Open Access Research Article
Phytochemical Screening And Anticancer Activity Of Cordia Myxa Extract On MCF-7 Cell Line (By-Priyanka Rani & Sanjiv Kumar)
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Article Details
Phytochemical
Screening And Anticancer Activity Of Cordia Myxa Extract On MCF-7 Cell Line
Authored By-1. Priyanka Rani1
2. Sanjiv Kumar
Department of Pharmaceutical
Sciences,
Chaudhary Bansi Lal University, Bhiwani-
127021 Haryana
Abstract:
Background: Breast cancer is a
malignant tumor (a collection of cancer cells) arising from the cells of the
breast. Although breast cancer predominantly occurs in women, it can also
affect men. While breast cancer rates are higher among women in more developed
regions, rates are increasing in nearly every region globally. Cordia myxa (CA)
widely known as Lasura consist of flavonoids,
triterpenes, tannins, alkaloids and saponins etc which are founded effective in
many studies for diseases as well as in cancer. The present study on the
malignant using cordia myxa extract has shown significant inhibition of cancer
cell of breast cancer with IC 50 of 222 on MCF-7 cell line. On the other hand
Doxorubicin which is an anticancer drug shown very promising inhibition of
MCF-7 cell line at IC 50 of 23.68.
Keywords: Breast carcinoma, MCF-7
cell line, Cordia myxa.
1. Introduction
Cancer is
a collective term for hordes of diseases characterized by invasion of
neighbouring parts of the body and / or pathological cells that can spread to
other organs, reproducing beyond their normal limits. Other common terms are
malignant tumors and neoplasms. Cancer affects almost all parts of the body,
and there are many anatomical and molecular subtypes that require specific
management strategies. Cancer is the second leading cause of death in the
world, and an estimated 9.6 million people will die by cancer in 2018. Prostate
cancer, lung cancer, stomach cancer, liver cancer, colorectal cancer are the
most common cancer among men, cervical cancer, lung cancer, colorectal cancer,
breast cancer, thyroid cancer are the most common among women (Bray F et al.,
2018) .
Breast
cancer is a malignant tumor (a collection of cancer cells) arising from the
cells of the breast. Although breast cancer predominantly occurs in women, it
can also affect men (Larsen MJ et al.,2014). Breast cancer is the most frequent
cancer among women, impacting 2.1 million women each year, and also causes the
greatest number of cancer-related deaths among women. In 2018, breast cancer is
the 2nd most frequent cancer with an estimated that 627,000 women died around
the world from breast cancer – that is approximately 15% of all cancer deaths
among women. While breast cancer rates are higher among women in more developed
regions, rates are increasing in nearly every region globally (Bray F et al.,2018) . Breast cancer can invasive to other body organ and cause
the lungs, liver, colon, rectal, vaginal and cervix cancer (Bargagna P et
al.,2007). Inflammatory Breast Cancer (IBC) and triple-negative breast cancer
are the invasive type of breast cancer while ductal carcinoma and lobular carcinoma
are the non-invasive type of breast cancer (Sun H et al.,2017). Early sign and
symptoms of breast cancer are typically not recognizable, but the most common
symptom is a lump or mass in the breast or underarm area, nipple discharge or
redness, changes in the breast skin texture such as puckering or dimpling (like
an orange skin), swelling of part of the breast (Hoadley KA et al., 2018).
Many
therapies are used such as surgery, radiation therapy, biological therapy
(targeted drug therapy), hormone therapy and chemotherapy for breast cancer but these are having more
serious side effects as well as very costly.
All these therapy have many serious side effects that’s why we are going
on herbal formulation. At present time natural treatments are used globally at
higher
rate as compare to allopathic medicines (Bargagna-Mohan P et al.,2007). Herbal treatments have similar
action to chemotherapy and radiation therapy. Combinational formulations of
herbals plants/oils are an attractive option to treat the cervical cancer and
breast cancer due to less side effects, less cost and gives the synergistic
effects for treating the cervical cancer and breast cancer. It might become a
satisfactory remedy for prevention and management of cervical cancer and breast
cancer (Shi YQ et al.,2009).
Cordia myxa (family-
Boraginaceae), about 300 species have been identified worldwide, mostly in
warmer regions . The tree keeps its leaves for most of the year. These are
broad, alternate, ovateelliptic shaped. The inflorescence carries numerous
white flowers. Fruits are round to ovoid shaped drupes, about 15–20 mm in
diameter, arranged in clusters. Their white-yellow color turns blackish when
dry. The pulp, very tough and mucilaginous, is edible and has a sweetish flavor
(K. Thirupathi et al.,2008) . The plant parts like fruits, leaves, stem
bark, seeds and roots of most species of plants of the genus Cordia, especially
Cordia dichotoma, C. myxa,C. oblique, C. verbenacea, C. martinicensis, C.
salicifolia, C. spinescens, C. latifolia, C. ulmifolia, among others, has long
been used in traditional medicine for cicatrizant, astringent,
antiinflammatory, anthelminthic, antimalarial, diuretic, febrifuge, appetite
suppressant, cough suppressant and to treat urinary infections, lung diseases
and leprosy (Enas R et al.,2019) . The pharmacological studies
carried out with extracts and purified compounds indicates that the plants of
Cordia species possess analgesic, anti-inflammatory, antimicrobial,
antiparasitic, insecticidal, cardiovascular, respiratory, gastrointestinal and
protective effects. Various compounds like flavonoids, triterpenes, tannins,
alkaloids and fatty acids possessing wide range of bioactivities were isolated
from different plant parts of Cordia species (Ali Esmail Al-Snafi 2016). The fruit has been widely used to
treat respiratory infections, coughing, and sore throat and used as a diuretic. The pulp is used to mature abscesses,
calm rheumatic pain, treat ringworm, and as an anthelminthic (Tarik Shwaish and
Faris JM Al-Imarah2017).
Flowers
Stem bark Ripened fruit
Experimental
2.1.
Material and method
2.1.1 Collection of
plant materials and sample preparation
The cordia myxa leaves were collected from herbal garden of Bank
colony, Bhiwani (Haryana). The plant was taxonomically identified by Dr. S.S
Yadav, Assistant professor, Department of Botany, Maharshi Dayanand University,
Rohtak (Haryana). The whole plant was washed with water and were chopped in
small pieces, dried at room temp, grinded in powder form.
2.1.2 Extraction procedure
The powdered sample was extracted
using Soxhlet apparatus.Hydroalcohol
(50% ethanol and 50% distilled water) solvent 50ml/g of sample weight was
measured and filled in to the apparatus. The time of extraction was 18 h for
each sample.A total of 50 g of dried powder was
packed in the timble of soxhlet apparatus and was extracted using hydroalcohol
refluxing at 50-70°C which yielded a dark brown extract. The stock extract was
putted in desiccator
2.1.3 Phytochemical screening of the extract by Qualitative
test
|
Sr. No
|
Test
|
Name of test
|
Ethanol extract of cordia myxa
|
|
1
|
Alkaloids
|
Dragendorff’s
test
|
+ve
|
|
2
|
Glycosides
|
Brontragar,s
|
+ve
|
|
3
|
Terpenoids
|
Noller test
|
-ve
|
|
4
|
Carbohydrates
|
Fehling test
|
+ve
|
|
5
|
Proteins
|
Ninhydrine test
|
+ve
|
|
6
|
Steroids
|
Salkowski reaction
|
+ve
|
|
7
|
Flavonoids
|
Shinoda test
|
+ve
|
|
8
|
Phenols
|
Lead acetate test
|
+ve
|
|
9
|
Saponins
|
Frothing
test
|
-ve
|
1. Alkaloids: Extract was treated with few drops Dragendroff’s
reagent(solution of potassium bismuth iodide) orange coloured precipitate
indicates a positive result.
2. Glycosides: the
extract boiled with dilute sulphuric acid, filtered and to the filtrate
benzene,or ether or chloroform is added and shaken well. The organic layer is
separated to which ammonia is added slowly.the ammoniacal layer shows pink to
red colour indicates the positive result.
3.Terpenoids : the
substance was warmed with tin and thionyl chloride . did not show pink colour indicates negative results.
4. Carbohydrates: These
two solutions should be stoppered and stored until needed. Mix 15 ml of
solution-A with 15 ml of solution -B add 2 ml of this mixture to an empty test
tube.Add 3 drops of the Extract to be tested to the tube. Place the tube in a water
bath at 60 c. Green suspension and red precipitate indicates a positive result.
5. Proteins: The extracts
were treated with a few drops of
Nin-hydrin reagent show yellow colour precipitate indicates a positive result.
6.Steriods: extract
treated with Salkowski reagent to show the pinkish or dark red colour indicates
the positive result.
7. Flavonoids: A few
drops of concentrated hydro chloride acid
were added to the extract; the formation of dark red color indicates the
positive result.
8.Phenols : 2 ml of test
solution and 2 ml of Sodium hydroxide
and boil for a minute. And add a drop of lead acetate solution. A Brown or a
black precipitate indicates a positive result.
9. Saponins: Extract was
mixed with water and shaken; formation of froth that’s stable for 15 minutes .
and then 1 ml extract was treated with 1 percent lead acetate solution .
formation of white colured precipitate not formed indicates negative result.
Materials & Method
1.MTT Powder (the solution is filtered through a
0.2 ? m filter and stored at 2–8 °C for frequent use or frozen for extended
periods)
2. DMSO (Dimethyl sulfoxide) can significantly
inhibit cancer cell invasion, migration, proliferation and colony formation
capabilities through upregulation of HLJ1 in a concentration- dependent manner
. DMSO led to a significant increase in the percentage of UV- induced apoptotic
cells.DMSO to arrest the cell cycle of cell lines at the G1phase.
3. CO2
incubator : CO2 incubator is used to culture cells to provide it with the
optimum temperature, moisture (sterile environment) to maintain optimum pH.
4. Tecan Plate reader: Easy to use multimode plate reader
affordable, measure absorbance, high performance detection solutions.
2.2.1.
Preparation of test solutions
For
cytotoxicity studies, 32mg/ml stocks were prepared using DMSO and serial two
fold dilutions were prepared from 320?g/ml to 10?g/ml using DMEM plain media
for treatment.
2.2.2 Cell
lines and culture medium
All
the cell lines were procured from ATCC, stock cells was cultured in DMEM
supplemented with 10% inactivated Fetal Bovine Serum (FBS), penicillin (100
IU/ml), streptomycin (100 ?g/ml) in a humidified atmosphere of 5% CO2
at 37oC until confluent. The cell was dissociated with cell
dissociating solution (0.2 % trypsin, 0.02 % EDTA, 0.05 % glucose in PBS). The
viability of the cells are checked and centrifuged. Further, 50,000 cells /well
was seeded in a 96 well plate and incubated for 24 hrs at 37oC, 5 %
CO2 incubator.
2.2.3
Anticancer Evaluation Assay
The
monolayer cell culture was trypsinized and the cell count was adjusted to
1.0 x 105 cells/ml using respective media containing 10 % FBS. To each well of the 96 well microtiter plate, 100 ?l of the diluted cell suspension (50,000 cells/well) was added. After 24 h,
1.0 x 105 cells/ml using respective media containing 10 % FBS. To each well of the 96 well microtiter plate, 100 ?l of the diluted cell suspension (50,000 cells/well) was added. After 24 h,
when
a partial monolayer was formed, the supernatant was flicked off, washed the
monolayer once with medium and 100 ?l of different test concentrations of test
drugs were added on to the partial monolayer in microtiter plates. The plates
were then incubated at
37oC for 24hrs in 5% CO2 atmosphere. After incubation the test solutions in the wells were discarded and 100 ?l of MTT (6 mg/10 ml of MTT in PBS) was added to each well. The plates were incubated for 4 h at 37oC in 5% CO2 atmosphere. The supernatant was removed and 100 ?l of DMSO was added and the plates were gently shaken to solubilize the formed formazan. The absorbance was measured using a microplate reader at a wavelength of 590 nm. The percentage growth inhibition was calculated using the following formula and concentration of test drug needed to inhibit cell growth by 50% (IC50) values is generated from the dose-response curves for each cell line.
37oC for 24hrs in 5% CO2 atmosphere. After incubation the test solutions in the wells were discarded and 100 ?l of MTT (6 mg/10 ml of MTT in PBS) was added to each well. The plates were incubated for 4 h at 37oC in 5% CO2 atmosphere. The supernatant was removed and 100 ?l of DMSO was added and the plates were gently shaken to solubilize the formed formazan. The absorbance was measured using a microplate reader at a wavelength of 590 nm. The percentage growth inhibition was calculated using the following formula and concentration of test drug needed to inhibit cell growth by 50% (IC50) values is generated from the dose-response curves for each cell line.
2.2.4. Calculating Inhibition:
% Inhibition = 100 – (OD of sample/OD of Control) x
100.
2.2.5 Result
and discussion
IC50
Value
The
half maximal inhibitory concentration (IC50) is a measure of the
effectiveness of a compound in inhibiting biological or biochemical function.
This quantitative measure indicates how much of a particular drug or other
substance (inhibitor) is needed to inhibit a given biological process (or component
of a process, i.e. an enzyme, cell, cell receptor or microorganism) by half.
The
IC50 of a drug can be determined by constructing a dose-response
curve and examining the effect of different concentrations of antagonist on
reversing agonist activity. IC50 values can be calculated for a
given antagonist by determining the concentration needed to inhibit half of the
maximum biological response of the agonist. IC50 values for
cytotoxicity tests were derived from a nonlinear regression analysis (curve
fit) based on sigmoid dose response curve (variable) and computed using Graph
Pad Prism 6 (Graph pad, SanDiego, CA, USA)
Nonlinear
regression
In
statistics, nonlinear regression is a form of regression analysis in which
observational data are modeled by a function which is a nonlinear combination
of the model parameters and depends on one or more independent variables. The
data are fitted by a method of successive approximations.
Table 1. Anticancer screening results of extracted compound
|
MCF-7
|
Standard
|
|||
|
Compound
Name
|
Conc.
µM
|
OD @
590nm
|
%
Inhibition
|
IC50 in
µM
|
|
Control
|
0
|
0.828
|
0.00
|
23.68
|
|
Doxorubicin
|
3.13
|
0.602
|
27.31
|
|
|
6.25
|
0.554
|
33.12
|
||
|
12.5
|
0.438
|
47.10
|
||
|
25
|
0.335
|
59.53
|
||
|
50
|
0.253
|
69.40
|
||
|
100
|
0.135
|
83.72
|
||
|
|
||||
The inhibition level increase with
concentration
Table 2. Anticancer screening results of extracted compound
|
MCF-7
|
Cordia myxa extract
|
|||
|
Compound
Name
|
Conc.
µg/ml
|
OD @
590nm
|
%
Inhibition
|
IC50
µg/mL
|
|
Control
|
0
|
0.828
|
0.00
|
222
|
|
Sample
|
25
|
0.693
|
16.28
|
|
|
50
|
0.602
|
27.26
|
||
|
100
|
0.510
|
38.41
|
||
|
200
|
0.435
|
47.49
|
||
|
400
|
0.286
|
65.50
|
||
|
800
|
0.186
|
77.50
|
||
Result increase with concentration
|
MTT assay using MCF-7
cells
0 1 2 3 4
Conc.?g/ml
(Log X)
|
Sample
|
|
MTT assay using MCF-7
cells
|
Doxorubicin
|
Fig 1
Fig 2
Conclusion
In present the extract has
shown IC50 value of 222?g/mL of inhibition in MCF-7 cells. Standard Doxorubicin
has showed 23.68?M inhibition in the same cell line.
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Article Information
Phytochemical Screening And Anticancer Activity Of Cordia Myxa Extract On MCF-7 Cell Line (By-Priyanka Rani & Sanjiv Kumar)
Authors: Priyanka Rani, Sanjiv Kumar
- Journal IJLRA
- ISSN 2582-6433
- Published 2022/08/30
- Volume 2
- Issue 7
About Journal
International Journal for Legal Research and Analysis
- Abbreviation IJLRA
- ISSN 2582-6433
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