Photoprotective activity of Buddleja scordioides

Article abstract of DOI:10.1016/j.fitote.2005.03.009

The purpose of this study was to determine the photoprotective properties of the methanolic extract of Buddleja scordioides, as well as verbascoside and linarin which were isolated from this extract, and linarin acetate prepared in the laboratory. The pho

Full text of DOI:10.1016/j.fitote.2005.03.009

Fitoterapia 76 (2005) 301–309
www.elsevier.com/locate/fitote
Photoprotective activity of Buddleja scordioides
a,
a
a
*
J.G. Avila Acevedo , C.M.C. Castan˜eda , F.J.C. Benitez ,
D.A. Dura´na, V.R. Barrosoa, C.G. Mart´ıneza, L.J.L. Mun˜oza,
C.A. Mart´ıneza, A. Romo de Vivarb
aLaboratorio de Fitoqu´ımica, Unidad de Biolog´ıa, Tecnolog´ıa y Prototipos (UBIPRO),
Universidad Nacional Auto´noma de Me´xico, FES Iztacala, Tlalnepantla, C. P. 54090, Me´xico
bInstituto de Qu´ımica, Universidad Nacional Auto´noma de Me´xico, Circuito Exterior,
Ciudad Universitaria, C. P. 04510, Me´xico
Received 5 June 2003; accepted in revised form 17 March 2005
Abstract
The purpose of this study was to determine the photoprotective properties of the methanolic
extract of Buddleja scordioides, as well as verbascoside and linarin which were isolated from this
extract, and linarin acetate prepared in the laboratory. The photoprotective effect of substances
against UV-B induced cellular death was evaluated by challenge experiments using Escherichia coli.
Verbascoside and linarin acetate showed the highest protection. The sun protection factor (SPF) of
the methanolic extract, linarin, linarin acetate, and verbascoside was evaluated by guinea pig
bioassays. Verbascoside showed the largest SPF measurement.
D 2005 Elsevier B.V. All rights reserved.
Keywords: Buddleja scordioides; Linarin; Verbascoside; Photoprotective activity; Sun protection factor
1. Introduction
Sun radiation constantly impacts the earth’s surface with approximately 50% of visible
light (400–800 nm), 40% of infrared radiation (IR) (1300–1700 nm), and 10% of
* Corresponding author. Tel.: +52 5623 1136; fax: +52 5623 1225.
E-mail address: tuncomaclovio@correo.unam.mx (J.G. Avila Acevedo).
0367-326X/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.fitote.2005.03.009

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J.G. Avila Acevedo et al. / Fitoterapia 76 (2005) 301–309
ultraviolet radiation (UV) (10–400 nm). UV is divided conventionally in UV-A (320–400
nm), UV-B (290–320 nm), UV-C (100–290 nm), and vacuo UV (10–100 nm) [1].
In the last decades UV-B radiation reached the planet with higher intensity as a
consequence of the ozone layer depletion in the upper atmosphere [2]. It has been reported
that adverse effects by UV-B radiation on the human skin include erythema (or sunburn),
accelerated skin ageing, and induction of skin cancer [3].
Sunscreens are chemicals that provide protection against the adverse effects of solar
and, in particular, UV radiation [4]. Studies in animals have shown that a variety of
sunscreens can reduce the carcinogenic and immunosuppressive effects of the sunlight [5].
Natural substances extracted from plants have been recently considered as potential
sunscreen resources because of their ultraviolet ray absorption on the UV region [6] and of
their antioxidant power [7]. Green tea polyphenols, Aloe barbadencis extract, and
aromatic compounds isolated from lichens are examples of natural substances evaluated
for their sunscreen properties [4,8,9].
Buddleja scordioides HBK (Buddlejaceae) is a shrub which grows in the Chihuahuan
desert [10]. Decoctions of this plant are used orally or topically for treating several
illnesses such as diarrhea, headache, and hurts. [11]. In the community of Doctor Arroyo,
town in Nuevo Leo´n, Me´xico, B. scordioides is known as bescobillaQ and rural laborers
use the water–alcohol extracts from aerial parts of this shrub as a sunscreen (personal
communication).
In the present study, we isolated two active compounds from B. scordioides and
evaluated their photoprotective activity using bacteria and guinea pig as experimental
biological systems.
2. Experimental
2.1. Plant material
The aerial parts of B. scordioides were collected in vicinity of Doctor Arroyo, Nuevo
Leo´n, Me´xico, in November 1998. A voucher specimen (Izta 26140) was deposited in Izta
Herbarium, FES-Iztacala-National University of Mexico.
2.2. Extraction procedure
Dried and ground aerial parts of B. scordioides (2000 g) were extracted with hexane,
EtOAc, and MeOH, successively. MeOH extract was concentrated in vacuo giving a
residue (15.15%). 3 g of the residue was employed for photoprotective experiments and
300 g was Si-gel CC eluting with CH2Cl2–MeOH 19:1 and increasing concentrations of
MeOH. Fractions CH2Cl2–MeOH (9:1 and 8:2) gave linarin (3 g) and verbascoside (11 g),
respectively.
Linarin (1). Identified by comparison with reported physical and spectroscopic data
[12,13].
Verbascoside (2). Identified by comparison with reported physical and spectroscopic
data [14].

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303
2.3. Linarin peracetate
Linarin (100 mg) treated with Ac2O–pyridine at 60 8C for 1 h and kept overnight at 25
8C gave linarin peracetate (1a), 68 mg. Identified by comparison with an authentic sample.
2.4. Animals
H-D guinea pigs of both sexes weighing 300–350 g were used. They were housed in
standard environmental conditions, fed with standard rodent diet with water ad libitum.
2.5. Photoprotective activity
2.5.1. Protective effect against UV-B induced cell death
A strain of Escherichia coli (ATCC 25922) was grown in heart and brain infusion
broth (Bioxon-112) until the culture reached a concentration of 107–108 cells/ml (O.D.
0.3 read at 550 nm). The bacteria were centrifuged 10 min at 6000 rev/min,
suspended in Ringer PBS (pH 7.0), and transferred into quartz cuvettes (Pye Unicam
B538751 A, thickness 1 mm, capacity 4 ml). Each photoprotective substance was
dissolved in an appropriated solvent (2 mg/ml) and put in a quartz cuvette. Cuvette
containing bacteria was placed behind the cuvette containing the photoprotective
substance, forming one experimental unit. The experimental units were irradiated with
an UV-B lamp (312 nm, Spectroline EB-280C) [15]. Irradiation dose was 0.60 J/cm2.
The survivor bacteria number was detected in accordance with the dilution method
[16] at different time periods. The substances employed were B. scordioides MeOH
extract, verbascoside in MeOH, linarin in DMSO, and linarin peracetate in CHCl3.
The positive control was escalol (ethylhexyl-methoxycinnamate, ISP VAN DIK) and
the negative controls were the solvents used for dissolving the photoprotective
substances.
2.5.2. Sun protection factor (SPF) measurements
Dorsal skin of guinea pigs was shaved with electric clippers (Oster Mod. 274-01)
and then depilated with lotion hair remover (Velvinette-Wella). The skin was rinsed
under warm tap water and dried with a towel. After 16 h, dorsal skin was treated
with 2 mg/cm2 of photoprotective substances or with vehicle (setting gel-Stil Net) or
was left untreated. Animals (5 for each group) were then wrapped with 7.5 cm wide
tape (Tuk M.R. #4) containing six exposure windows (three windows on each side
of the spinal line) of 2.0 cm2. After 15 min animals were placed under a bank of 5
UV-B lamps (312 nm, Spectroline EB-280C) positioned 15 cm above their backs.
The irradiation at this distance was of 0.60 J/cm2 measured with a Spectroline model
DM-300HA research radiometer. Irradiation times of the six exposure windows on
each animal were set to bracket the suspected SPF of the substance being tested.
Exposure windows were covered with opaque tape at the end of each time point. At
the completion of irradiations, tape was removed from animals. Erythema was scored
(0–3 grading scale, with half-grade increments) 24 h later, using non-exposed
adjacent skin on each animal as a non-UV control (score=0). A grade of 1.0

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10
8
6
4
2
0
0
20
40
60
80
100
120
140
time (minutes)
Fig. 1. Photoprotection of methanolic extract of B. scordioides on E. coli against UV-B (312 nm) irradation
(K =0.06, R2 =0.97).
(detectable redness over the entire exposure area) was considered to be 1 Minimal
Erythema Detectable (1 MED). The SPF was then calculated from the ratio: (UV
dose to achieve 1 MED with photoprotective substance)/(UV dose to achieve 1 MED
without photoprotective substance) [17]. The erythema responses to UV exposure of
vehicle-treated and untreated skin were identical (SPF=1). Twenty-four hours after
SPF determination, biopsies were taken from irradiated zones at the maximum
irradiation time without MED of each treatment, then the animals were killed.
Samples of dorsal skin tissue were fixed in 10% buffered formalin, embedded in
paraffin, and sectioned at 6 to 8 Am. Sections were stained with hematoxylin and
eosin dyes (H&E stain).
2.6. Statistical analysis
In the study of photoprotective effect with bacteria, the mortality rate (K) and
determination coefficient (R2) were obtained using the mortality Gomperts function. In
10
8
6
4
2
0
0
1
2
3
4
5
time (minutes)
Fig. 2. Cellular death of E. coli exposed to UV-B irradation (312 nm) without protection (K =1.44, R2 =0.95).

J.G. Avila Acevedo et al. / Fitoterapia 76 (2005) 301–309
305
10
8
6
4
2
0
0
50
100
150
200
250
300
time (minutes)
Fig. 3. Photoprotection of escalol on E. coli against UV-B irradation (312 nm) (K =0.060, R2 =0.99).
the SPF determination, the statistical significance among treatment groups at specific
time points was determined using the non-parametric Friedman test. The significant
difference between irradiation times was determined by the Wilcoxon test.
3. Results
3.1. Protective effect against UV-B induced cell death
The protective effect against UV-B induced cell death were evaluated using E. coli
(ATCC 25922) as a cell model. The results showed that the methanolic extract of B.
scordioides possesses a pronounced photoprotective activity compared to the negative
control even if resulted less active than escalol as a positive control. In fact, the bacteria
population (i108 cells/ml), protected with methanolic extract of B. scordioides, reached
cell death at 37–65 min interval (Fig. 1); negative and positive control, in turn (Figs. 2 and
3), reached cell death at 180 s and 65–130 min intervals, respectively. Linarin (1) and
verbascoside (2) were isolated from the methanolic extract of B. scordioides, and linarin
peracetate (1a) was prepared in our laboratory. These compounds were tested for their
10
8
6
4
2
0
0
50
100
150
200
250
300
time (minutes)
Fig. 4. Photoprotection of linarin acetate against UV-B (312 nm) irradation on E. coli (K =0.019, R2 =0.99).

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J.G. Avila Acevedo et al. / Fitoterapia 76 (2005) 301–309
10
8
6
4
2
0
0
50
100
150
200
250
300
time (minutes)
Fig. 5. Photoprotection of verbascoside against UV-B (312 nm) irradation on E. coli (K =0.025, R2 =0.99).
protective effect against UV-B induced cell death. Figs. 4–6 show the photoprotective
effect. Linarin peracetate and verbascoside protected bacteria more efficiently than
positive control and methanolic extract. Bacterial population protected by these
compounds reached cell death until 125–250 min interval, while bacteria protected with
linarin reached cell death until 40–80 min interval.
Linarin (1), verbascoside (2) and linarin peracetate (1a) are reported in Fig. 7.
3.2. Sun protection factor (SPF)
The SPF values of the tested substances were determined on guinea pigs (Table 1).
Negative control (guinea pigs protected only with vehicle) showed perceptible erythema at
20F2 min; this time was considered as 1 SPF. Methanolic extract had the smallest SPF,
probably due to low concentration of the photoprotective compounds in the extract.
Verbascoside was the substance with the highest photoprotective activity, its SPF was of
24F0.7 retarding the erythema appearance until 440F14 min. Linarin acetate protected
10
8
6
4
2
0
0
20
40
60
80
100
time (minutes)
Fig. 6. Photoprotection of linarin against UV-B (312 nm) irradation on E. coli (K =0.051, R2 =0.96).

J.G. Avila Acevedo et al. / Fitoterapia 76 (2005) 301–309
307
RO
O
O
OR
RO
RO
O
O
OR
O
O
RO
RO
RO
1: R=H
1a: R=Ac
OH
OH
HO
CH2 CH2
O
O
HO
H
O
HO
C
C
CO
HO
O
O
OH
H
OH OH
2
Fig. 7. Linarin (1), linarin peracetate (1a), verbascoside (2).
skin in similar manner than positive control (escalol). The SPF obtained for linarin was of
9F0.3.
According to Friedman test, there were significant differences in each treatment.
Wilcoxon test showed significant differences between negative control and experimental
Table 1
Sun protection factor (SPF) in guinea pigs
Tested (2 mg/cm2)
SPF
1
Exposition time without
erythema (min)
Control B. scordioides
MeOH extract
Verbascoside
Linarin
20F2
40F1.8
440F14
160F6
80F4
3F0.09
24F0.7
9F0.3
5F0.2
5F0.1
Linarin acetate
Escalol
80F2

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J.G. Avila Acevedo et al. / Fitoterapia 76 (2005) 301–309
Table 2
Statistical significance according to Friedman and Wilcoxon tests
Friedman test:
MeOH extract of B. scordioides Verbascoside
Linarin
Linarin acetate
Escalol
v2 =10.78, p b0.05
v2 =37.5,
p b0.005
v2 =28.27,
p b0.005
v2 =28.27,
p b0.005
v2 =28.27,
p b0.005
Wilcoxon test:
MeOH extract of B. scordioides Verbascoside
Linarin
Linarin acetate
Escalol
MED
P-value
MED P-value
MED P-value
MED P-value
MED P-value
1-2
0.046
1-2
1-4
1-8
0.025
0.025
0.025
1-2
1-4
1-8
1-9
0.025
0.046
0.025
1-2
1-4
1-5
0.025
0.046
1-2
1-4
0.025
1-3
0.083 n.s.
0.083 n.s.
0.157 n.s.
0.157 n.s.
0.025
1-8
0.317 n.s. 1-5
0.500 n.s. 1-8
0.083 n.s.
0.083 n.s.
1-16
1-20
1-16 0.025
1-22 0.025
0.157 n.s. 1-8
1-16 0.157 n.s. 1-16 0.500 n.s.
1-24 0.083 n.s. 1-22 0.317 n.s.
1-32 0.570 n.s. 1-28 0.059 n.s.
n.s.=no significant difference.
substances during the time of effective photoprotection (Table 2), consequently the SPF
values obtained from UV-B absorbing substances are reliable.
4. Discussion
The results showed that methanolic extract of B. scordioides possesses a photo-
protective activity as showed on the effect against UV-B induced cell death. Also, linarin
and verbascoside isolated from the extract and linarin peracetate prepared in our laboratory
protected bacteria efficiently.
In the in vivo test methanolic extract showed the smallest sun protection factor (SPF)
probably due to low concentration of the photoprotective compounds in the extract. In
contrast with photoprotection in bacteria experiments, linarin had larger SPF than linarin
peracetate; probably linarin peracetate is absorbed across the skin due to its high lipofilicity.
In all cases methanolic extract, isolated compounds, and positive control protected the
skin tissue from irradiation burn because there was no evidence of histological changes,
while negative control exhibited cellular (vacuolization) and vascular inflammatory
alterations (edema), as well as stratum corneum detachment (data not shown).
Verbascoside was the best photoprotector compound because the protection against
cellular death and SPF values were greater than those obtained from linarin, linarin
peracetate, and escalol. In accordance with Elmets and Young [4], the sunscreens should
have two activities: first, furnish a SPF higher than 15 and, second, have antioxidant
properties. Verbascoside could be a good sunscreen because its SPF is higher than 15, and
it has antioxidant and wound healing properties [1].
It is probable that phenolics compounds such as verbascoside and linarin provide
chemical defense against UV radiation in B. scordioides, because similar substances in
some other plant species fulfill such functions [18].

J.G. Avila Acevedo et al. / Fitoterapia 76 (2005) 301–309
309
In summary, our study clearly correlates the popular use of B. scordioides with its
photoprotective effect. The methanolic extract of B. scordioides has photoprotective
activity; this may be explained by the presence of absorbing UV-B compounds in the
extract such as verbascoside and linarin. Results show that verbascoside has photo-
protection quality better than the other tested compounds. The advantage of verbascoside
could be represented by the good extinction coefficient and by their antioxidant properties.
Acknowledgements
We thank Alejandrina Acosta, Carmen Ma´rquez, and Roc´ıo Patin˜o for their technical
assistance. We are grateful to Dr. Mar´ıa Edith Lo´pez V. for the plant identification. This
work was partially financed by DGAPA-UNAM (grant IN-210197) and PAEP-UNAM.
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