Antioxidant, Cytotoxic and Antibacterial Activity and Total Phenol Contents of the Roots and the Shoots of Euphorbia macrostegia and Euphorbia microsciadia

Journal of Environmental Treatment Techniques

2021, Volume 9, Issue 1, Pages: 328-334

J. Environ. Treat. Tech.

ISSN: 2309-1185

Journal web link: http://www.jett.dormaj.com

https://doi.org/10.47277/JETT/9(1)334

Antioxidant, Cytotoxic and Antibacterial Activity

and Total Phenol Contents of the Roots and the

Shoots of Euphorbia macrostegia and Euphorbia

microsciadia

1,2

Somayeh Zare , Niloofar Moheimanian , Omidreza Firuzi , Amir Reza Jassbi

Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

Students Research Committee, Shiraz University of Medical Sciences, Shiraz, Iran

Received: 06/01/2020

Accepted: 13/04/2020

Published: 20/03/2021

Abstract

The roots and the shoots of Euphorbia macrostegia and E. microsciadia were extracted using different solvents; dichloromethane

(

DCM), methanol (MeOH) and MeOH: water (80:20) and the extracts were screened for their cytotoxic, antioxidant and antimicrobial

properties as well as total phenolic content. Cytotoxicity of the extracts was evaluated against human acute lymphoblastic leukemia

MOLT-4) cells by MTT reduction assay. The extracts were also subjected to the 2,2'-diphenyl-1-picrylhydrazyl (DPPH) free radical

(

scavenging and Folin-Ciocalteu total phenolic assays. The MeOH extract of the shoots of E. microsciadia and DCM extract of the roots

of E. macrostegia were the most cytotoxic ones with IC50 values of 10.5 ± 2.6 and 7.0 ± 1.2 µg/mL, respectively. The MeOH extract of

the shoots of E. microsciadia showed considerable antioxidant activity (IC50 = 9.95 ± 1.00 µg plant extracted to scavenge 1 mL of a 0.1

mM DPPH solution), which was consistent with its highest phenolic content (288.50 ± 29.38 mg equivalent of gallic acid in 1g dry plant

extract: mg EG/g PE). Determination of minimum inhibitory concentrations (MICs) using the broth dilution method, confirmed that all

the extracts from the plants gave various degrees of antibacterial activity against all tested microorganisms. In thin layer chromatography

(

TLC) investigations, some compounds previously isolated from Euphorbia species inclduing cycloartenol, 24-methylenecycloartan-3β-

ol, β-sitosterol and euphol were tested and suggested to be responsible for the above-mentioned biological activities in the plants.

Therefore, we suggest E. macrostegia and especially E. microsciadia as new sources for isolation and identification of various bioactive

compounds.

Keywords: Euphorbia microsciadia, Euphorbia macrostegia, anticancer effect, biological activity, radical scavenging, natural

compounds and phytochemical analyses

attributed to the

presences

quercetin-3-β-O-

Introduction

galactopyranoside, while myricetin-3-β-O-galactopyranoside

was less active constituent among the isolated flavonol

glycosides from the aerial part of the plant (6). On the other

hand, a dichloromethane (DCM) extract of E. macrostegia

showed cytotoxicity against two cancer cell lines; MDA-MB-

Euphorbia is the largest genus of the family Euphorbiaceae

spurges), with more than 2000 known species and is

(

characterized by the presence of milky latex and unique flower

structures. It has about 80 species in Iran, two of which are

called locally "Dena and Persian spurge", E. microsciadia

Boiss. and E. macrostegia Boiss., respectively. They grow in

the mountainous area of Iran (1, 2). Various species of the

genus Euphorbia have shown different biological activities

including enzyme inhibition and cytotoxic activity (3). In

addition to their biological activities, these plants are

ecologically important in the flora of Iran as weeds, anti-

vegetative and poisonous plants (2).

It has been previously shown that a butanol/hexane extract

of the aerial parts of E. microsciadia had the ability to modulate

T-cell responses that suggest its possible beneficial effect on

immune host defense (4). A MeOH extract of E. microsciadia

showed stimulatory effects on the proliferation of the

lymphocytes and a n-hexane layer of the MeOH extract had

significant strong antiproliferative effect against tumor cells

8 and MCF-7 (7). Four cycloartane triterpenoids were isolated

from the DCM extract. Two of which; cycloart-23(E)-ene-

β,25-diol and cycloart-23(Z)-ene-3β, 25-diol, showed the

strongest cytotoxicity against the above mentioned cell lines,

respectively. In addition to the cytotoxic activity, the tyrosinase

inhibitory activity of constituents of E. macrostegia has also

been reported recently (8). Among 10 compounds identified in

the

plants’

extract,

2-(4-hydroxyphenyl)-

ethylhentriacontanoate, hentriacontan-1-ol, lupenone and

cycloart-22-ene-3,25-diol were detected as the most active

tyrosinase inhibitors with IC50s of 71.4-78.6 μM. Also, recently

we isolated three triterpenoids; 24-methylenecycloartan-3β-ol,

butyrospermol and cycloartenol and three diglycerides, 1,2-di-

O-α-linolenoyl-sn-glycerol, 1-O-linoleoyl-3- O-palmitoyl-sn-

glycerol and 1-O-α-linolenoyl-2-O-palmitoyl-sn-glycerol from

(

5). The immunosuppressive activity of the plant's extract was

Corresponding Author: Amir Reza Jassbi: Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical

Sciences, Shiraz, Iran. Tel: +98-71-32303872, E-mail: jassbiar@sums.ac.ir

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Journal of Environmental Treatment Techniques

2021, Volume 9, Issue 1, Pages: 328-334

the hexane soluble part of MeOH/DCM extracts of E.

macrostegia Boiss. (9).

In this study, we report here antimicrobial potential, DPPH

radical scavenging activity, total phenolic contents and

cytotoxicity of different extracts of the roots and shoots of E.

macrostegia and E. microsciadia.

of mobile phase were examined to obtain optimum retention

factor (R

thymol-sulfuric acid (0.5 g thymol in 95 mL EtOH and 5 mL

97% H SO ) and vanillin-sulfuric acid (0.3 g vanillin: 28 ml

) and resolution. In addition, two TLC reagents

ethanol: 1 ml sulfuric acid), were sprayed followed by heating

the developed TLCs to distinguish different phytochemical

classes such as glycosides and terpenes in different colors (12).

Materials and Methods

.1 General Experimental Procedures

RPMI 1640 (cell culture medium), fetal bovine serum

(

FBS), trypsin, and phosphate buffered saline (PBS) were

purchased from Biosera (Ringmer, UK). Chloramphenicol and

-(4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium

bromide (MTT) were obtained from Sigma-Aldrich (St Louis,

MO, USA) and penicillin ⁄ streptomycin were purchased from

Invitrogen (San Diego, CA, USA). Doxorubicin and Folin-

Ciocalteu reagents were obtained from EBEWE Pharma

(

Unterach, Austria) and Fluka, respectively. 2,2'-diphenyl-1-

picrylhydrazyl (DPPH), Vanillin and p-iodonitrotetrazolium

violet (INT) were purchased from Merck chemical companies.

All the solvents were purchased from Merck. Thymol was

purchased from Riedel-de Haen (Germany).

.2 Plant material

Euphorbia microsciadia Boiss. and Euphorbia

Figure 1: Structures of some compounds previously isolated from

Euphorbia plants

macrostegia Boiss. were collected in July 2012 from the Dena

◦

mountain (N 30 52′ E 51 31′, 2980 m altitude), Yasuj, Iran. The

plants were identified by Mr. Mehdi Zare and Dr. Mojtaba

Asadollahi, plant taxonomists, in Medicinal and Natural

Products Chemistry Research Center (MNCRC), Shiraz

University of Medical Sciences, Iran. A voucher specimen for

E. macrostegia (PC-91-4-1-1.2) and E. microsciadia (PC-91-

.5 Cytotoxic bioassay

Human acute lymphoblastic leukemia (MOLT-4) cells

were obtained from the National Cell Bank of Iran, Pasteur

Institute, Tehran, Iran. MTT assay was performed to assess

viability after the exposure of cells to the extracts (13, 14). The

cells were cultured in RPMI 1640 medium supplemented with

-2-1.2) has been deposited in the herbarium of MNCRC.

00 units⁄mL penicillin-G, 10% FBS, and 100 µg⁄mL

streptomycin and maintained at 37 C in humidified air

containing 5% CO . The experiments were performed in 96-

.3 Extraction procedure

The shade-dried powdered shoots and roots of E.

well microplates and 3000 cells were seeded in each well. The

wells containing growth medium alone were used as blank for

background correction. After overnight incubation at 37°C,

half of the growth medium was removed and medium

supplemented with different concentrations of extracts were

added in triplicate. The extracts of E. microsciadia and E.

macrostegia were first dissolved in DMSO and then diluted

several times in complete growth medium. Maximum

concentration of DMSO in the wells did not exceed 0.5%, a

concentration that showed no cytotoxicity in the cells. Standard

cytotoxic agents; cisplatin and doxorubicin were used as

positive controls. After incubation for further 72 h, the medium

was removed and MTT was added to each well at a final

concentration of 0.5 mg⁄mL and plates were incubated for

another 4 h at 37°C. In the end, formazan crystals were

dissolved in 200 μl DMSO. The optical density was evaluated

at 570 nm using a microplate reader (Bio-Rad, Model 680) with

background correction at 655 nm. The percentage of viability

compared to control wells was calculated for each

concentration of the extracts and IC50 values were estimated

with the software Curve Expert (for Windows, version 1.34).

Each experiment was repeated at least 3 times and data were

presented as mean ± S.E.M.

microsciadia (120 g) and E. macrostegia (120 g) were

extracted twice separately and sequentially in 1500 mL of

DCM, MeOH and 80% MeOH, by maceration for 24 h. at room

temperature. Each filtered extract was concentrated to remove

traces of the solvents under reduced pressure using a rotary

evaporator to afford the respective dried solvent extracts. The

weight of the shoots and the roots extracts of the plants were

respectively as follows: DCM (1.04 and 5.8 g), MeOH (12.4

and 4.2 g), and 80% MeOH (4.16 and 1.44 g) for E.

microsciadia and DCM (2.4 and 1.44 g), MeOH (0.8 and 2.8

g), and 80% MeOH (4.08 and 1.04 g) for E. macrostegia.

.4 Preparation of the extracts, pure compounds, and TLC

conditions

The dried MeOH and 80% MeOH extracts were dissolved

in MeOH to a concentration of 5 mg/mL for TLC analysis. Also

for the bioassays, the extracts were prepared in their extracting

solvents in different concentrations. Pure phytochemicals,

previously isolated from Euphorbia plants in our group, named

cycloartenol (1), 24-methylenecycloartan-3β-ol (2), β-

sitosterol (3) and euphol (4), were selected for the assessment

of their existence in the above extracts (Figure 1). They were

extracted from the hexane soluble part of methanol-

dichloromethane extracts of the aerial parts of E. macrostegia

.6 DPPH radical scavenging activity

The antioxidant activities of all extracts of E. microsciadia

(

1 and 2) (9), methanol-dichloromethane extracts of E.

erythradenia (3) (10), and acetone extract of the roots of E.

microsciadia (4) (11). They were dissolved in MeOH (5

mg/mL) for spotting on TLC plates. We analyzed the chemical

constituents of the extracts compared to the standard

phytochemicals 1-4 using pre-coated TLC plates (silica gel 60

F254, 0.25 mm film thickness, Merck). Different composition

and E. macrostegia were determined according to the modified

method that we have previously described (9, 15-17). Briefly,

μL of the extracts was mixed with 195 μL of 0.1 mM DPPH

in 96-well microplate. After incubation in the dark at room

temperature for 30 minutes, the absorbance of the reaction

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2021, Volume 9, Issue 1, Pages: 328-334

mixture was measured at λ 517 nm using a Bio-Rad microplate

reader. Inhibition ratio (percent) was calculated from the

following equation:

micro-dilution (NBMD) assays (19). To perform the test, the

stock solution of plant extracts were serially diluted in dimethyl

sulfoxide (DMSO) to afford different concentrations of test

samples. Chloramphenicol solution in DMSO was also

prepared as the positive control. Breifly, 5 µL of the tests

sample solutions was added to 95 µL of the fresh media and

DPPH radical scavenging activity (%) = [(AbsControl - AbsSample

AbsControl] × 100

)

100 µL of bacterial suspension culture (OD = 0.1 at 600 nm) in

We used butylated hydroxytoluene (BHT) and quercetin as

a 96-well microplate. After 24 h incubation at 37 °C in a

shaking incubator, 10 µL of a 0.5% INT solution in water was

added to each well. Afterwards, at the above-mentioned culture

conditions, the microplates were incubated for further 30 min.

Finally, the MIC was calculated as the minimum concentration

of the test sample extract or antibacterial standard inhibiting the

growth of bacterial strain by discoloration of the purple INT

solution (6).

the standards radical scavenger. The IC50 values were

calculated by linear regression equations of the DPPH

inhibition percentage from different concentrations of the

extracts and the standards, using Microsoft Excel and Curve

Expert statistical programs (15).

.7 Determination of total phenolic content

The concentration of total phenolics in the DCM, MeOH

and 80% MeOH extracts were determined separately by the

Folin-Ciocalteu colorimetric method as described previously

Results and Discussion

Cytotoxicity of DCM, MeOH and 80% MeOH extracts of

(

18). Briefly, in each test plate, 5 μL of the 10 mg/mL plant

the roots and shoots of E. macrostegia and E. microsciadia

were tested against MOLT-4 cells (Table 1). Among the tested

extracts of E. microsciadia, the DCM extracts showed no

activity. The MeOH and 80% MeOH extracts of the shoots of

this plant with IC50 values of 10.5 ± 2.6 and 17.1 ± 2.9 µg/mL,

respectively, showed stronger activity compared to the same

extracts from the roots with the IC50 values of 46.0 ± 3.5 and

extracts or the standard gallic acid solution, 158 μL distilled

water and 10 μL Folin-Ciocalteu reagent were added and the

solution was shaken briefly on a vortex mixer well, then after

.5 min incubation at room temperature 30 μL of a 0.25%

sodium carbonate was added to each solution. The reaction

mixtures were kept in the dark at room temperature for 2 h and

the absorbance of the solutions were measured at λ 765 nm

against the blank. The concentrations of the total phenolics

were measured against a series of gallic acid standard solutions

and expressed as mg equivalent of gallic acid in 1 g plant

extract (mg EG/g PE) (18).

40.0 ± 3.7 µg/mL, respectively. Unlike E. microsciadia, all the

three extracts of E. macrostegia showed strong activity against

MOLT-4 cells with IC50 values in the range of 7.0- 38.4 μg/mL.

Unlike E. microsciadia, the root extracts of E. macrostegia

exhibited lower IC50s compared to the extracts from the shoots

of this plant.

The MeOH and 80% MeOH extracts of the shoots of E.

microsciadia showed the highest radical scavenging potentials

in the DPPH free-radical test, with IC50 values of 9.95 ± 1.00

and 10.82 ± 1.64 µg/mL, respectively (Table 2). Their activities

are more than that measured for butylated hydroxytoluene

.8 Antibacterial minimum inhibitory concentration using

nutrient broth microdilution

To examine the antibacterial activity of the plant extracts,

four Gram-negative bacteria (Escherichia coli: PTCC1330,

Klebsiella pneumoniae: PTCC1053, Pseudomonas aeruginosa:

PTCC 1074, and Salmonella typhi: PTCC1609) and three

Gram-positive bacteria (Staphylococcus aureus: PTCC1112,

Staphylococcus epidermidis: PTCC1114, Bacillus subtilis:

PTCC1023) were chosen to measure the minimum inhibitory

concentrations (MIC) of the active extracts using nutrient broth

(

BHT: IC50ꢀ=ꢀ51.09 ± 1.35 μg/mL, P < 0.05) but less than the

quercetin’s radical scavenging potential with an IC50 value of

.0 ± 0.16 µg/mL (P < 0.05). The other extracts exhibited

radical scavenging activity in the range of 28.51- 85.22 μg/mL.

Table 1: Cytotoxic activity of different extracts of E. macrostegia and E. microsciadia

MOLT-4 IC50 (µg/mL)

MeOH extract)

MOLT-4 IC50 (µg/mL)

(80% MeOH extract)

MOLT-4 IC50 (µg/mL)

(DCM extract)

Sample name

(

E. macrostegia, shoots

E. macrostegia, roots

E. microsciadia, shoots

E. microsciadia, roots

Doxorubicin (nM)

Cisplatin (µM)

34.1 ± 4.2

7.9 ± 1.0

10.5 ± 2.6

46.0 ± 3.5

17.2 ± 2.0

3.1 ± 0.7

38.4 ± 3.2

34.8 ± 3.3

17.1 ± 2.9

40.0 ± 3.7 (11)

13.4 ± 2.3

7.0 ± 1.2

>100

Table 2: DPPH radical scavenging activity and total phenolic contents (mg EG/g PE) of different extracts of E. macrostegia and E.

microsciadia

DPPH IC50 (µg/mL)

MeOH extract)

Total phenol

(MeOH extract)

78.94 ± 3.89

71.65 ± 1.01

288.50 ± 29.38

37.59 ± 2.07

DPPH IC50 (µg/mL)

(80% MeOH extract)

28.51 ± 2.37

35.55 ± 3.14

10.82 ± 1.64

85.22 ± 2.98

51.09 ± 1.35

2 ± 0.16

Total phenol (80%

MeOH extract)

147.12 ± 5.46

132.53 ± 4.43

203.61± 28.58

18.29 ± 1.45

Sample name

(

E. macrostegia, shoots

E. macrostegia, roots

E. microsciadia, shoots

E. microsciadia, roots

BHT

47.64 ± 4.74

41.68 ± 2.97

9.95 ± 1.00

45.39 ± 2.93 (20)

51.09 ± 1.35

2 ± 0.16

Quercetin

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2021, Volume 9, Issue 1, Pages: 328-334

Table 3: Minimum inhibitory concentrations (mg/mL) of different extracts of E. macrostegia and E. microsciadia by nutrient-broth

micro-dilution bioassay

Microorganisms (rows)

Plants

S.a.

S.e.

B.s.

S.t.

P.a.

E.c.

K.p.

E. macrostegia, shoots (DCM extract)

E. macrostegia, shoots (MeOH extract)

E. macrostegia, shoots (80% MeOH extract)

E. macrostegia, roots (DCM extract)

E. macrostegia, roots (MeOH extract)

E. macrostegia, roots (80% MeOH extract)

E. microsciadia, roots (DCM extract)

E. microsciadia, roots (MeOH extract)

E. microsciadia, roots (80% MeOH extract)

E. microsciadia, shoots (DCM extract)

E. microsciadia, shoots (MeOH extract)

E. microsciadia, shoots (80% MeOH extract)

Chloramphenicol

1.25

2.5

1.25

2.5

1.25

2.5

1.25

2.5

1.25

2.5

1.25

2.5

1.25

2.5

1.25

2.5

1.25

2.5

1.25

2.5

0.05

0.0125 0.025

0.0125 0.05

0.05 0.05

NA: not active (>5). S.a., Staphylococcus aureus; S.e., Staphylococcus epidermidis; B.s., Bacillus subtilis; S.t., Salmonella typhi;

P.a., Pseudomonas aeruginosa; E.c., Escherichia coli; K.p., klebsiella pneumoniae

0.12

0.10

0.08

0.06

MeOH extracts

0% MeOH extracts

0.04

0.02

0.00

100

200

300

Total phenolic content (mg EG/g PE)

Figure 2: Correlation graphs for DPPH 1 /IC50 values and total phenolic contents for MeOH and 80% MeOH extracts

The 80% MeOH extract of the roots of E. microsciadia had

the weakest antioxidant activity against DPPH radicals, as the

result of the highest IC50 value (85.22 ± 2.98 µg/ml). The

increasing order of total phenol contents (18.29 ± 1.45 to

DCM extract of E. macrostegia was only active at MIC 1.25

mg/mL against the growth of B. subtilis, E. coli, and K.

pneumonia. Generally, among the microorganisms, B. subtillis

was the most susceptible ones to almost all extracts.

88.50 ± 29.38 mg EG/ g PE) of the plant extracts were in

We have analyzed the bioactive plant extracts using TLC

in comparison to Euphorbia derived phytochemicals. The TLC

analyses of DCM, MeOH and 80% MeOH of the roots and the

shoots of E. macrostegia and E. microsciadia, resulted in

several purple colored spots after vanillin-sulfuric acid reagent

treatments after development by chloroform: acetone (95:5)

agreement with the decreasing order of the IC50 (85.22 ± 2.98

to 9.95 ± 1.00 μg/mL) of DPPH test (Figure 2). The most

prominent total phenolic contents was measured in the shoots

of E. microsciadia, while the roots of E. microsciadia had the

least TP contents. Antimicrobial activity of the DCM, MeOH

and 80% MeOH extracts of the plants were measured against

four different Gram-negative and three Gram-positive bacteria

mobile phase (Figure 3). Two spots with R values between

0.45 and 0.80, were present in all extracts, while the

chromatogram of the methanol roots extract of E. macrostegia

was the most chemically diverse among them. On the other

hand, all of the tested extracts were analyzed by silica-gel TLC

using more polar mobile phase (chloroform: formic acid:

methanol: water (5:0.2:3:0.5)).

(

Table 3). The MeOH extracts of the shoots and roots of E.

macrostegia, and methanol and 80% MeOH extracts of the

roots of E. microsciadia, were the most active antibacterial

extracts that inhibited the growth of all tested microorganisms

at MIC values between 1.25-5 mg/mL (Table 3). In addition,

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2021, Volume 9, Issue 1, Pages: 328-334

Figure 3: Spots in daylight after spray with vanillin-sulfuric acid using silica gel- TLC plates and chloroform: acetone (95:5) mobile phase. Spots are the

MeOH extracts of the shoots (A) and the roots (B) of E. microsciadia, the MeOH extracts of the shoots (C) and the roots (D) of E. macrostegia, the 80%

MeOH extracts of the shoots (E) and the roots (F) of E. microsciadia, the 80% MeOH extracts of the shoots (G) and the roots (H) of E. macrostegia,

DCM extracts of the shoots (I) and the roots (J) of E. microsciadia, and DCM extracts of the shoots (K) and the roots (L) of E. microsciadia

phases (Figure 6, (2), (3)). In literature, compound 1 and 4

showed antioxidant and cytotoxic activity, while compound 3

showed antibacterial activity (20-24). Compounds 1 and 2 are

present in almost all extracts in different ratios (Figure 6 (1),

(

3)), while the presence of compound 3 was confirmed in the

MeOH extracts of the shoots of E. microsciadia, the roots of E.

macrostegia, 80% MeOH extracts of the roots of E.

microsciadia and the shoots of E. macrostegia and all the DCM

extracts. To distinguish compounds 1 and 2, we used silica gel

TLC plates impregnated with 5% AgNO

acetone (97:3) as the mobile phase, followed by treatment with

the thymol-sulfuric acid reagent spray. In AgNO -silica gel

, using chloroform:

TLC plate, compound 1 is clearly present in all MeOH and 80%

MeOH extracts of the roots of the plants (Figure 6, (2)).

Figure 4: Spots in daylight after spray with thymol using a TLC plate

and chloroform: formic acid: methanol: water (5:0.2:3:0.5). The

extracts numbers are the same as described in Figure 3

Then, after visualizing the chromatogram by thymol spray

reagent followed by heating, all the TLCs except DCM one,

exhibited two major pink colored spots that suggested the

glycosides character of compounds (Figure 4). While, the pink

colored compounds were appeared as green-gray color spots

Figure 5: Spots in daylight after spray with vanillin-sulfuric acid

reagent using an TLC plate and chloroform: formic

acid:methanol:water (5:0.2:3:0.5). The extract numbers are the same as

described in Figure 3

(

except the 80% extracts of roots and shoots of E. macrostegia)

when the chromatogram sprayed with vanillin-sulfuric acid

reagent (Figure 5). Co-TLC analyses of the extracts along with

common phytochemicals 1, 2, 3 and 4 were performed using

silica gel (Figure 6, (1)) and AgNO - silica gel stationary

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Figure 6: (1) Spots in daylight using chloroform: acetone (97:3) as the mobile phase (left) spray with vanillin-sulfuric acid reagent using an TLC plate,

2) and (3) spray with thymol reagent using AgNO coated TLC plate. cycloartenol [1] 24-methylenecycloartan-3β-ol [2], β-sitosterol [3] and euphol [4].

The extract numbers are the same as described in Figure 3

(

Conclusion

Authors’ contribution

All authors of this study have a complete contribution for

data collection, data analyses and manuscript writing.

The MeOH and DCM extracts of the roots of E.

macrostegia are good candidates to isolate cytotoxic

compounds. While the shoots DCM, and 80% MeOH extracts

of the plant are suitable for extraction of cytotoxic and radical

scavengers. However, the shoots of E. microsciadia is the best

for exploring cytotoxic, and antioxidants when the plant is

extracted with MeOH and 80% MeOH. The shoots MeOH

extracts and roots MeOH and 80% MeOH extracts of E.

macrostegia are the best for exploring antibacterial agents,

while the last two solvent extracts of E. microsciadia shoots are

preferred for isolating moderate antibacterial agents. The

above-mentioned results indicated that the semi-polar to polar

substances are responsible for the studied biological activity of

the shoots of E. microsciadia in addition to the nonpolar to

polar phytochemicals in both roots and shoots of E.

macrostegia. It seems that terpenoid and glycosylated

phytochemicals are the major phytochemicals in the DCM,

MeOH and 80% MeOH extracts of E. macrostegia and E.

microsciadia, which need to be isolated and identified. The

antibacterial, cytotoxic and antioxidant activity of the plant

extracts may be attributed to the presence of compounds 1-4 as

these activities have been reported for them previously in the

literature. For instance, compound 1 and 4 showed antioxidant

and cytotoxic activity, while compound 3 showed antibacterial

activity (12, 14, 19, 21, 22). However, the other compounds

detected in the TLCs maybe isolated, identified, and further

tested for the above biological activities in future.

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We are grateful to Mr. Mehdi Zare and Dr. Mojtaba

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