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Rev. Biol. Trop. (Int. J. Trop. Biol.) • Vol. 69(1): 317-330, March 2021

Low cytotoxicity, and antiproliferative activity on cancer cells,

of the plant Senna alata (Fabaceae)

Amir Modarresi Chahardehi

, Hasni Arsad

, Noor Zafirah Ismail

& Vuanghao Lim

1. Integrative Medicine Cluster, Advanced Medical and Dental Institute, Universiti Sains Malaysia, 13200, Bertam,

Penang, Malaysia; amirmch@gmail.com, hasniarsad@usm.my, piecesnzi@gmail.com, vlim@usm.my

Received 04-VI-2020. Corrected 23-XI-2020. Accepted 14-XII-2020.

ABSTRACT. Introduction: The leaves of Senna alata from the Fabaceae family have been used in folk

medicine for the cure of skin disease. In this study, we tested the extract and fractions on brine shrimp lethal-

ity test and antiproliferative activity on cancer and normal cell lines. Objective: In this study, we assessed the

cytotoxicity of S. alata using brine shrimp test and two cell lines. Methods: The 80 % ethanolic leaf extract

and its fractions were examined for possible cytotoxic effect using sulforhodamine B (SRB) cytotoxicity assay

towards breast cancer (MCF-7), normal (MCF10A) cell lines, and brine shrimp lethality test (BSLT). Results:

The brine shrimp lethality bioassay exhibits no cytotoxicity even at high concentration (5 000 µg/mL). The LC

for dichloromethane, chloroform, butanol, and aqueous were > 1 000 µg/mL (non-toxic). The IC

for in vitro

SRB cytotoxicity against MCF-7 for n-hexane was 0.013 µg/mL, which was considered highly toxic, while

dichloromethane and chloroform recorded at 47.11 and 57.61 µg/mL, respectively after 72 hours exposure time

although there was no cytotoxicity found on the normal cell line. Conclusion: This study shows that S. alata

crude ethanolic leaf extract and its fractions potentially contain significant bioactive compounds that are safe

from adverse effects, which proves the therapeutic application of S. alata in traditional remedy.

Key words: cytotoxicity; brine shrimp; Senna alata; breast cancer.

Natural products have been recognized as

the source of medicinal substances and struc-

tural sustainability for several years (Beutler,

2019). The natural resources of medicinal

plants are precious phytochemicals that are

often employed for the treatment of different

diseases (Al-Ansari et al., 2019), especially for

cancer treatment. Plants as natural resources

are used for several years provide poten-

tial chemical therapeutics in cancer treatment

and interest in nature (Akindele et al., 2015).

Hence, phytochemicals cover a wide range

of chemical spaces for the discovery of drugs

(Mohanraj et al., 2018). Phytochemicals have

various pharmacology mechanisms, including

stimulating enzymes such as glutathione trans-

ferase or preventing cell proliferation (Shareef,

Ashraf, & Sarfraz, 2016).

Over a million women with breast can-

cer are identified per year around the globe

(Shareef et. al., 2016); therefore, breast cancer

has been the second most common cause of

death for women (Azamjah, Soltan-Zadeh, &

Zayeri, 2019; Levitsky & Dembitsky, 2014).

Since mammography is not available for rou-

tine screening, late stages of breast cancer are

usually investigated (Shareef et al., 2016). The

function of flavonoids in cancer prevention

has been documented (Elsyana, Bintang, &

Priosoeryanto, 2016). Their ability and healing

Modarresi Chahardehi, A., Arsad, H., Zafirah Ismail, N., & Lim, V. (2021). Low

cytotoxicity, and antiproliferative activity on cancer cells, of the plant Senna alata

(Fabaceae). Revista de Biología Tropical, 69(1), 317-330. DOI 10.15517/rbt.

v69i1.42144

ISSN Printed: 0034-7744 ISSN digital: 2215-2075

DOI 10.15517/rbt.v69i1.42144

318

Rev. Biol. Trop. (Int. J. Trop. Biol.) • Vol. 69(1): 317-330, March 2021

potential have been separately documented

worldwide, indicating that plants could become

a prospective source of new medicines (Idris,

Wintola, & Afolayan, 2019).

Cassia alata L. (also recognized as Senna

alata) is a shrub that belongs to the Fabaceae

family (sub-family Caesalpinioideae), which

is distributed in the intertropical region (Saito

et al., 2012). This plant is popularly known as

the candle bush and also ringworm tree due

to its folk medicine, which is referenced in

the complete flower head (Hennebelle, Weni-

ger, Joseph, Sahpaz, & Bailleul, 2009). It is

originally from Central America, primarily

found in the Caribbean region, and has also

been spread to several tropical climates on all

continents (Hennebelle et al., 2009). Senna

alata has been utilized primarily for traditional

medicine against skin infection, and constipa-

tion (Elsyana et al., 2016; Hennebelle et al.,

2009) and lately has been suggested for the

cosmetic industry as a natural product (Elsyana

et al., 2016). Extracts of S. alata are consid-

ered to possess antibacterial activity; however,

some other antibacterial effects such as preven-

tion bacterial adhesion and biofilm formation

besides specific compounds and mechanisms

of action are not discovered properly (Saito

et al., 2012). This plant possesses potential

insecticidal, fungicidal (Iyengar, Rama, & Rao,

1995; Palanichamy & Nagarajan, 1990), anti-

inflammatory (Abatan, 1990), antimicrobial

(Ibrahim & Osman, 1995; Khan, Kihara, &

Omoloso, 2001), wound healing (Palanichamy,

Bhaskar, Bakthavathsalam, & Nagarajan, 1991)

and antitumor activity (Olarte, Herrera, Villase-

nor, & Jacinto, 2013; Pamulaparthi & Nanna,

2015; Karchesy, Kelsey, Constantine, & Karch-

esy, 2016). S. alata leaf extract is traditionally

used for treating any type of diseases (Olarte

et al., 2013), which is rich in polyphenols and

anthraquinones (Fernand et al., 2008). The

extensive use of S. alata has been encouraged

to look for its pharmaceutically significant

compounds in traditional medicine in several

research studies (Saito et al., 2012). Tradition-

ally, this plant used for treatment of cancer in

Cameroon (mostly breast cancer) (Tene, Tala,

Tatong, & Tchuente, 2017). Research on plant

chemistry showed that the leaves of S. alata

include saponins, anthraquinones, tannins,

terpenes, alkaloids, and steroids (Prasenjit,

Tanaya, Sumanta, Basudeb, & Kumar, 2016).

This significant worldwide herbal medicine has

been used historically as an anti-helminthic,

anti-inflammatory, uterus illness (Heyde, 1990)

and bacterial infection (Igoli, Igwue, & Igoli,

2004; Panda, Padhi, & Mohanty, 2011; Prom-

gool, Pancharoen, & Deachatai, 2014; Prasenjit

et al., 2016).

Meyer et al. (1982) identified the brine

shrimp lethality bioassay (BSLA) as a par-

ticular test that was able to detect screening the

range of crude plant extracts in herbal medicine

for cytotoxicity in a simple, quick and exten-

sive bioassay for bioactive compounds of natu-

ral product (Meyer et al., 1982; Karchesy et al.,

2016; Henry, 2017). The brine shrimp lethality

test (BSLT) is the primary anticancer test pro-

cess (Prasetyo, Sidharta, Hartini, & Mursyanti,

2019). However, there is a significant correla-

tion between BSLT toxicity and cytotoxicity

in certain cell lines, but this approach is not

unique to anticancer activity (Asnaashari et al.,

2017). The previous result showed that LC

value on brine shrimp larvae for ethanol extract

of S. alata was 7.7 µg/mL (Logarto, Silva,

Guerra, & Iglesias, 2001). The previous study

showed that ethanol extract brought more reli-

able activity than other extracts (Panda et al.,

2011). In recent years, GC-MC has developed

as a primary technical tool for the secondary

profiling of metabolites in both plant and non-

plant organisms (Kanthal, Dey, Satyavathi,

& Bhojaraju, 2014). Thus, the aim of analys-

ing 80 % ethanolic extract of this study was,

therefore, to detect potential chemicals and to

separate the compounds and to identify them

by GC-MS application (Kanthal et al., 2014).

Also, the ethanolic extract of S. alata can cause

significant toxic effects on rats due to the pres-

ence of some compounds like emodin, aloe-

emodin, kaempferol and rhein (Yagi, Tigani, &

Adam, 1998). Fernand and colleagues assessed

the range of phenolic compounds for S. alata

between 81.2 to 106.0 % (Fernand et al., 2008).

319

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Therefore, the present study focused on the

antioxidant, antiproliferative, and cytotoxicity

induced by extract/fractions in breast cancer

(MCF-7) cells and normal human mammary

epithelial (MCF10A) cells.

MATERIALS AND METHODS

Plant materials: The leaves of S. alata

were obtained from Penang Golf Resort

(5°31’13.8” N & 100°26’35.4” E) in Bertam

(North of Penang State) in November 2019.

The plant was identified by a botanist, and a

voucher specimen kept at the Herbarium Unit,

School of Biological Sciences, Universiti Sains

Malaysia, Penang Island, Malaysia.

Extraction Procedure: The leaves (3 Kg)

of S. alata were thoroughly washed with double

distilled water, dried at room temperature, and

pulverized using a mechanical blender (Retsch,

ZM200, Germany) at Cluster of Integrative

Medicine Laboratory, Advanced Medical and

Dental Institute, Universiti Sains Malaysia.

Then the powder plants weighed 100 g in each

Erlenmeyer flask. Each flask was macerated

with hydroalcoholic (80 % ethanol) containing

400 mL of solvent. Maceration was done for

three days with mechanical stirring (Bioteck,

Elx808) with a constant speed of 150 rpm. The

solvent changed daily with a new hydroalco-

holic solvent, and residues were macerated in

the respective solvent for the next day to reach

exhaustive extraction (up to 3 days). After

maceration, filtration was performed using

Whatman filter paper (150 mm). The rotavapor

(Eyela, Japan) was used to concentrate the total

filtrate of alcoholic extract to dryness. The

concentrated extract was removed from the

round bottom flask into a weighed small glass

bottle as crude 80 % ethanolic extract. This

crude extract was then fractioned by liquid-

liquid extraction using separation funnel and

resulted in n-hexane, dichloromethane, chloro-

form, butanol, and aqueous fractions. Vacuum

evaporator was used to evaporate each of the

extract and fractions. The concentrated extracts

were frozen at -2 °C until further application.

The yield of each extract was measured and

kept until further use. Fifty milligrams of dried

samples from maceration, including crude 80

% ethanol extract, n-hexane, dichloromethane

(DCM), chloroform, butanol and aqueous were

dissolved with 100 % DMSO in 1 mL tube,

then sonicated to dissolve the dried samples.

ABTS scavenging activity: The antioxi-

dant activity of various concentration (10,

5, 2.5, 1.25, 0.625, 1563, 0.078 mg/mL) of

S. alata 80 % ethanol extract and Trolox was

determined by using ABTS assay. ABTS free

radical scavenging was carried out as previous-

ly explained (Re et al., 1999). Firstly, the stock

solutions of 7 mM ABTS solution and 2.45

mM of potassium persulfate solution was pre-

pared and combined to make the working solu-

tion ABTS

•+

at an 8:12 (v/v) ratio. Then was

maintained in the dark at room temperature for

16 to 18 h. The solution was then blended by

mixing 4.0-4.5 mL ABTS radical solution with

250 mL distilled water to give an absorbance

of 0.70 ± 0.02 at 734 nm. Next, 100 µL extract

(0.078 to 10 mg/mL) in absolute ethanol was

applied to 180 µL of ABTS

•+

working reagent

in a 96-well plate. At room temperature, for 45

minutes the 96-well plate was incubated, and

the absorbance was recorded at 734 nm. Tripli-

cate tests have been performed. The scavenging

capacity was analyzed as a scavenging activity.

Scavenging activity (%)

The percentage of ABTS extract scaveng-

ing activity was compared with the percentage

of Trolox. A graph of percent inhibition against

concentration was used to establish IC

Gas chromatographic-mass spectrom-

etry (GC-MS) analysis for crude extract:

Elmer Clarus Mass Spectrometer together with

the Agilent Gas Chromatography (Santa Clara,

CA, U.S.A.) was performed for GC-MS to ana-

lyze the 80 % ethanol extract. A 10 µL syringe

was used to inject one microliter (1 µL) into

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the chromatogram system. The Helium gas

transported the analyte in the column at a flow

rate of 1.2 mL/min. During the examination, a

split ratio of 5:1 was performed. Temperature

of the injector has been scheduled at 220 °C.

The analytes are extracted from capillary col-

umn model Agilent 19091S-433 with HP-5MS,

0.25 mm × 30 m × 0.25 film width. Initially the

temperature of the oven had been adjusted at 70

°C for 2.00 min, heating up to 280 °C at 10 °C/

min. It took 32.5 min overall. The energy used

for ionisation was 60.922eV. Mass measure-

ment was conducted at 300 °C. Identification

of compounds was achieved by contrasting the

mess spectra with the MS library.

Brine shrimp lethality test (BSLT): The

cytotoxicity activity of extract and fractions

was used using BSLT method. This test was

performed following the mentioned protocol

by Meyer et al. (1982) and McLaughlin, Rog-

ers, & Anderson (1998) with a bit modifica-

tion. The larvae of brine shrimp were used as

research specimen. Cysts were put and hatched

at room temperature for 48 hours with a con-

tinuous supply of oxygen, and there is a lamp

above the tank’s open side which attracts the

hatched shrimps near the wall of the tank, and

then incubate for 25-27 °C. The shrimp became

matured as nauplii after 48 hours and ready for

the experiment. The artificial seawater has been

prepared to produce a 38 g/L concentration

by dissolving the sea salt, then the unwanted

particles were extracted to eliminate them. The

number of dead and surviving brine shrimp

nauplii was calculated in every well after 6

and 24 hours of incubation under light. Potas-

sium dichromate was dissolved in artificial

seawater as a positive control, functioned like

a positive control between 0.01 to 3.00 µg/mL

concentrations. Larvae from the first day were

transferred to the 24-well plates (10 per each

well). All the extract and fractions dissolved in

saline water and dimethyl sulfoxide (DMSO).

As a negative control, a saline media contain-

ing DMSO (1 %) were used. Ten nauplii are

counted under a dissecting microscope (Meiji

Techno, 10X) and then transferred with the aid

of Pasteur pipette to each well; for each well a

volume of 2 mL has been retained in order to

achieve the required concentration for extract.

The experiment performed nine concentration

of samples (5000, 2500, 1250, 625, 312, 156,

78.1, 39.06, 19.50 µg/mL). Each concentration

was conducted in three replicates. When larvae

did not show any motion for 10 seconds of

monitoring, they were supposedly dead (Meyer

et al., 1982). Samples of LC

(lethal concen-

tration 50 %) higher than 1000 µg/mL is found

to be toxic to brine shrimp. The surviving

larvae were recorded after 6 and 24 hours of

sample exposure. Statistical analysis was used

to determine the mortality rate and the lethal

concentrations of S. alata extract resulting to

50 % mortality of the brine shrimp (LC

Mortality rate (%)

Cytotoxicity using Sulforhodamine B

(SRB) assay: Sulphorhodamine B (SRB) assay

has been used to determine the cytotoxicity

activity of S. alata extracts using breast car-

cinoma cell line (MCF-7) and normal human

mammary epithelial cells (MCF10A) from

American Type Culture Collection (ATCC,

Manassas, VA, USA). MCF-7 and MCF10A

cell lines were cultured in RPMI 1640 and

Dulbecco’s Modified Eagle Medium (DMEM)

medium, respectively, containing 10 % (v/v)

fetal bovine serum (FBS) and 1 % (v/v) peni-

cillin-streptomycin (PS) (Invitrogen Co., Carls-

bad, CA, USA). Briefly, 1×10

cells/well of

MCF-7 and MCF10A were separately seeded

in 96-well plates in a triplicate row and loaded

100 µL culture medium (RPMI 1640 and

DMEM, for MCF-7 and MCF10A cell lines,

respectively). Microplates were incubated at 37

°C, 5 % CO

, 95 % air, and humidity about 100

%. On the following day, the cells were treated

with seven concentrations of extracts (0, 9.38,

18.75, 37.50, 75.00, 150.00, and 300.00 µg/mL)

for 24, 48, and 72 hrs. Following these hours,

the plate containing extract concentration was

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incubated, and finally, the test ended by adding

cold TCA. 50 µL of cold 30 % (w/v) TCA (at

final concentration, 10 % TCA) was applied for

in-situ cell fixation with incubation at 4 °C for

30 minutes. Then, the supernatant solution has

been discarded, microplates were rinsed with

tap water five times and kept for air-dried. Sul-

forhodamine B (SRB) (50 µL) at 0.4 % (w/v)

in 1 % acetic acid was loaded and incubated for

30 minutes at room temperature. Once staining

is finished, loose dyes have been retrieved, and

the remaining dyes have been removed using

five times washing with 1 % acetic acid. After

the plates were air-dried at room temperature,

and then bounded stain with a 10 mM Tris base.

The optical density (OD) of the plate wells

has been measured with a microplate reader

(Biotek, Elx808) at 570 nm, and the data were

held. The percentage survival (viability) of

treated cells over the control cells ×100 (T/C)

was calculated as cell viability.

% Cell viability

A linear regression of absorbance against

the examined concentrations was calculated

the concentration at which cell proliferation is

inhibited by 50 % (IC

Cell imaging: The high-resolution cell

microscopes were demonstrated after 72 hours

of incubation of cancer cell line and 24 hours

for normal cell line, capturing and tracking

images using an inverted phase-contrast micro-

scope (Olympus, CKX41) of each concentra-

tion for clearly visible cell viability and cell

morphology evaluation.

Statistical analysis: Statistical analysis

was performed using GraphPad Prism Ver.8

(GraphPad Software, 1996). The means of

three replicates are shown in all analytical data

(mean ± standard deviation). P ≤ 0.05 was con-

sidered statistically significant.

RESULTS

The residue of the plant was then extracted

with n-hexane, dichloromethane (DCM), chlo-

roform, butanol and water (aqueous) subse-

quently in the same way to give 80 % EtOH

(10.9 %, yield: 43.9 g), n-hexane (0.22 %,

yield: 0.89 g), DCM (0.05 %, yield: 0.18 g),

chloroform (0.02 %, yield: 0.09 g), butanol

(0.13 %, yield: 0.51 g) and aqueous (0.16 %,

yield: 0.65 g) for using 400 g powder leaves

of S. alata. This study demonstrates that the

ABTS assay IC

values of Trolox as positive

control and 80 % ethanol extract were 0.092

± 0.02 and 5.59 ± 1.50 mg/mL, respectively

(Table 1).

The findings of the GC-MS study of

Senna alata ethanolic extract contribute to

many compounds being identified. The mass

spectrometry attached to the GC classifies

these substances. The GC-MS spectrum and the

potential cytotoxicity of 80 % ethanol extract

(crude extract) to evaluate the biomass chemi-

cal groups revealed the existence of various

compounds with different retention time, as

shown in Fig. 1 and Table 2.

The big fragments of the compound into

small compounds lead to peaks with varying

ratios of m/z. These mass spectra are the com-

pound fingerprint detectable in the data library.

In this analysis, the formula and struc-

ture of 20 biomolecules can be predicted.

Further study can proceed to the isolation

of bioactive compounds, and their structural

clarification and evaluation and screening of

pharmaceutical activity will be useful for fur-

ther drug research. GC-MS investigated ste-

roids (ɣ-sitosterol), linear alkanes (undecane,

octadecane, eicosane), esters (ethylparaben,

TABLE 1

Antioxidant activity of 80 % ethanolic extract

of S. alata using ABTS assay

ABTS (Radical scavenging assay) mg/mL

80 % Ethanol extract Trolox

value 5.59 ± 1.50 0.092 ± 0.02

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benzoic acid, hexadecanoic acid, ethyl ester,

hexadecanoic acid, methyl ester), tocopherol

(vitamin E and β-tocopherol) as well as fatty

acid such as n-hexadecanoic acid and octadeca-

noic acid in the 80 % EtOH extract.

Only two hours after an interaction with

the higher potassium dichromate concentration,

there was a fatal effect in the brine shrimp.

The LC

value for potassium dichromate was

43.76 μg/mL for the corresponding regression

line and showed toxic signs (LC

against

the brine shrimp was less than 1 000 µg/mL).

Due to high toxicity on A. salina cysts, potas-

sium dichromate has shown limited hatching

success. The median lethal concentration of

the brine shrimp lethality assay (LC

) for

Senna alata leaf extract/fractions are shown

in Table 3.

Fig. 1. GC-MC chromatogram of 80 % ethanolic extract of Senna alata.

TABLE 2

Compound investigated in the 80 % ethanol extract of Senna alata in GC-MS

RT Name of compound

Molecular

formula

Molecular

weight (g/mol)

Percentage

Compound

nature

5.32 1,3,5-Triazine-2,4,6,-triamine C

126.11 80 Cyanamide

5.59 Undecane C

156.31 94 Alkane

7.73 Phenol,2-propyl- C

O 136.19 87 Phenylpropanes

8.52 Cycloheptasiloxane, tetradecamethyl- C

519.07 91 Cyclomethicone

8.72 Ethylparaben C

152.15 93 Ester

8.79 Benzoic acid, 4-ethoxy, ethyl ester C11H14O3 194.23 87 Ester

9.00 Beta-D-Glucopyranoside, methyl C

194.18 80 Glucoside

10.43 Cyclononasiloxane, octadecamethyl- C18H54O9Si9 667.40 91 Polysiloxane

10.96 Hexadecanoic acid, methyl ester C

270.45 93 Ester

11.11 n-Hexadecanoic acid C

256.42 97 Saturated fatty acid

11.28 Hexadecanoic acid, ethyl ester C

284.47 93 Ester

11.88 Phytol C

O 296.50 90 Alcohol

12.00 9,12,5-octadecatrienoic acid, methyl ester C

292.50 83 Ester

12.06 Octadecanoic acid C

284.48 95 Saturated fatty acid

16.76 Octadecane C

254.50 96 Alkane

16.77 Eicosane C

282.50 98 Alkane

19.04 Beta-tocophenol C

416.70 83 Tocopherol

19.50 Eicosane C

282.50 91 Alkane

20.44 Vitamin E C

430.71 97 Tocopherol

24.62 Gamma.sitosterol C

432.70 98 Steroid

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Of the six extracts tested, 2 exhibited no

toxicity to the brine shrimps. These included 80

% ethanolic crude extract and n-hexane frac-

tion, in which no mortality was observed dur-

ing screening. Dichloromethane, chloroform,

butanol, and aqueous fractions showed an LC

value higher than 1 000 µg/mL. There was no

cytotoxic effect on any of the concentrations of

the candle bush 80 % ethanol extract and hex-

ane fraction using the BSLT method and, brine

shrimp were still moved vigorously. However,

the other four extracts showed practically non-

toxic (LC

> 1 000 µg/mL) to brine shrimps.

These extracts were aqueous, dichloromethane,

chloroform, and butanol with LC

values

between 1 034-2 428 µg/mL (after 24 hrs).

TABLE 3

Cytotoxicity activity of various extracts

of Senna alata on brine shrimp

Extract/fraction

(µg/mL)

6 hrs

(acute)

24 hrs

(chronic)

80 % EtOH (Crude extract) ND ND

Hexane ND ND

Dichloromethane ND 1 432

Chloroform 2 520 1 214

Butanol 1 447 1 034

Aqueous 5 053 2 428

ND = not determined; LD

value for potassium dichromate

was 43.76 µg/mL.

Note: The brine shrimp mortality percentage were

measured as mean ± SD.

In the present study, the cytotoxic effect

(IC

) of the crude ethanol and fractioned

extracts (hexane, dichloromethane, chloroform,

butanol and aqueous) were identified on one

human cancer cells (MCF-7) and one normal

non-cancer cells (MCF10A) using the SRB

assay. 80 % EtOH extract did not show toxicity

on both cell lines (Fig. 2A). Hexane fraction

of S. alata exhibited an excellent inhibition

towards MCF-7 cells with IC

of 0.013 µg/mL

at 72 h, in comparison to IC

values of 48 h

(Fig. 2B). It is interesting to note that this frac-

tion did not show cytotoxicity against MCF-7

cells at 24 hours. Others, such as dichlorometh-

ane and butanol against MCF-7 cell line after

72 hours with IC

values of 47.11 and 57.61

µg/mL, respectively, have been shown to have

significant cytotoxic activity (Table 2; Fig. 2B,

Fig. 2C, Fig. 2E). Generally, the 80 % EtOH,

chloroform, and aqueous exhibited weaker

cytotoxicity profile against the MCF-7 cell line

(IC

> 100 µg/mL). The viability of untreated

control cells corresponds to 100 % because all

extracts had no cytotoxic effect on the normal

cell, though we did not analyze the selectivity

index (SI). Although, most importantly, all the

extracts did not show the cytotoxic effect on

MCF10A as normal human mammary epithe-

lial cells. In addition, IC

values were deter-

mined for SRB assay, extracts and the results

are tabulated (Table 4), and also in Fig. 2.

Treated cells were observed for the

morphological feature using a bright-field

TABLE 4

Inhibition concentration (IC

) of various extracts of Senna alata against breast cancer (MCF-7)

and normal human mammary epithelial cells (MCF10A)

Extract/fraction

MCF-7 MCF10A

(µg/mL)

24 hrs 48 hrs 72 hrs 24 hrs

80 % EtOH > 100 > 100 > 100 ND

Hexane ND 9.626 0.013 ND

Dichloromethane > 100 60.03 47.11 ND

Chloroform > 100 > 100 > 100 ND

Butanol 89.30 41.98 57.61 ND

Aqueous > 100 > 100 > 100 ND

ND = not detected.

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microscope (Olympus, CKX41) at 4X and

10X magnification. MCF-7 and MCF10A cells

treated with various extract/fractions and then

observed after 72 h incubation (Fig. 3). The

results only showed for hexane (Fig. 3A, Fig.

3D, Fig. 3G), DCM (Fig. 3B, Fig. 3E, Fig. 3H)

and butanol (Fig. 3C, Fig. 3F, Fig. 3I) fractions.

Significant phenotypic differences were

observed in the presence of Senna alata

extracts as cancer cell line was incubated

(Fig. 3). From cell photographs at first day (24

hours) that the cells treated with fractions in

Fig. 3B and Fig. 3C the cells and their volume

started to decrease and round shape in contrast

to the control of the MCF-7 cells treated with

Tamoxifen which were a simple function of

apoptosis (figure not shown). After 48 and 72

hours, cells became cluster together, exhibited

membrane blebbing (Fig. 3D, Fig. 3E, Fig. 3F,

48 hrs), and began to detach from the dish (Fig.

3H, Fig. 3I, 72 hrs). Normal MCF10A cells,

by contrast, have not shown those significant

morphologic changes (data not shown). This

indicates that S. alata is effective and reason-

ably non-toxic for folk/conventional drugs and

appropriate for cancer treatment.

Fig. 2. In vitro cytotoxic activity of various extracts in MCF-7 cells (Human breast cancer cells) by SRB assay at different

times of exposure (24, 48 and 72 hours). All the values are mean ± SD of three samples. A. 80 % Ethanolic extract, B.

Hexane, C. Dichloromethane, D. Chloroform, E. Butanol, F. Aqueous fraction.

325

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DISCUSSION

Breast cancer is the world’s second most

fatal illness for women (Kamalanathan & Nata-

rajan, 2018). Several other findings have shown

that numerous medicinal plants can be used to

prevent the growth of human breast cancer

(Kamalanathan & Natarajan, 2018). However,

a collection of antioxidant compounds exists

in herbs, fruits and plants have already shown

that breast cancer cells are destroyed by them

without no toxic effect on normal cells (Raj,

Ireland, Ouhtit, Gaur, & Abdraboh, 2015). Both

BSLT and ABTS (antioxidant assay) are easy

to handle, low cost, and use small quantities

of test equipment (Peteros & Uy, 2010; Asna-

ashari et al., 2017). The ABTS radical-scav-

enging measuring technique, a popular method

utilized to test the antioxidant activity, gains

from adopting a hydrogen ion from the antioxi-

dant, decolorizing its blue colors, as ABTS free

radicals become steady (Lee, Oh, Cho, & Ma,

2015). The ABTS assay seems to be more sen-

sitive than DPPH assay in detecting antioxidant

activity due to extreme faster reaction kinetics,

and its reaction to antioxidants is stronger (Lee

et al., 2015), and The ABTS radical is signifi-

cantly more water-soluble than DPPH (He et

al., 2010). Although the antioxidant activity of

leaf extract from S. alata fractionation obtained

a new indole alkaloid, 1-(4′-hydroxyphenyl)-

2,4,6-trihydroxy-indole-3-carboxylic acid that

exhibited strong antioxidant potential with an

of 0.0311 μM ± 0.002 (Olarte, Her-

rera, Villasenor, & Jacinto, 2010). In other

study, ethanol extract from leaves of this plant

showed 67% of the antioxidant activity (Sagnia

Fig. 3. Morphological changes of MCF-7 and MCF10A cells treated with extract/fractions of Senna alata L. during 24,

48 and 72 h. IC

calculated with the SRB assay evaluating dose-responsive curves. Various cell forms shown on MCF-7

and MCF10A, treated with S. alata during 72 h. Vehicle DMSO is used to treat control cells. A. MCF-7 cells with hexane

treatment at 24 hrs; B. Cells with dichloromethane treatment at 24 hrs; C. MCF-7 cells with butanol treatment at 24 hrs;

D. MCF-7 cells with hexane treatment at 48 hrs; E. MCF-7 cells with dichloromethane treatment at 48 hrs; F. MCF-7 cells

with butanol treatment at 48 hrs; G. MCF-7 cells with hexane treatment at 72 hrs; H. MCF-7 cells with dichloromethane

treatment at 72 hrs; I. MCF-7 cells with butanol treatment at 72 hrs; J. MCF10A cells untreated as control after 72 hrs; and

K. MCF-7 cells untreated as a control after 72 hrs.

326

Rev. Biol. Trop. (Int. J. Trop. Biol.) • Vol. 69(1): 317-330, March 2021

et al., 2014). Also, the hexane extract of S.

alata showed no free radical scavenging activ-

ity (Jacinto, Olarte, Galvez, Villasenor, & Pez-

zuto, 2005). To identify bioactive compounds

from 80 % ethanolic extract of S. alata, our

GC-MS result confirmed the study by Ali et

al. (2017), which found the same compounds

mostly, fatty acids composition from leaves of

Senna alata (Ali et al., 2017).

It indicated that the brine shrimp lethality

test was helpful in assessing the toxicity of the

plant extract (Sahgal et al., 2010). This proce-

dure involves exposure of brine shrimp larvae

to plant extract in saline media, and the death

of larvae is measured after one day (Mayilsamy

& Geetharamanan, 2016). Logarto has shown

that a strong link was found between the LC

of the brine shrimp lethality test and LD

the acute oral toxicity test in mice (r = 0.85; P

< 0.05) (Logarto et al., 2001). Upon 24 hours

of treatment, Artemia salina larvae with LC

;

if the sample extract is LC

< 1 000 μg/mL, its

toxicity is high, and the cytotoxicity is expect-

ed to occur. The level of toxicity would have

an anticancer effect on extracts (Prasetyo et al.,

2019). Evaluating the efficiency of hatching

cysts concerning the time of exposure showed

that extracts had notable hatching success

after 36-48 hours, which would be the greatest

hatching time for brine shrimp (Meyer et al.,

1982; Braguini, Pires, & Alves, 2018).

The method of Meyer et al., graded as

toxic (LC

value < 1 000 μg/mL) and non-toxic

(LC

value > 1 000 μg/mL) for crude extracts

and pure materials (Meyer et al., 1982; Naher

et al., 2019). Another study revealed that seed

extract showed more toxic than leaf extract of

S. alata showed LC

value at 4.31 and 5.29

ppm, respectively, from the result of the brine

shrimp lethality test (Rahman, 2004). Also,

the LC

value of the C. alata seed oil extract

was at 250 µg/mL (Mannan et al., 2011),

and 7.74 µg/mL (Parra, Yhebra, Sardiñas, &

Buela, 2001). This suggests that these fractions

may contain no cytotoxic compound. Brine

shrimp mortality was predicted to be related

to bioactive compounds and not malnutrition

after exposure to dichloromethane, chloroform,

butanol, and aqueous fractions. However, the

percentage of deaths as time and concentra-

tion was increased for these fractions and the

existence of toxic compounds in the fractions,

which requires further examination, may lead

to that effect. Several studies showed a strong

correlation with different tumor cell lines in the

BSLT (Elsyana et al., 2016). In BSLT, the cyto-

toxicity activity of the extract is determined by

a 50 % death response to brine shrimp (LC

)

(Elsyana et al., 2016). Based on our hexane

fraction results from MCF-7 cell line, and

according to Elsyana et al., compared this frac-

tion with other extracts and fractions contain-

ing flavonoids and triterpenoids, the maximum

cytotoxic activity was reported by hexane

fraction (Elsyana et al., 2016). Also, Olarte

and colleagues (Olarte et al., 2013) found out

that the hexane extract from S. alata showed

the highest growth inhibition against MCF-7

cell line among three other extracts with IC

value 16 µg/mL which confirm our present

study with IC

values 9.63 and 0.01 µg/mL for

48 and 72 hrs, respectively. However, based

on the National Cancer Institute guideline

(NCI, USA) that 30 µg/mL is the higher IC

ranges assumed reasonable for purification

of an extract (Akindele et al., 2015). Another

study revealed that hexane fraction of S. alata

possessed cytotoxic effect against lung cancer

cell (A549) and ovarian cancer cells (OV2008)

(Levy & Carley, 2012). Also, ethyl acetate

extract of S. alata by other studies showed 50

% inhibition (GI

) value at 5.90 µg/mL against

the MCF-7 cell line (Onyegeme-Okerenta,

2018). On the other hand, the chloroform frac-

tion showed anticancer activity against MCF-7

with IC

value 37.4 µg/ mL (Ali et al., 2017).

According to other studies, chloroform extract

from the stem of three species from Cassia

sp., namely, C. glauca, C. obtusifolia and

C. sophera showed high cytotoxicity against

MCF-7 cell line (Shankar & Surekha, 2017).

Emodin was previously separated from S. alata

leaves (Prasenjit et al., 2016; Ali et al., 2017)

and showed anticancer activity (Hsu & Chung,

2012). These findings revealed that there is

a direct connection between the brine shrimp

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lethality test and in vitro cytotoxicity towards

the S. alata extracts. In the present study, we

displayed that hexane and butanol fractions

induce apoptosis in MCF-7 human breast cells

in a time- and concentration-dependent basis,

which is similar with previous studies using

different extracts of S. alata (Olarte et al.,

2013; Onyegeme-Okerenta, 2018). Our find-

ing indicates that S. alata extracts cytotoxicity

is performed through apoptotic cell death in

tumor cells. In a study by Olarte and colleagues

that they treated hexane fraction with MCF-7

cell line. The MCF-7 cells rounded up and

missed contact with adjacent cells between

12-24 hrs (Olarte et al., 2013).

The anticancer function of flavonoids and

triterpenoids, according to their antioxidant

characteristics, is consistent with their capacity

to scavenge free radicals, to suppress radical

oxygen species (ROS), enzymes and to prevent

cells and extracellular compound oxidation

(Elsyana et al., 2016). Flavonoids and triter-

penoids were concentration-dependent toxic

to the brine shrimp and, therefore, could have

resulted in the death of brine shrimp (Elsyana

et al., 2016). Several studies have shown that

flavonoids can prevent the proliferation and

delay of tumor cells (Razak et al., 2019).

Assessment of bioactive compounds such as

flavonoids, alkaloids, glycosides, carbohy-

drates, protein, saponins, triterpenoids, and

amino acids indicated the existence of most of

the component in polar extracts such as etha-

nol, methanol and aqueous extracts comparison

with nonpolar extracts such as petroleum ether

and chloroform. Though, all extracts possessed

flavonoids, phenols, and tannins (Panda et

al., 2011). Because of its perfect fundamental

chemistry to free radical scavenging activities,

phenols are a significant class of antioxidants

(Chaudhary et al., 2015). However, S. alata

extracts showed potential cancer cell inhibition

and reduced the risk of further proliferation

based on the results of the SRB assay. Jacinto

et al. (2005) identified a high cancer chemo

preventive ability while S. alata hexane leaf

extract was found to cause a particular activ-

ity of the quinone reductase similar to the

bromoflavone as a chemo preventive agent

(Jacinto et al., 2005). More pharmacological

and phytochemical tests are worthwhile in this

research to establish the exact principal cyto-

toxicity compound reaction.

The result of this study shows S. alata

could be an outstanding lead in the progress of

breast cancer anticancer agents (IC

< 100 µg/

mL), which did not exhibit toxicity on normal

cell line as well. Interestingly, in contrast to

SRB assay results, the S. alata extract/fractions

exhibited non-toxic activity (LC

> 1 000

µg/mL) was assessing using the brine shrimp

lethality test as a primary assay for anticancer

activity. The source of organic antioxidants

is available and provides significant medical

benefits. It could be inferred from GC-MS

findings that S. alata contains many bioactive

compounds. Our laboratory is also investigat-

ing further research to clarify the mechanism of

action of apoptosis in breast cancer and bioac-

tive compounds, which will be published in a

future manuscript.

Ethical statement: authors declare that

they all agree with this publication and made

significant contributions; that there is no con-

flict of interest of any kind; and that we fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are

fully and clearly stated in the acknowledge-

ments section. A signed document has been

filed in the journal archives.

ACKNOWLEDGMENTS

This study was supported in part by the

FRGS grant (203.CIPPT.6711684) from Min-

istry of Education (MOE) Government of

Malaysia.

RESUMEN

Baja citotoxicidad, y actividad antiproliferativa

sobre las células cancerosas, de la planta Senna alata

(Fabaceae). Introducción: Las hojas de Senna alata de la

familia Fabaceae se han utilizado en la medicina popular

para la cura de enfermedades de la piel. En este estudio,

328

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probamos el extracto de la planta en líneas celulares norma-

les y cancerosas. Objetivo: Evaluamos la citotoxicidad de

S. alata usando una prueba del camarón Artemia y la activi-

dad antiproliferativa. Métodos: El extracto de hoja etanóli-

co al 80 % y sus fracciones se examinaron en busca de un

posible efecto citotóxico utilizando un ensayo de citotoxi-

cidad de sulforrodamina B (SRB) frente a líneas celulares

de cáncer de mama (MCF-7), normales (MCF10A) y prue-

ba de letalidad del camarón Artemia (BSLT). Resultados:

El bioensayo de letalidad del camarón Artemia no presenta

citotoxicidad incluso en alta concentración (5 000 µg/mL).

La CL50 para diclorometano, cloroformo, butanol y acuoso

fue > 1000 µg/mL (no tóxico). La CI50 para la citotoxici-

dad in vitro de SRB contra MCF-7 para n-hexano fue de

0.013 µg/mL, que se consideró altamente tóxica, mientras

que el diclorometano y el cloroformo registraron 47.11 y

57.61 µg/mL, respectivamente, después de 72 horas de

tiempo de exposición, aunque no hubo citotoxicidad encon-

trada en la línea celular normal. Conclusión: Este estudio

muestra que el extracto de hoja etanólico crudo de S. alata

y sus fracciones contienen potencialmente compuestos bio-

activos significativos que están a salvo de efectos adversos,

lo que demuestra la aplicación terapéutica de S. alata como

remedio tradicional.

Palabras clave: citotoxicidad; Artemia; Senna alata; cán-

cer de mama.

REFERENCES

Abatan, M.O. (1990). A note on the anti-inflammatory

action of plants of some Cassia species. Fitoterapia,

61, 336-338.

Akindele, A.J., Wani, Z.A., Sharma, S., Mahajan, G., Satti,

N.K., Adeyemi, O.O., . . . Saxena, A.K. (2015). In

vitro and In vivo anticancer activity of root extracts of

Sansevieria liberica gerome and labroy (Agavaceae).

Evidence-Based Complementary and Alternative

Medicine, 2015, 560404. DOI: 10.1155/2015/560404

Al-Ansari, M., Al-Humaid, L.A., Vijayaraghavan, P.,

Ravindran, B., Chang, S.W., Agastian, P., . . . Balamu-

ralikrishnan, B. (2019). Identification of phytochemi-

cal components from Aerva lanata (Linn.) medicinal

plants and its in-vitro inhibitory activity against drug

resistant microbial pathogens and antioxidant pro-

perties. Saudi Journal of Biological Sciences, 26(6),

1129-1133. DOI: 10.1016/j.sjbs.2019.02.010

Ali, M.I., Aboul-Enein, A.M., Mohamed, S.M., Elella,

F.M.A., Mohammed, M.M.D., & Hamed, A.R.

(2017). Phytochemical, cytotoxicity and antioxidant

investigation of Cassia alata leaves growing in

Egypt. Journal of Innovations in Pharmaceutical and

Biological Sciences (JIPBS), 4(4), 97-105.

Asnaashari, S., Delazar, A., Asgharian, P., Lotfipour, F.,

Moghaddam, S.B., & Afshar, F.H. (2017). In-vitro

bioactivity and phytochemical screening of extracts

from rhizomes of Eremostachys azerbaijanica rech.

f. growing in Iran. Iranian Journal of Pharmaceutical

Research, 16(1), 306-314.

Azamjah, N., Soltan-Zadeh, Y., & Zayeri, F. (2019). Global

trend of breast cancer mortality rate: A 25-year study.

Asian Pacific Journal of Cancer Prevention, 20(7),

2015-2020. DOI: 10.31557/APJCP.2019.20.7.2015

Beutler, J.A. (2019). Natural products as a foundation for

drug discovery. Current Protocols in Pharmacology,

86(1), e67. DOI: 10.1002/cpph.67

Braguini, W.L., Pires, N.V., & Alves, B.B. (2018). Phyto-

chemical analysis, antioxidant properties and brine

shrimp lethality of unripe fruits of Solanum viarum.

Journal of Young Pharmacists, 10(2), 159-163.

Chaudhary, S., Chandrashekar, K.S., Pai, K.S.R., Setty,

M.M., Devkar, R.A., Reddy, N.D., & Shoja, M.H.

(2015). Evaluation of antioxidant and anticancer

activity of extract and fractions of Nardostachys jata-

mansi DC in breast carcinoma. BMC Complementary

and Alternative Medicine, 15(1), 50. DOI: 10.1186/

s12906-015-0563-1

Elsyana, V., Bintang, M., & Priosoeryanto, B.P. (2016).

Cytotoxicity and antiproliferative activity assay of

Clove Mistletoe (Dendrophthoe pentandra (L.) Miq.)

Leaves Extracts. Advances in Pharmacological Scien-

ces, 2016, 3242698. DOI: 10.1155/2016/3242698

Fernand, V.E., Dinh, D.T., Washington, S.J., Fakayode,

S.O., Losso, J.N., van Ravenswaay, R.O., & Warner,

I.M. (2008). Determination of pharmacologically

active compounds in root extracts of Cassia alata

L. by use of high-performance liquid chromato-

graphy. Talanta, 74(4), 896-902. DOI: 10.1016/j.

talanta.2007.07.033

GraphPad Software. (1996). GraphPad Prism version 8.00

for Windows. Retrieved from https://www.graphpad.

com/scientific-software/prism

He, W., Liu, X., Xu, H., Gong, Y., Yuan, F., & Gao, Y.

(2010). On-line HPLC-ABTS screening and HPLC-

DAD-MS/MS identification of free radical scaven-

gers in Gardenia (Gardenia jasminoides Ellis) fruit

extracts. Food Chemistry, 123(2), 521-528.

Hennebelle, T., Weniger, B., Joseph, H., Sahpaz, S., &

Bailleul, F. (2009). Senna alata. Fitoterapia, 80(7),

385-393. DOI: 10.1016/j.fitote.2009.05.008

Henry, Y.W. (2017). Brine Shrimp Cytotoxic Activity of

Morinda elliptica Leaves and Root Crude Extracts.

Borneo Journal of Resource Science and Technology,

7(1), 43-46. DOI: 10.33736/bjrst.390.2017

Heyde, H. (1990). Medicinal plants in Suriname. Parama-

ribo, Suriname: Mungra & Madarie.

329

Rev. Biol. Trop. (Int. J. Trop. Biol.) • Vol. 69(1): 317-330, March 2021

Hsu, S.C., & Chung, J.G. (2012). Anticancer potential of

emodin. BioMedicine, 2(3), 108-116. DOI: 10.1016/j.

biomed.2012.03.003

Ibrahim, D., & Osman, H. (1995). Antimicrobial acti-

vity of Cassia alata from Malaysia. Journal of

Ethnopharmacology, 45(3), 151-156. DOI:

10.1016/0378-8741(94)01200-J

Idris, O.A., Wintola, O.A., & Afolayan, A.J. (2019). Eva-

luation of the bioactivities of Rumex crispus L. leaves

and root extracts using toxicity, antimicrobial, and

antiparasitic assays. Evidence-Based Complementary

and Alternative Medicine, 2019, 6825297. DOI:

10.1155/2019/6825297

Igoli, J.O., Igwue, I.C., & Igoli, N.P. (2004). Traditional

medicinal practices among the Igede people of Nige-

ria. Journal of Herbs, Spices & Medicinal Plants,

10(4), 1-10. DOI: 10.1300/J044v10n04_01

Iyengar, M.A., Rama, M.P., & Rao, G. (1995). Antifungal

activity of Cassia alata, leaf extract. Indian Drugs,

32(5), 230-231.

Jacinto, S.D., Olarte, E.I., Galvez, M., Villasenor, I.M., &

Pezzuto, J.M. (2005). Leaf extracts from Cassia alata

L. (“Akapulko”) induces quinone reductase and com-

petes for estrogen receptor binding indicating cancer

chemopreventive property. Philippine Agricultural

Scientist, 88(2), 175-178.

Kamalanathan, D., & Natarajan, D. (2018). Anticancer

potential of leaf and leaf-derived callus extracts of

Aerva javanica against MCF-7 breast cancer cell line.

Journal of Cancer Research and Therapeutics, 14(2),

321-327. DOI: 10.4103/0973-1482.171210

Kanthal, L.K., Dey, A., Satyavathi, K., & Bhojaraju,

P. (2014). GC-MS analysis of bio-active com-

pounds in methanolic extract of Lactuca runcinata

DC. Pharmacognosy Research, 6(1), 58-61. DOI:

10.4103/0974-8490.122919

Karchesy, Y.M., Kelsey, R.G., Constantine, G., & Kar-

chesy, J.J. (2016). Biological screening of selected

Pacific Northwest forest plants using the brine shrimp

(Artemia salina) toxicity bioassay. Springerplus, 5,

510. DOI: 10.1186/s40064-016-2145-1

Khan, M.R., Kihara, M., & Omoloso, A.D. (2001). Anti-

microbial activity of Cassia alata. Fitoterapia, 72(5),

561-564. DOI: 10.1016/S0367-326X(00)00335-X

Lee, K.J., Oh, Y.C., Cho, W.K., & Ma, J.Y. (2015). Antioxi-

dant and anti-Inflammatory activity determination of

one hundred kinds of pure chemical compounds using

offline and online screening HPLC assay. Evidence-

Based Complementary and Alternative Medicine,

2015, 165457. DOI: 10.1155/2015/165457

Levitsky, D.O., & Dembitsky, V.M. (2014). Anti-breast

cancer agents derived from plants. Natural Products

and Bioprospecting, 5(1), 1-16. DOI: 10.1007/

s13659-014-0048-9

Levy, A.S., & Carley, S. (2012). Cytotoxic activity of

hexane extracts of Psidium guajava L (Myrtaceae)

and Cassia alata L (Caesalpineaceae) in Kasumi-1

and OV2008 cancer cell lines. Tropical Journal of

Pharmaceutical Research, 11(2), 201-207.

Logarto, A., Silva, R., Guerra, I., & Iglesias, L. (2001).

Comparative study of the assay of Artemia salina L.

and the estimate of the medium lethal dose (LD50

value) in mice, to determine oral acute toxicity of

plant extracts. Phytomedicine, 8(5), 395-400. DOI:

10.1078/0944-7113-00044

Mannan, A., Kawser, M.J., Ahmed, A.A., Islam, N.N.,

Alam, S.M., Khan, M.A.E., & Gupta, S.D. (2011).

Assessment of antibacterial, thrombolytic and cyto-

toxic potential of Cassia alata seed oil. Journal of

Applied Pharmaceutical Science, 1(9), 56.

Mayilsamy, M., & Geetharamanan, K. (2016). Cytotoxic

activity of certain medicinal plants extracts against

sea monkey: Artemia salina. Journal of Medicinal

Herbs and Ethnomedicine, 2, 19-25.

McLaughlin, J.L., Rogers, L.L., & Anderson, J.E. (1998).

The use of biological assays to evaluate botanicals.

Drug Information Journal, 32(2), 513-524. DOI:

10.1177/009286159803200223

Meyer, B.N., Ferrigni, N.R., Putnam, J.E., Jacobsen, L.B.,

Nichols, D.E., & McLaughlin, J.L. (1982). Brine

shrimp: a convenient general bioassay for active

plant constituents. Planta Medica, 45(5), 31-34. DOI:

10.1055/s-2007-971236

Mohanraj, K., Karthikeyan, B.S., Vivek-Ananth, R.P.,

Chand, R.P.B., Aparna, S.R., Mangalapandi, P., &

Samal, A. (2018). IMPPAT: A curated database of

Indian medicinal plants, phytochemistry and thera-

peutics. Scientific Reports, 8(1), 4329. DOI: 10.1038/

s41598-018-22631-z

Naher, S., Aziz, M.A., Akter, M.I., Rahman, S.M.M.,

Sajon, S.R., & Mazumder, K. (2019). Anti-diarrheal

activity and brine shrimp lethality bioassay of metha-

nolic extract of Cordyline fruticosa (L.) A. Chev. lea-

ves. Clinical Phytoscience, 5(1), 15. DOI: 10.1186/

s40816-019-0109-z

Olarte, E.I., Herrera, A.A., Villasenor, I.M., & Jacinto, S.D.

(2010). Antioxidant activity of a new indole alkaloid

from Cassia alata L. Philippine Agricultural Scien-

tist, 93(3), 250-254.

Olarte, E.I., Herrera, A.A., Villasenor, I.M., & Jacinto, S.D.

(2013). In vitro antitumor properties of an isolate

from leaves of Cassia alata L. Asian Pacific Journal

of Cancer Prevention, 14(5), 3191-3196.

Onyegeme-Okerenta, B. (2018). Ethyl acetate extract of

Senna alata (L) Roxb increases cytotoxicity in the

330

Rev. Biol. Trop. (Int. J. Trop. Biol.) • Vol. 69(1): 317-330, March 2021

human breast, prostate and colorectal cancer cells.

Journal of Cancer Treatment and Research, 6(3),

44-53.

Palanichamy, S., Bhaskar, E.A., Bakthavathsalam, R., &

Nagarajan, S. (1991). Wound healing activity of Cas-

sia alata. Fitoterapia, 62(1), 153-156.

Palanichamy, S., & Nagarajan, S. (1990). Antifungal

activity of Cassia alata leaf extract. Journal of

Ethnopharmacology, 29(3), 337-340. DOI:

10.1016/0378-8741(90)90043-S

Pamulaparthi, A., & Nanna, R.S. (2015). Determination of

anticancer activity of aqueous leaf extracts of Senna

alata using MTT assay. Journal of Cancer Science

and Therapy, 7, 10. DOI: 10.4172/1948-5956.C1.055

Panda, S.K., Padhi, L.P., & Mohanty, G. (2011). Antibac-

terial activities and phytochemical analysis of Cassia

fistula (Linn.) leaf. Journal of Advanced Pharma-

ceutical Technology & Research, 2(1), 62-67. DOI:

10.4103/2231-4040.79814

Parra, A.L., Yhebra, R.S., Sardiñas, I.G., & Buela, L.I.

(2001). Comparative study of the assay of Artemia

salina L. and the estimate of the medium lethal dose

(LD50 value) in mice, to determine oral acute toxicity

of plant extracts. Phytomedicine, 8(5), 395-400.

Peteros, N.P., & Uy, M.M. (2010). Antioxidant and cyto-

toxic activities and phytochemical screening of four

Philippine medicinal plants. Journal of Medicinal

Plants Research, 4(5), 407-414.

Prasenjit, M., Tanaya, G., Sumanta, G., Basudeb, B., &

Kumar, M.P. (2016). Isolation and characterization

of a compound from the leaves of Cassia alata Linn.

ECronicon Chemistry, 2(2), 138-144.

Prasetyo, A., Sidharta, B.R., Hartini, Y.S., & Mursyanti,

E. (2019). Toxicity of bioactivity compound from

endophytic fungi isolated from red ginger (Zingiber

officinale var. rubrum) utilizing brine shrimp lethality

assay. Biogenesis: Jurnal Ilmiah Biologi, 7(1), 30-37.

DOI: 10.24252/bio.v7i1.6000

Promgool, T., Pancharoen, O., & Deachatai, S. (2014).

Antibacterial and antioxidative compounds from

Cassia alata Linn. Songklanakarin Journal of Scien-

ce and Technology, 36(4), 459-463.

Rahman, Ì. (2004). Brine shrimp toxicity of leaf and seed

extracts of Cassia alata Linn, and their antibacterial

potency. Journal of Medical Sciences, 4(3), 188-193.

Raj, M., Ireland, S., Ouhtit, A., Gaur, R., & Abdraboh, M.

(2015). Complementary/alternative medicine strate-

gies for prevention and or cure of breast cancer: a

review. Women’s Health International, 1(2), 111.

Razak, N.A., Abu, N., Ho, W.Y., Zamberi, N.R., Tan, S.W.,

Alitheen, N.B., ... Yeap, S.K. (2019). Cytotoxicity

of eupatorin in MCF-7 and MDA-MB-231 human

breast cancer cells via cell cycle arrest, anti-angioge-

nesis and induction of apoptosis. Scientific Reports,

9(1), 1514. DOI: 10.1038/s41598-018-37796-w

Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang,

M., & Rice-Evans, C. (1999). Antioxidant acti-

vity applying an improved ABTS radical cation

decolorization assay. Free Radical Biology and

Medicine, 26(9-10), 1231-1237. DOI: 10.1016/

s0891-5849(98)00315-3

Sagnia, B., Fedeli, D., Casetti, R., Montesano, C., Falcioni,

G., & Colizzi, V. (2014). Antioxidant and anti-inflam-

matory activities of extracts from Cassia alata, Eleu-

sine indica, Eremomastax speciosa, Carica papaya

and Polyscias fulva medicinal plants collected in

Cameroon. PLoS One, 9(8), e103999. DOI: 10.1371/

journal.pone.0103999

Sahgal, G., Ramanathan, S., Sasidharan, S., Mordi, M.N.,

Ismail, S., & Mansor, S.M. (2010). Brine shrimp

lethality and acute oral toxicity studies on Swietenia

mahagoni (Linn.) Jacq. seed methanolic extract.

Pharmacognosy Research, 2(4), 215-220. DOI:

10.4103/0974-8490.69107

Saito, S.T., Trentin, S., Macedo, A.J., Pungartnik, C.,

Gosmann, G., Silveira, D., . . . Brendel, M. (2012).

Bioguided fractionation shows Cassia alata Extract

to inhibit Staphylococcus epidermidis and Pseudo-

monas aeruginosa growth and biofilm formation.

Evidence-Based Complementary and Alternative

Medicine, 2012, 867103. DOI: 10.1155/2012/867103

Shankar, B.N., & Surekha, R.D. (2017). Cytotoxicity of

stem extracts of selected Cassia species against Hela

and breast cancer cell lines in vitro. Asian Journal of

Pharmaceutical and Clinical Research, 10(3), 80-82.

DOI: 10.22159/ajpcr.2017.v10i3.11991

Shareef, M., Ashraf, M.A., & Sarfraz, M. (2016). Natural

cures for breast cancer treatment. Saudi Pharma-

ceutical Journal, 24(3), 233-240. DOI: 10.1016/j.

jsps.2016.04.018

Tene, O., Tala, V.R.S., Tatong, N., & Tchuente, K. (2017).

Ethnobotanical uses, phytochemical and pharma-

cological profiles, and toxicity of Cassia alata L.

An overview. Medicine and Medical Sciences, 4(2),

16-24.

Yagi, S.M., Tigani, S.E., & Adam, S.E.I. (1998).

Toxicity of Senna obtusifolia fresh and fermen-

ted leaves (kawal), Senna alata leaves and some

products from Senna alata on rats. Phytotherapy

Research, 12(5), 324-330. DOI: 10.1002/(sici)1099-

1573(199808)12:5<324::Aid-ptr300>3.0.Co;2-2