening and In-vitro Antioxidant and Antiproliferative Activity of Aqueous Leaf Extract of Ximtenia americana against Non- Small Cell Lung Cancer
Abstract
Introduction: Cancer is a general term applied of se-
ries of malignant diseases that may affect different
parts of body. These diseases are characterized by
rapid and uncontrolled formation of abnormal cells,
which may mass together to form a tumor or prolif-
erate throughout the body by the process of metas-
tasis. The main forms of cancer treatment for cancer
in humans are surgery, radiation and drugs (chemo-
therapeutic agents) can often provide temporary re-
lief of symptoms, prolongation of life and occasion-
ally cures. Cancer continues to represent the largest
cause of mortality in the world and claims over 6
million lives every year [1]. In developing countries
since from following decades, the numerous of peo-
ple with cancer will continue to increase may be due
to life style, nutrition and environmental conditions
[2-4]. In many countries cancer is 2nd leading cause
of death after heart diseases [5]. Lung, colorectal
and stomach cancer are among the five most com-
mon cancers in the world for both men and women
[6]. Lung cancer is the leading cause of cancer deaths
worldwide. American cancer society estimated in
2016, about 1 of 4 cancer deaths are from lung can-
cer. Every year, more people die of lung cancer than
of colon, breast and prostate cancers. Furthermore if
you consider 5 year survival rate for lung cancer pa-
tients it drops from 54% to 4% in patients with meta-
static lung cancer [7].
However, most of the anticancer drugs currently
used such as doxorubicin, paclitaxel give rise to un-
desirable side effects such as cardio toxicity and tu-
mor drug resistance [8]. Since from ancient time’s
plant secondary metabolites and their semi synthetic
derivatives continue to play an important role in the
treatment of cancer as novel drugs [9,10] and 60% of
currently used anticancer agents are derived in one
way or another from natural sources [11]. Plant de-
rived natural products such as flavonoids, terpenes,
alkaloids and phenols are gaining more importance
due to their diverse pharmacological properties in-
cluding cyto-toxic and cancer chemo protective ef-
fects [12].
Plants are the rich sources of secondary metabo-
lites such as alkaloids, phenols, flavonoids, tannins,
saponins, glycosides, terpenoids etc. that possess a
wide array of biological properties including antibac-
terial, antifungal, antioxidant and anticancer [13].
Phytochemicals and even the whole plant extracts
are known to prevent arrest or reverse the cellular
and molecular processes of carcinogenesis due to
its multiple intervention strategies [14] because of
these reason herbal medicines making an impact on
both world health and international trade. Medicinal
plants continue to play a central role in the health
care system of the large proportions of the world’s
population [15].
However, till-date a systematic study on biological ac-
tivities of chemical constituents present in X. amer-
icana is still not agreeable [23,24]. The extensive lit-
erature survey exposed that only few reports exist
on this plant leaves, but no information are available
on anticancer activity in particular with lung cancer.
Henceforth, present study was undertaken and made
an attempt to identify Phytochemicals and invitro an-
tioxidant and antiproliferative activity of aqueous ex-
tract of Ximenia americana.
Ximenia americana leaves were collected from Kar-
natak University Campus, Dharwad, India in the
month of June, 2017. The leaves were identified and
authenticated by Dr. Kotresha K., Department of Bot-
any, Karnatak Science College, Dharwad, Karnataka,
India. A voucher specimen (N0-01/2016) was depos-
ited at the Department of Botany, Karnatak Science
College, Dharwad, Karnataka. Fresh disease free
plant material was washed under running tap water,
shade dried and pulverized to fine powder using me-
chanical grinder. The powder was stored in airtight
containers at room temperature for further use.
Chemicals: 3-(4,5–dimethyl thiazol–2–yl)–5–diphen-
yltetrazolium bromide (MTT), Fetal Bovine serum
(FBS), Phosphate Buffered Saline (PBS), Dulbecco’s
Modified Eagle’s Medium (DMEM) and Trypsin were
obtained from Sigma Aldrich Co, St Louis, USA.EDTA,
Glucose and antibiotics from Hi-Media Laboratories
Ltd., Mumbai. Dimethyl Sulfoxide (DMSO) and Propa-
nol from E.Merck Ltd., Mumbai, India.
Cell lines: A549 &NCI-H460non small cell lung can-
cer cell lines were procured from National Centre for
Cell Sciences (NCCS), Pune, India. Stock cells were
cultured in DMEM supplemented with 10% inacti-
vated Fetal Bovine Serum (FBS), penicillin (100 IU/
ml), streptomycin (100 μg/ml) and amphoteri¬cin B
(5 μg/ml) in a humidified atmosphere of 5% CO2 at
37°C until confluent.
Crude extraction: The 100 g of dried X. americana leaf
material was extracted with distilled water using Sox-
hlet apparatus for 4-6 hrs at 40-500°C. The extractant
solvent was evaporated using rotary evaporator and
the resultant slurry of crude extract was thoroughly
dried and weighed. The extract was freeze-dried at
−200°C and stored at 40°C until use. The yield was
found to be 8% w/w with reference to the air dried
plant material.
Phytochemical analysis: The crude powder of Xime-
nia americana was qualitatively tested for different
phytochemical constituents namely alkaloids, flavo-
noids, glycosides, phenols, lignin’s, saponins, sterols,
tannins, anthraquinone and reducing sugar by fol-
lowing the standard procedure [25].
Estimation of flavonoids content: The flavonoids
content in the plant extracts was estimated accord-
ing [26] with quercetin as reference standard. It is
an aluminium chloride colorimetric method in which
each extract (0.5 ml) separately mixed with 1.5 ml
of methanol, 0.1 ml of 10% aluminium chloride, 0.1
ml of 1M potassium acetate and 2.8 ml of distilled
water. The reaction mixture was kept at room tem-
perature for 30 min; the absorbance of the reaction
mixture was measured at 415 nm using a UV-VIS
Spectrophotometer. The value of optical density was
used to calculate the flavonoids content present in
the sample and the calibration curve was plotted by
using quercetin solutions at concentrations 12.5 to
100 μg/ml in methanol.
The antioxidant activity of Ximenia americana was
evaluated with ascorbic acid as a standard based on
their ability to scavenge the hydrogen peroxide [27].
0.6 ml of 4mM H2O2 solution in phosphate buffer
(pH-7.4) was added to 0.5 ml of known concentration
of standard ascorbic acid and to tubes containing dif-
ferent concentrations ranging from 100 μl to 500 μl
of plant extracts in phosphate buffer (pH-7.4). Absor-
bance of the solution was measured at 230 nm after
10 min against the blank solution containing phos-
phate buffer without hydrogen peroxide. Control was
prepared by replacing the sample or standard with
phosphate buffer. All samples were assayed in tripli-
cates. The percentage of inhibition was calculated by
using formula method.
The cells were dissociated with TPVG solution (0.2%
trypsin, 0.02% EDTA, 0.05% glucose in PBS). The stock
cultures were grown in 25 cm2 culture flasks and all
experiments were carried out in 96 microtitre plates
(Tarsons India Pvt. Ltd., Kolkata, India).
For Cytotoxicity studies, each weighed test drugs
were separately dissolved in DMSO and volume was
made up with DMEM supplemented with 2% inacti-
vated FBS to obtain a stock solution of 1 mg/ml con-
centration and sterilized by filtration. Serial two fold
dilutions were prepared from this for carrying out
cytotoxic studies.
The effect of aqueous extract of leaves extract of Xi-
menia americana on the viability of non-small cell
lung cancer (A549 &NCI-H460) cells was determined
using the standard colorimetric MTT assay using the
3-(4,5-dimethylthiazol- 2-yl)-2,5-dimethyl tetrazoli-
um bromide dye (Sigma, St. Louis, MO, USA) [28]. The
monolayer cell culture was trypsinized and the cell
count was adjusted to 1.0 × 105 cells/ml using DMEM
containing 10% FBS and seeded to 96-well microtiter
plates (Falcon, Becton– Dickinson, Franklin Lakes, NJ,
USA). After 24 h of plating, cells were serum starved
for 24 hr. In the present study untreated A549 and
NCI-H460 cell lines taken as control group, A549 and
NCI-H460 cell lines treated with standard drug Oxal-
iplatin considered as positive control group whereas
aqueous extract of X americana Figure 1 treated A549
and NCI-H460 cell lines taken as treated group. In the
present study different concentration of standard
drug and aqueous extract of X. americana were taken
to study morphological changes as well as cell growth
inhibition in both A549 and NCI-H460 non-small cell
lung cancer cell lines Figure 2. Morphological studies
revealed that compared with control group, treat-
ed group and positive control group showed signif-
icant increase in detached cells in culture medium.
The cells displayed as turgid and shrunken in shape
compared to untreated control cells. Morphological
changes in nucleus representing apoptosis. Where-
as normal cells appeared as regular and normal in
shape, in case of treated group chromatin conden-
sation, elongation of cells and decrease in cell count
and density were observed which are the character-
istic features of apoptosis. Microscopic examination
revealed that morphological changes and shrinkage
of cells leading to cell apoptosis induced by the aque-
ous extract of Ximenia americana (Figures 3 and 4).
