Anal. Methods Environ. Chem. J. 5 (4) (2022) 55-65

Research Article, Issue 4

Analytical Methods in Environmental Chemi s try Journal

Journal home page: www.amecj.com/ir

AMECJ

In-vitro evaluation of photoprotection, cytotoxicity and

phototoxicity of aqueous extracts of Cuscuta campe s tris

and Rosa damascene by MTT method and UV spectroscopy

analysis

Payam Khazaeli a,b, Atefeh Ameri b, Mitra Mehrabani c, Morteza Barazvana,d, Marzieh Sajadi Bami a,

and Behzad Behnam c,a,e,*

a Pharmaceutics Research Center, Ins titute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran

b Pharmaceutical sciences and cosmetic products research center, Kerman University of Medical Sciences, Kerman, Iran

c Herbal and Traditional Medicines Research Center, Kerman University of Medical Sciences, Kerman, Iran

d S tudents Research Committee, Faculty of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran

e Extremophile and Productive Microorganisms Research Center, Kerman University of Medical Sciences, Kerman, Iran

AB S TRACT

Applying sunscreen is essential for protecting the skin from UV’s

acute and chronic eects. Some of these products on the market

display side eects and are expensive. There is a great demand for

eective, cheap, safe, and herbal sunscreens with a wide range of sun

protection activities. This s tudy aimed to evaluate the photoprotection,

cytotoxicity, and phototoxicity of aqueous extracts of Cuscuta

campe s tris (CC-AE) and Rosa damascena (RD-AE). The maceration

method prepared the CC-AE and RD-AE from the aerial branch. In-

vitro photoprotection was evaluated by determining the sun protective

factor (SPF) of CC-AE and RD-AE by a UV-visible spectrophotometer.

The cytotoxicity and phototoxicity s tudies were assessed using the

MTT assay on 3T3 cells. In the nal, the PIF (Photo Inhibitor Factor)

was calculated. The SPF values of CC-AE and RD-AE were found

at 11.10±0.05 and 1.36±0.04, respectively, at the concentration of 0.2

mg mL-1. The half maximal eective concentration (EC50) of CC-AE

and RD-AE was obtained at 35.05±0.91 µg mL-1 and 40.7±0.87 µg

mL-1, respectively. The phototoxicity analysis showed that CC-AE

and RD-AE had low PIF values and were considered as the probable

phototoxic. Overall, regarding the considerable SPF and PIFs values

plus the anti-inammatory and antioxidant properties of these extracts,

they can be evaluated for further pharmaceutical formulations.

Keywords:

Cuscuta campe s tris,

UV-visible spectrophotometer,

Rosa damascene,

Sun protective factor,

Phototoxicity

ARTICLE INFO:

Received 29 Jul 2022

Revised form 12 Oct 2022

Accepted 15 Nov 2022

Available online 30 Dec 2022

*Corresponding Author: Behzad Behnam

Email: behnamb@kmu.ac.ir

https://doi.org/10.24200/amecj.v5.i04.202

------------------------

1. Introduction

Solar ultraviolet (UV) radiation such as UVA (320–

400 nm) and UVB (~295–320 nm) have acute and

chronic inuences on the skin; they might nally

cause cancers of the skin [1]. UVB radiation can

cause acute eects such as erythema and edema,

and chronic eects such as immunosuppression and

carcinogenesis [2, 3]. However, UVA radiation can

induce tanning by the oxidation of melanin, and

photoaging by the de s truction of dermal s tructures, as

well as leading to damage of the macromolecules, and

oxidative s tress by the production of reactive oxygen

species (ROS) [2, 3]. The main de s tructive factors of

UV radiation on the skin are free radicals including

superoxide anions, hydroxyl radicals, singlet oxygen,

hydrogen peroxide, ferric ion, nitric oxide, etc. [3].

Photoprotection which is caused with using of

sunscreen, prevents the acute and chronic eects of

UV radiation. UV protectors are classied as UV

lters and UV absorbers based on types of cosmetic

materials [2, 4]. UV lters are divided into two classes

according to their chemical s tructure and mechanism

of action: inorganic, such as titanium dioxide and

zinc oxide, are of low irritation potential and exhibit

photo s tability and wide-ranging absorption spectra

and organic such as UVA, UVB, and broadband

absorbers that absorb the radiations based on their

chemical s tructure. The lter’s ability of organic

lters is classied as a photo s table, photo-un s table,

and photoreactive lters [2, 4, 5]. The sunscreens’

formulations that protect the skin from harmful UV

rays could be introduced as physical and chemical

sunscreens by blocking, reecting, scattering and

absorbing the UV rays [2, 5]. The ecacy rate of

sunscreens is usually measured by the sun protection

factor (SPF) e s timation, which represents an

accepted global characteri s tic of protection from

erythema after exposure to simulated solar radiation

[6]. Generally, the components of sunscreens have

shown side eects such as disruption in the endocrine

sy s tem and changes in the hypothalamic-pituitary-

thyroid (HPT) axis. In addition, they could be caused

reproductive homeo s tasis during long-term use [4, 7].

However, some sunscreens may have environmental

toxicity eects and can have detrimental eects on

the ecosy s tem [2, 4]. Be s t sunscreens should have

several characteri s tics, including safe, non-toxic, and

photo- s table, and be able to protect the skin from

UVA and UVB rays [4]. The natural photoprotectants

can be included the obtained extracts of plants such

as aloe vera, pomegranate, rambutan, grape, tomato,

the green tea, and the oils obtained from soybean,

olive, coconut, almond, and jojoba as well as the

mycosporine-like amino acids (MAA), etc. [5,

8-10]. Several s tudies have described the use of

plant extracts with photoprotection properties. For

example, Rangel et al [2] assessed the photoprotective

capability of extracts from red macroalgae. Permana

et al [11] showed a potential absorption of UVA and

UVB radiation by the hydrogel-containing propolis

extract-loaded phytosome and indicated their high

SPF value of them [11]. Natural combinations have

shown the desirable SPF and anti-inammatory

and antioxidant properties [9, 12]. Rosa damascena

mill, commonly known as Gole Mohammadi in Iran

[13], showed several medicinal properties including

antiviral, antimicrobial, antioxidant, antitussive,

hypnotic, anti-diabetic, and sedative eects on the

respiratory sy s tem [14]. This plant contains dierent

chemical compounds such as tannins, polyphenols,

carotenoids, quercetin, eugenol, citronellol, geraniol,

liquiritin, etc. [14, 15]. Generally, the extracts of

rose petals have shown high antioxidant activity

that correlated to the total phenolic, and avonoid

contents of rose [16, 17]. The analgesic and anti-

inammatory eects of rose have also been reported

[18-20]. The hydroalcoholic extract of R. damascene

can signicantly reduce edema, which may be

mediated by the inhibition of acute inammation

[13]. Cuscuta campe s tris Yuncker with the common

name dodder has analgesic, antipyretic, anti-

inammatory, and anti-cancer properties [21, 22].

This holoparasitic plant has been applied to treat

a liver injury, cancer prevention, sciatica, scurvy,

and scrofula derma [22-24]. Based on reported

works, polyphenolic compounds such as quercetin,

sinapic acid, kaempferol, isorhamnetin hesperidin,

and eugenol were identied in extracts from C.

campe s tris [25, 26]. The ethyl acetate extract of the

plant has the s tronge s t antioxidant eect due to the

highe s t content of avonoid compounds kaempferol

and quercetin [22]. A review of the literature did not

expose any previous s tudies on the photoprotective,

cytotoxicity, and phototoxicity activities of the

aqueous extract of Cuscuta campe s tris (CC-AE) and

Rosa damascena (RD-AE) plants by MTT method

and UV spectroscopy analysis. Generally, the UV/

visible spectrophotometric method were applied

to analyze the UV radiation protection capability

for probable sunscreen applications [2, 6]. One of

the mo s t important issue in pharmaceutical circles

is to optimizing the wright method for analyzing

the active ingredients in bulk drug materials, their

impurities and decompositions sub s tances, and

also pharmaceutical formulations and biological

Anal. Methods Environ. Chem. J. 5 (4) (2022) 55-65

products. Spectrophotometry is the quantitative

measurement of the reection or transmission

properties of a material as a function of wavelength.

The use of UV-Vis spectrophotometry, especially in

the analysis of pharmaceutical forms, has increased

rapidly in recent years [2, 3, 26]. In vitro methods

for evaluating the sunscreen potentials of materials

are generally of two types. Methods that involve

measuring the absorption or transmission of UV

radiation through sunscreen product lms on quartz

plates or bio-membranes, and methods in which the

absorption characteri s tics of sunscreen agents are

determined based on spectrophotometric analysis of

dilute solutions [2, 3, 26].

In the present s tudy, the UV absorption of each

sample was obtained and the Mansur equation was

applied to nd the nal SPF. Afterwards the eects

of extracts were evaluated in vitro in 3T3 cells to

obtain their probable photo-toxic or photo-protective

behaviors.

2. Materials and Methods

2.1. Chemicals

Trypsin, phosphate-buered saline (PBS), and

3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-

tetrazolium bromide (MTT) were supplied by

Sigma company (S t. Louis, MO, USA). Fetal bovine

serum (FBS), Dulbecco’s modied Eagle’s medium

(DMEM), and Penicillin-S treptomycin solution

(100X) were obtained from Borna Pouyesh Gene

Company (BPGene Co., Kerman, Iran).

2.2. Extracts preparation

The plants (C. Campe s tris and R. Damascena) were

collected from Mahan, Kerman, Iran (30.0630°

N, 57.2875° E). The plants were then identied

by Dr. Mitra Mehrabani and kept in the Faculty of

Pharmacy herbarium (Kerman University of Medical

Sciences, Kerman, Iran). The aerial branches of

plants were washed three times with deionized water

and dried at room temperature. The dried aerial

branches were ground with a mill to obtain a ne

powder. The extracts of plants were prepared using

the maceration method. For this purpose, 10 g of the

ne powder was combined with the deionized water

(100 mL) in a laboratory ask with a volume of 500

mL. The mixture was heated at 80 °C for 30 min and

ltrated through Buchner funnel linked with Watman

lter paper (No.1). Finally, the ltrate was freeze-

dried (freeze dryer FD-550 purchased from Tokyo

Rikakikai Co., Ltd, Japan) [27]. The dried aqueous

extract of C. campe s tris and R. damascena were

labeled as CC-AE and RD-AE, respectively, and the

SPF of compounds was determined by UV-visible

spectrophotometer.

2.3. Determination of UV absorption spectra by

UV-vis spectrophotometer

The characterizations of UV absorption spectra

were carried out by analyzing an aqueous extract of

C. campe s tris and R. damascena at concentrations

20000, 10000, 5000, 2500, and 1250 μg mL-1. The

UV spectra were recorded using a Synergy TM 2

multi-mode microplate reader (BioTek In s truments,

Inc., Winooski, VT, USA) from 200 to 900 nm.

2.4. Determination of photoprotection activity of

plants extracts

The procedure of Khazaeli and Mehrabani [28],

with some modications, was used to measure

the photoprotection activity of plant extracts. For

this purpose, the obtained aqueous extracts of C.

campe s tris and R. damascene were individually

scanned in the range from 337.5 nm to 292.5 nm

with interval ve nm using a double beam UV/Vis

spectrophotometer (Optizen 3220 UV). Then, in vitro

SPF was measured by the following equation I [29].

(Eq.I)

Where T(λ), E(λ), and ε(λ) represents the

transmittance of the sample at λ, the spectral

irradiance of terre s trial sunlight at λ, and the

erythemal action spectrum at λ, respectively. The

E(λ) × ε(λ) values are showed in Table 1, the T(λ)

was three times measured and the obtained means

were applied to e s timate the SPF value for each

extract. Afterward, the graph relationship of SPF

versus LnC was used to calculate SPF in 2.0 mg

mL-1 solution for each extraction.

Analysis of CC-AE and RD-AE by MTT and UV spectroscopy Payam Khazaeli et al

2.5. Cell culture

The mouse embryonic brobla s t cells (3T3)

(ATCC Number: IBRCC10100) were provided by

the Iranian Biological Resource Center (IBRC) in

Tehran, Iran. The cell line was cultured in DMEM

medium supplemented with 10% (v/v) FBS, 100 U

mL-1 penicillin, and 100 µg mL-1 s treptomycin and

incubated at 37 °C in a 5% CO2 incubator [30].

2.6. Cytotoxicity assay

Based on methods reported in the literature [31-

34], in the exponential growth s tage, the cells were

harve s ted and seeded into 96-well tissue culture

plates (approximately 104 cells per well). After 24 h,

the samples of serial concentrations of CC-AE and

RD-AE (at the nal concentration range of 3.9–125

µg mL-1) were separately poured into the desired

wells. After 24 h, the medium in each well was

switched with 20 µL of MTT solution (5 mg mL-1)

and plates were incubated at 37 °C for a further 3

h. For dissolving the formazan cry s tals, the culture

media were removed from the wells and 100 μL of

fresh DMSO was added to each well of the plate.

The optical density of nal solutions was then read at

570 nm using a Synergy TM 2 multi-mode microplate

reader (BioTek In s truments, Inc., Winooski, VT,

USA). Doxorubicin (12 µg mL-1) was applied as a

positive control. All experiments were repeated in

triplicate on dierent days and EC50 values were

determined and analyzed by non-linear regression

analysis (SPSS software, SPSS inc., Chicago) and

the data were reported as mean (m±SD).

2.7. Evaluation of phototoxicity and

determination of PIF factor

For the purpose of evaluation of phototoxicity in the

presence and absence of UVA radiation [35], cells

were prepared as described in the cytotoxicity assay

into two plates (A and B). Plate A was exposed to

UVA light (1.8 mW cm-2) for 60 min. After 60 min,

the medium was discarded and the fresh medium

was added. Plate B was used as a non-irradiated

control. Both plates were incubated for 24 h at 37

°C in a 5% CO2 incubator. Afterward, the medium

in each well was discarded and MTT solution (20

µL, 5 mg mL-1) was added. Plates were incubated at

37 °C for 3 h and following the culture, media were

removed from the wells and 100 μL of fresh DMSO

was added to each well to dissolve the formazan

cry s tals. The absorption was then measured at 570

Anal. Methods Environ. Chem. J. 5 (4) (2022) 55-65

Table 1. Normalized product function used in the calculation of SPF.

Wavelength (nm) E(λ) × ԑ(λ)

292.5 1.139

297.5 6.510

302.5 10.00

307.5 3.577

312.5 0.973

317.5 0.567

322.5 0.455

327.5 0.289

332.5 0.129

337.5 0.046

E(λ): the spectral irradiance of terre s trial sunlight at each wavelength.

ԑ(λ): the erythemal action spectrum at each wavelength.

nm, and the EC50 values were e s timated. The PIF

(Photo Inhibitor Factor) was determined based on

below equation II:

(Eq.II)

In compliance with the OECD TG 432 [36], the

below Table was considered for analyzing the PIF

values (Table 2).

2.8. Investigationofprotectiveeectsof

plantextractsagainstofphototoxiceectsof

chlorpromazine

In assessing of the ability of plant extracts to

prevent of the phototoxic eects of chlorpromazine

(CPZ), two culture plates (A and B) were seeded

with about 104 cells per well. Then, the CC-AE and

RD-AE were prepared at a concentration of 31.25

µg mL-1. The concentrations of chlorpromazine

were also trained at the range of 0.1, 0.5, and

1 µg mL-1. After 24 h, the culture media on the

cells were evacuated and 100 µL of the prepared

concentration of extracts and 100 µL of the

concentrations of chlorpromazine were separately

added into the desired wells of the culture plates.

Subsequently, the plate A was exposed to UVA light

(1.8 mW cm-2) for 60 min. Over time, the cultural

media were removed and the fresh media were

added. The plate B was maintained in darkness (as

a non-irradiated control). The culture plates (A and

B) were incubated at 37 °C for 24 h in a 5% CO2

incubator. The subsequent s teps were performed as

in Section 2.8. All experiments were repeated three

times in dierent days. Then, the cell viabilities

(%) were determined, and data were s tated as mean

results (m±SD).

2.9. S tatis tical analysis

Experimental data are presented as the mean

(m±SD) with at lea s t three determinations for

independent experiments. All data were analyzed

by non-linear regression analysis (SPSS software,

SPSS inc., Chicago) and the p-valve (p< 0.05) was

considered to be s tati s tically signicant.

3. Results and Discussion

3.1. UV absorption spectra and critical

wavelength

The UV absorption spectra of CC-AE and RD-AE

are shown in Figure 1. The max absorbance of CC-

AE (at 2500 µg mL-1) and RD-AE (at 2500 µg mL-1)

Table 2. The categorization of phototoxicity s tages

based on PIF values.

PIF value Type of hazard

PIF < 2 Non phototoxic

PIF > 2 and < 5 Probable phototoxic

PIF > 5 Potential phototoxic

Fig. 1. The UV absorption spectra of aqueous extracts of Rosa damascena

and Cuscuta campe s tris at concentration 2500 μg mL-1

Analysis of CC-AE and RD-AE by MTT and UV spectroscopy Payam Khazaeli et al

were at 240 nm and 320 nm, respectively (Fig. 1).

3.2. In vitro SPF assessment by UV

Spectrophotometry analysis

The SPF is a quantitative capacity of the eciency

of a sunscreen product. To prevent sunburn and

other skin damage, a sunscreen product should

have a broad absorption of between 290 and 400

nm. Antioxidants from natural resources, especially

plants, might be oered as novel potentials for the

treatment and prevention of diseases caused by UV

rays. There are reports on the correlation between

antioxidant activity and SPF values [2, 37]. Based

on previous reports of the excellent antioxidant

activity of CC-AE and RD-AE plants [16, 17, 25],

the current s tudy inve s tigated the SPF values of

aqueous extracts of plants by UV spectrophotometry

applying Mansur mathematical equation [6]. In

Table 3, the SPF values measured using the UV

transmission spectra of CC-AE and RD-AE are

li s ted. As shown in Table 3, the SPF values obtained

at 2 mg mL-1 were 11.10±0.05 and 1.36±0.04 for

CC-AE and RD-AE, respectively. Ebrahimzadeh

et al [38] assessed the SPF values of extracts from

Sambucus ebulus, Zea maize, Feijoa sellowiana, and

Crataegus pentagyna and reached the highe s t value

(SPF = 24.47) using ultrasonic extract of Crataegus

pentagyna. They also reported that there is a good

correlation between SPF and phenolic contents.

Hashemi et al [37] reported the highe s t SPF values

(0.841 and 0.717) for Cucumis melo leaf ultrasonic

extract and Artemisia absinthium shoots methanolic

extract, respectively. Da Silva Fernandes et al [36]

obtained a low SPF (2.5±0.3) for an aqueous fraction

(AF) from Antarctic moss Sanionia uncinata;

however, the SPF values increased more than three

times in association with UV-lters with AF. The

highe s t value (25.8±0.3) was reported in AF plus

3-(4 methylbenzylidene)-camphor [36]. In another

s tudy, the sunscreen formulations prepared by using

the combination of organic UV lters (w/w %),

and Olea europaea leaf extract (OLE, w/w %) and

measured in vitro photoprotective ecacy using a

UV transmittance analyzer for the determination of

SPF values [7]. The SPF values 56±3, 42± 5, and

21±2 were obtained by formulations that contained

5%, 3%, and 1% OLE, respectively [7]. Therefore,

the association of UV lters with dierent plant

extracts can be increased the eciency of sunscreen

formulations [7, 36].

3.3. Phototoxicity Analysis

The toxicity eects of the CC-AE and RD-AE on

the 3T3 cell line were analyzed using the MTT-based

colorimetric te s t after 24 h; however, phototoxicity

was evaluated by comparing the dierence in toxicity

between the sample plate that was not exposed to UVA

light and the sample plate exposed to UV light. The

half-maximal eective concentration (EC50), without

UVA light, for 3T3 cell line treated with CC-AE,

RD-AE, and was measured to be 35.05±0.91 µg

mL-1, 40.7±0.87 µg mL-1, and 16.79±0.35 µg mL-1,

respectively (Table 4). According to analyses of

Anal. Methods Environ. Chem. J. 5 (4) (2022) 55-65

Table 3. Calculation of SPF of the aqueous extracts of plants

in dierent concentrations by UV–visible spectrophotometry

Plant Concentration of aqueous extract SPFa

Cuscuta campes tris 10 0.070±0.04

50 2.000±0.05

500 7.450±0.04

2000 11.10±0.05

Rosa damascena 10 0.027±0.04

50 0.110±0.05

500 1.072±0.05

2000 1.360±0.04

a Data represent means±SE (n=3).

the PIF, CC-AE (PIF=3.55) and RD-AE (PIF=2.35)

were exhibited as probable phototoxic in the te s ted

doses (Table 2). Chlorpromazine (PIF=35.59) was

a potential phototoxic hazard and results were

obtained for the cell viability with a dierence

approximately 35-fold in EC50 values, with and

without UV light (Table 4). Amaral et al [39]

presented that the IC50 values for Caryocar

brasiliense supercritical CO2 extract (CBSE) in

the phototoxicity assay considered 6.50% w/v

in dark conditions and 35.53% w/v in irradiated

conditions. According to the PIF value, the CBSE

not exhibited phototoxic potential (PIF=0.18).

Da Silva Fernandes et al [36] reported that the

AF presented non-phototoxic (PIF=1.089) and

the AF in mixtures with UV lters did not oer

any phototoxic potential (PIF < 2). Svobodová

et al [40] assessed the phototoxic potential of

silymarin, an identical extract of the seeds of

Silybum marianum, and its bioactive components.

The obtained results showed that silymarin and its

major component had no phototoxicity. Nathalie et

al [35] assessed the phototoxic of some essential

oils and showed that the PIF values of lemongrass

oil, orange oil, and CPZ were 2.34, 2.21, and 31.24,

respectively, as probably phototoxic hazard by 3T3/

MTT procedure [35]. Consequently, in the present

s tudy, C. campe s tris and R. damascene aqueous

extracts can be identied as probable phototoxic

ingredients; however, additional inve s tigations are

needed to evaluate the health risks associated with

them in vivo.

3.4. AnalysisandEvaluationofprotectiveeects

of plant extracts on prevention of phototoxic

eectsofchlorpromazine

The eect of combinations of C. campe s tris aqueous

extract, and/or R. damascena aqueous extract and

chlorpromazine as s trong phototoxic sub s tance were

assessed using the MTT-assay on the 3T3 cell line. These

experiments were evaluated using the combination of a

concentration of CC-AE or RD-AE (31.25 µg mL-1) with

three concentrations of CPZ (0.1, 0.5 and, 1 µg mL-1) in

the presence and absence of UVA light. The obtained

results of cell viability (%) are shown in Table 5. After

24 h, the measured cell viabilities (%) for the 3T3

cell line treated with a combination of the CC-AE

and the dierent ranges of CPZ were 53.70±1.51%,

49.15±1.01%, and 43.67±1.2%, respectively, in the

absence of UVA light; however, the measured cell

viabilities (%) were 49.59±2.00%, 45.44±1.51%,

and 37.47±0.93%, for similar concentrations, in

the presence of UVA light (Table 5). The measured

cell viabilities (%) for the s tudied concentration

of RD-AE on the dierent concentrations of CPZ

were 51.29±1.13%, 46.43±1.64%, and 41.82±0.86,

respectively, in the absence of UVA light. Measured

cell viabilities were 43.36±1.02%, 35.53±1.33%, and

47.78±2.1%, respectively, in the presence of UVA

light (Table 5). Generally, in the fact of UVA light, the

measured cell viabilities of CPZ alone were lower than

the combination of CC-AE and CPZ. The measured

cell viabilities of CPZ alone at concentrations 0.5 µg

mL-1 and 1 µg mL-1 were higher than the combination

of RD-AE and CPZ (Table 5).

Table 4. Evaluation of the cytotoxicity and phototoxicity of the aqueous extracts of plants

and chlorpromazine in murine brobla s ts cell (3T3)

Sample UV radiationaEC50

bPIF

Cuscuta campes tris aqueous extract

(CC-AE)

- 35.05±0.91 3.55

+ 9.86±0.61

Rosa damascena aqueous extract

(RD-AE)

- 40.7±0.87 2.35

+ 17.31±0.22

Chlorpromazine

(CPZ)

- 16.79±0.35 35.59

+ 0.467±0.06

a – or + represents the te s ts performed with and without UV light.

b Data represent the mean±SD of three experiments in dierent days

Analysis of CC-AE and RD-AE by MTT and UV spectroscopy Payam Khazaeli et al

4. Conclusion

Ultraviolet rays cause numerous injuries to the

skin, so there is a vital need to protect it again s t

its harmful eects. Natural materials usually have

the ability to protect again s t the toxic eects of

ultraviolet rays. Based on favorable antioxidant and

anti-inammatory properties of Cuscuta campe s tris

(CC-AE) and Rosa damascena (RD-AE) plants,

the current s tudy inve s tigated the photoprotection,

cytotoxicity and phototoxicity activities of

aqueous extracts of CC-AE and RD-AE in mouse

brobla s t cells (3T3 cells) by MTT method and

UV spectroscopy analysis. In this research, the

SPF values of CC-AE and RD-AE were evaluated

by UV–visible spectrophotometry applying the

Mansur equation. At the concentration of 0.2 mg

mL-1, the SPF values of CC-AE and RD-AE were

11.10±0.05 and 1.36±0.04, respectively. The EC50

of CC-AE and RD-AE was 35.05±0.91 µg mL-1 and

40.7±0.87 µg mL-1, respectively. The PIF values for

CC-AE and RD-AE are in the range of probable

phototoxic materials (PIF > 2 and < 5), but as these

numbers are decient and near the range of non-

phototoxic, they could be hypothesized for future

anti-solar formulations. Moreover, in the presence

of UVA light, the measured cell viabilities of CPZ

alone were lower than the combination of CC-AE

and CPZ. Overall, the presented data in this report

showed that RD-AE, with SPF and PIF of 11 and

2.35 and various prominent biological eects,

could be regarded as an ecient natural product to

be considered in sunscreen formulations.

5. Acknowledgements

We thank the Pharmaceutics Research Center,

In s titute of Neuropharmacology, Kerman

University of Medical Sciences (Kerman, Iran) for

their support (Grant number: 95000346).

Anal. Methods Environ. Chem. J. 5 (4) (2022) 55-65

Table 5. Evaluation of protective eects of plant extracts on prevention of phototoxic eects

of chlorpromazine in murine brobla s ts (3T3).

Plant

CAE* Chlorpromazine

UV radiation a

Cell viability b

(%)

Cuscuta campes tris 31.25 1 - 53.70±1.51

31.25 0.5 - 49.15±1.01

31.25 0.1 - 43.67±1.20

31.25 1 + 49.59±2.00

31.25 0.5 + 45.44±1.51

31.25 0.1 + 37.47±0.93

Rosa damascena 31.25 1 - 51.29±1.13

31.25 0.5 - 46.43±1.64

31.25 0.1 - 41.82±0.86

31.25 1 + 43.36±1.02

31.25 0.5 + 35.53±1.33

31.25 0.1 + 47.78±2.10

Chlorpromazine - 1 - 55.22±1.09

- 0.5 - 53.60±2.11

- 0.1 - 53.60±1.21

- 1 + 47.66±1.21

- 0.5 + 38.04±0.98

- 0.1 + 32.86±0.88

* CAE: Concentration of aqueous extract

a – or + represents the te s ts performed with and without UVA light

6. References

[1] S.R. Varma, T.O. Sivaprakasam, I.

Arumugam, N. Dilip, M. Raghuraman, K.

Pavan, M. Raq, R. Paramesh, In vitro anti-

inammatory and skin protective properties

of Virgin coconut oil, J. Tradit. Complement.

Med., 9 (2019) 5-14. https://doi.org/10.1016/j.

jtcme.2017.06.012.

[2] K.C. Rangel, L.Z. Villela, K.d.C. Pereira,

P. Colepicolo, H.M. Debonsi, L.R. Gaspar,

Assessment of the photoprotective potential

and toxicity of Antarctic red macroalgae

extracts from Curdiea racovitzae and Iridaea

cordata for cosmetic use, Algal Res., 50

(2020) 101984. https://doi.org/10.1016/j.

algal.2020.101984.

[3] G. Bhandari, S. Baurai, Assessment of in

vitro sun protection factor of plant extracts

by ultraviolet spectroscopy method, Univ. J.

Phytochem. Ayurvedic Heights, 2 (2020) 20-25.

https://doi.org/10.51129/ujpah-2020-29-2(3).

[4] M. Majeed, S. Majeed, R. Jain, L. Mundkur,

H.R. Rajalakshmi, P. Lad, P. Neupane, A

randomized s tudy to determine the sun

protection factor of natural ptero s tilbene

from pterocarpus marsupium, Cosmetics,

7 (2020) 16. https://doi.org/10.3390/

cosmetics7010016.

[5] D. Bhattacharjee, S. Preethi, A.B. Patil, V.

Jain, A comparison of natural and synthetic

sunscreen agents: a review, Int. J. Pharm.

Sci. Res., 13 (2021) 3494-3505. https://doi.

org/10.31838/ijpr/2021.13.01.524.

[6] T. Sharma, V. Tyagi, M. Bansal, Determination

of sun protection factor of vegetable and fruit

extracts using UV–Visible spectroscopy: A

green approach, Su s tain. Chem. Pharm., 18

(2020) 100347. https://doi.org/10.1016/j.

scp.2020.100347.

[7] A.C. Da Silva, J.P. Paiva, R.R. Diniz, V.M.

Dos Anjos, A.B.S. Silva, A.V. Pinto, E.P.

Dos Santos, A.C. Leitão, L.M. Cabral, C.R.

Rodrigues, Photoprotection assessment

of olive (Olea europaea L.) leaves extract

s tandardized to oleuropein: In vitro

and in silico approach for improved

sunscreens, J. Photochem. Photobiol.

B, 193 (2019) 162-171. https://doi.

org/10.1016/j.jphotobiol.2019.03.003.

[8] M. Radice, S. Manfredini, P. Ziosi, V. Dissette,

P. Buso, A. Fallacara, S. Vertuani, Herbal

extracts, lichens and biomolecules as natural

photo-protection alternatives to synthetic UV

lters. A sy s tematic review, Fitoterapia, 114

(2016) 144-162. https://doi.org/10.1016/j.

tote.2016.09.003.

[9] B. Prasanth, A. Soman, J. Jobin, P.S. Narayanan,

A.P. John, Plants and phytocon s tituents

having sunscreen activity, World J. Curr.

Med. Pharm. Res., 2 (2020) 14-20. https://doi.

org/10.37022/WJCMPR.2020.02019.

[10] F. Merlin, W. Ratnasooriya, R. Pathirana,

In vitro inve s tigation of sunscreen activity

and evaluation of phytochemical prole

of methanolic leaf extract of Rauvola

tetraphylla, J. Pharmacogn. Phytochem., 9

(2020) 2063-2067. https://www.phytojournal.

com/

[11] A.D. Permana, R.N. Utami, A.J. Courtenay,

M.A. Manggau, R.F. Donnelly, L. Rahman,

Phytosomal nanocarriers as platforms for

improved delivery of natural antioxidant

and photoprotective compounds in propolis:

An approach for enhanced both dissolution

behaviour in biorelevant media and skin

retention proles, J. Photochem. Photobiol.,

205 (2020) 111846. https://doi.org/10.1016/j.

jphotobiol.2020.111846.

[12] M. Hupel, N. Poupart, E.A. Gall, Development

of a new in vitro method to evaluate the

photoprotective sunscreen activity of plant

extracts again s t high UV-B radiation, Talanta,

86 (2011) 362-371. https://doi.org/10.1016/j.

talanta.2011.09.029.

[13] M.H. Boskabady, M.N. Shafei, Z. Saberi,

S. Amini, Pharmacological eects of Rosa

damascena, Iran. J. Basic Med. Sci., 14 (2011)

295. https://ijbms.mums.ac.ir/

[14] M. Mahboubi, Rosa damascena as holy

ancient herb with novel applications, J. Tradit.

Analysis of CC-AE and RD-AE by MTT and UV spectroscopy Payam Khazaeli et al

Complement. Med., 6 (2016) 10-16. https://

doi.org/10.1016/j.jtcme.2015.09.005.

[15] N. Kumar, B. Singh, V.K. Kaul, Flavonoids from

Rosa damascena Mill, Nat. Prod. Commun.,

1 (2006) 1934578X0600100805. https://doi.

org/10.1177/1934578X0600100805.

[16] N.G. Baydar, H. Baydar, Phenolic compounds,

antiradical activity and antioxidant capacity

of oil-bearing rose (Rosa damascena Mill.)

extracts, Ind. Crops Prod., 41 (2013) 375-380.

https://doi.org/10.1016/j.indcrop.2012.04.045.

[17] N. Yassa, F. Masoomi, R. Rohani, Chemical

composition and antioxidant activity of the

extract and essential oil of Rosa damascena

from Iran, Population of Guilan, DARU J.

Pharm. Sci.,, 17 (2009) 175-180. http://daru.

tums.ac.ir/index.php/daru/article/view/541

[18] V. Hajhashemi, A. Ghannadi, M. Hajiloo,

Analgesic and anti-inammatory eects of

Rosa damascena hydroalcoholic extract and

its essential oil in animal models, Iran. J.

Pharm. Res., 9 (2010) 163. https://pubmed.

ncbi.nlm.nih.gov/24363723/

[19] G. Lati, A. Ghannadi, M. Minaiyan, Anti-

inammatory eect of volatile oil and

hydroalcoholic extract of Rosa damascena

Mill. on acetic acid-induced colitis in rats,

Res. Pharm. Sci., 10 (2015) 514. https://www.

rpsjournal.net/

[20] F. Fatemi, A. Golbodagh, R. Hojihosseini, A.

Dadkhah, K. Akbarzadeh, S. Dini, M.R.M.

Malayeri, Anti-inammatory eects of

deuterium-depleted water plus rosa damascena

mill. Essential oil via cyclooxygenase-2

pathway in rats, Turk. J. Pharm. Sci., 17

(2020) 99. https://doi.org/10.4274/tjps.

galenos.2018.24381.

[21] M. Behbahani, Evaluation of in vitro

anticancer activity of Ocimum basilicum,

Alhagi maurorum, Calendula ocinalis and

their parasite Cuscuta campe s tris, PloS One,

9 (2014) e116049. https://doi.org/10.1371/

journal.pone.0116049.

[22] E.K. Selvi, H. Turumtay, A. Demir, E.A.

Turumtay, Phytochemical proling and

evaluation of the hepatoprotective eect of

Cuscuta campe s tris by high-performance

liquid chromatography with diode array

detection, Anal. Lett., 51 (2018) 1464-1478.

https://doi.org/10.1080/00032719.2017.1382

502.

[23] M. Moradzadeh, A. Hosseini, H. Rakhshandeh,

A. Aghaei, H.R. Sadeghnia, Cuscuta

campe s tris induces apoptosis by increasing

reactive oxygen species generation in human

leukemic cells, Avicenna J. Phytomed., 8

(2018) 237. https://ajp.mums.ac.ir/

[24] W.H. Peng, Y.W. Chen, M.S. Lee, W.T. Chang,

J.C. Tsai, Y.C. Lin, M.K. Lin, Hepatoprotective

eect of Cuscuta campe s tris Yunck. whole

plant on carbon tetrachloride induced chronic

liver injury in mice, Int. J. Mol. Sci., 17 (2016)

2056. https://doi.org/10.3390/ijms17122056.

[25] E. Jafari, A. Bahmanzadegan, G. Ghanbarian,

V. Rowshan, Antioxidant activity and total

phenolic content from aerial parts of three

Cuscuta species, Anal. Chem. Lett., 5 (2015)

377-384. http://dx.doi.org/10.1080/22297928

.2016.1143394.

[26] M.M. Akiner, E.K. Selvİ, M. Öztürk, I. Güney,

U. Asu, Toxic ecacy of Cuscuta campe s tris

Yunck.(Solanales: Convolvulaceae) and

Lupinus albus L.(Fabales: Fabaceae) plant

crude extracts again s t nymphs and adults

of Orosanga japonica (Melichar, 1898)

(Hemiptera: Ricaniidae) under laboratory

conditions, Turk. Entomol. Derg., 45

(2021) 65-75. https://doi.org/10.16970/

entoted.743439.

[27] P. Khazaeli, M. Mehrabani, M.R. Heidari,

G. Asadikaram, M.L. Naja, Prevalence of

aatoxin contamination in herbs and spices in

dierent regions of Iran, Iran. J. Public Health,

46 (2017) 1540. https://ijph.tums.ac.ir/index.

php/ijph

[28] P. Khazaeli , M. Mehrabani, Screening of sun

protective activity of the ethyl acetate extracts

of some medicinal plants, Iran. J. Pharm.

Res., 1 (2010) 5-9. https://doi.org/10.22037/

IJPR.2010.738.

Anal. Methods Environ. Chem. J. 5 (4) (2022) 55-65

[29] [29] J.D.S. Mansur, M.N.R. Breder, M.C.D.A.

Mansur, R.D. Azulay, Determinaçäo do fator

de proteçäo solar por espectrofotometria, An.

Bras. Dermatol., 61 (1986) 121-124. https://

www.medscape.com/viewpublication/2466_33

[30] M. Mo s tafavi, I. Shari, S. Farajzadeh, P.

Khazaeli, H. Shari, E. Pourseyedi, S. Kakooei,

M. Bamorovat, A. Keyhani, M.H. Parizi,

Niosomal formulation of amphotericin B alone

and in combination with glucantime: In vitro

and in vivo leishmanicidal eects, Biomed.

Pharmacother., 116 (2019) 108942. https://

doi.org/10.1016/j.biopha.2019.108942.

[31] B. Behnam, M. Rezazadehkermani, S.

Ahmadzadeh, A. Mokhtarzadeh, S.N.

Nematollahi-Mahani, A. Pardakhty,

Microniosomes for concurrent doxorubicin

and iron oxide nanoparticles loading;

preparation, characterization and cytotoxicity

s tudies, Artif. Cells Nanomed. Biotechnol., 46

(2018) 118-125. https://doi.org/10.1080/2169

1401.2017.1296850.

[32] E. Mohammadi, M. Zeinali, M.

Mohammadi-Sardoo, M. Iranpour, B.

Behnam, A. Mandegary, The eects of

functionalization of carbon nanotubes on

toxicological parameters in mice, Hum. Exp.

Toxicol., 39 (2020) 1147-1167. https://doi.

org/10.1177/0960327119899988.

[33] R. Mohammadinejad, A. Dehshahri, H.

Sassan, B. Behnam, M. Ashrazadeh,

A. Samareh Gholami, A. Pardakhty, A.

Mandegary, Preparation of carbon dot as a

potential CRISPR/Cas9 plasmid delivery

sy s tem for lung cancer cells, Minerva

Biotecnol., 32 (2020) 106-113. https://doi.

org/10.23736/S1120-4826.20.02618-X.

[34] M. Moballegh-Nasery, A. Mandegary, T.

Eslaminejad, M. Zeinali, A. Pardakhti, B.

Behnam, M. Mohammadi, Cytotoxicity

evaluation of curcumin-loaded abody-

decorated liposomes again s t brea s t cancerous

cell lines, J. Liposome Res., 31 (2021) 189-

194. https://doi.org/10.1080/08982104.2020.

1755981.

[35] D. Nathalie, G. Yannick, B. Caroline, D.

Sandrine, F. Claude, C. Corinne, F. Pierre-

Jacques, Assessment of the phototoxic hazard

of some essential oils using modied 3T3

neutral red uptake assay, Toxicol. In Vitro,

20 (2006) 480-489. https://doi.org/10.1016/j.

tiv.2005.08.018.

[36] A. Da Silva Fernandes, L.B. Brito, G.A.R.

Oliveira, E.R.A. Ferraz, H. Evangeli s ta,

J.L. Mazzei, I. Felzenszwalb, Evaluation

of the acute toxicity, phototoxicity and

embryotoxicity of a residual aqueous fraction

from extract of the Antarctic moss Sanionia

uncinata, BMC Pharmacol. Toxicol., 20

(2019) 1-10. https://doi.org/10.1186/s40360-

019-0353-3.

[37] Z. Hashemi, M.A. Ebrahimzadeh, M. Khalili,

Sun protection factor, total phenol, avonoid

contents and antioxidant activity of medicinal

plants from Iran, Trop. J. Pharm. Res., 18

(2019) 1443-1448. https://doi.org/10.4314/

tjpr.v18i7.11.

[38] M.A. Ebrahimzadeh, R. Enayatifard, M.

Khalili, M. Ghaarloo, M. Saeedi, J.Y.

Charati, Correlation between sun protection

factor and antioxidant activity, phenol and

avonoid contents of some medicinal plants,

Iran. J. Pharm. Res., 13 (2014) 1041. https://

brieands.com/journals/iranian-journal-of-

pharmaceutical-research/

[39] L.F. Amaral, P. Moriel, M.A. Foglio, P.G.

Mazzola, Evaluation of the cytotoxicity

and phototoxicity of Caryocar brasiliense

supercritical carbon dioxide extract, BMC

Complement. Altern. Med., 14 (2014) 1-6.

https://doi.org/10.1186/1472-6882-14-450.

[40] A.R. Svobodová, B. Zálešák, D. Biedermann,

J. Ulrichová, J. Vo s tálová, Phototoxic

potential of silymarin and its bioactive

components, J. Photochem. Photobiol. B,

156 (2016) 61-68. https://doi.org/10.1016/j.

jphotobiol.2016.01.011.

Analysis of CC-AE and RD-AE by MTT and UV spectroscopy Payam Khazaeli et al