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Research Project
Cytinus hypocistis L. L.: a case study for the development of an innovative, safe, and effective anti-photoaging cosmeceutical formulation
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Publications
Morphometric measurements in HPV-transgenic mice after topical application of Cytinus hypocistis (L.) extract
Publication . Silva, Ana Rita; Medeiros, Catarina; Ferreira, Teresa; Barros, Lillian; Ferreira, Isabel C.F.R.; Pires, Maria João; Costa, Rui Miguel Gil; Gama, Ana; Oliveira, Paula A.
Human papillomavirus (HPV) infections are associated with the development of several anogenital cancers.
K14HPV16 transgenic model is an animal model that mimics HPV-induced lesions. Cytinus hypocistis (L.)
is an endophytic parasitic plant that has been shown to possess several antibacterial and antiproliferative
properties1. During antiproliferative studies in animal models, the toxicological analysis is also required.
Therefore, the present research aims to study the toxic effects of applying a topical formulation enriched with
Cytinus hypocistis extracts (FCH) on K14HPV16 mice.
Three different concentrations of FCH were used to enrich a base cream, the phenolic compounds were
analyzed by HPLC-DAD-ESI/MS and were applied to the animal’s ears for 28 days. Thirty female mice were
equally divided into six groups (G) (n=5): G1 (HPV16+-C1); G2 (HPV16+-C2); G3 (HPV16+-C3); G4 (HPVC3);
G5 (HPV+-control); and G6 (HPV-control). The animals were kept under controlled conditions and
biological variables were registered throughout the study. In the end, animals were sacrificed, and the organs
collected and weighed.
The main phenolic compound present in the base cream enriched with C. hypocistis was galloyl-bis-HHDPglucose.
Food and water intake were constant throughout the trial. G3 presented a higher relative weight of
the liver and lungs than the control animals, and a significant difference in body weight relatively to G4
(p<0.05). The body weight loss and the increase in liver weight observed in HPV+ mice treated with they
may reflect a negative impact of the extract on these already fragile animals. However, overall, the FCH was
well tolerated by the animals. Although the highest concentration of the extract had a negative impact on the evaluated morphometric
parameters, this does not necessarily imply that the extract is toxic, which can be corroborated with
histological analysis. Further studies are needed to understand whether different doses of this extract would
cause distinct effects.
Cytinus hypocistis (L.) L. extract effects in an animal model of papillomavirus-induced neoplasia
Publication . Medeiros, Catarina; Silva, Ana Rita; Ferreira, Tiago; Barros, Lillian; Neuparth, Maria João; Peixoto, Francisco P.; Bastos, Margarida M.S.M.; Medeiros, Rui; Costa, Rui Miguel Gil; Pires, Maria João; Faustino-Rocha, Ana; Gama, Adelina; Oliveira, Paula A.
Infections with certain types of papillomavirus, such as the human papillomavirus 16 (HPV16), are associated with the development of preneoplastic lesions and cancers of the anogenital, and head and neck regions. Cytinus hypocistis (L.) L. extracts are composed of substances presenting antiproliferative, antioxidant, anti-inflammatory and antibacterial properties, which might be promising as new therapeutic compounds. This study analysed the influence of topical application of an extract obtained from C. hypocistis (CH) on K14-HPV16 and FVB/n mice to evaluate its therapeutic and toxicological properties. To achieve the study goals, 30 female mice, 33–37 weeks old, were divided into six groups (n=5/group): I (HPV+CH3.1); II (HPV+CH6.2); III (HPV+CH12.4); IV (FVB/ n+CH12.4); V (HPV+control) and VI (FVB/n+control). CH was applied topically to both ears for 28 days. After this period, all animals were sacrificed for samples collection. Skin lesions were classified histologically. Toxicological parameters included haematological and biochemical blood markers, and hepatic oxidative stress analysis. Transgenic animals showed a decrease in mean body weight regardless of the concentration of extract applied. The extract had no influence on physiological parameters, organ weight, or biochemical and oxidative stress parameters. Histology demonstrated the presence of proliferative epithelial lesions in the skin and oral mucosa of K14-HPV16 mice, with no association with the application of this extract. Overall, the application of CH extract had no influence on the skin lesions and was well tolerated by the animals in these concentrations.
Metabolomic approach to assess the effect of the foraging year on the anti-elastase activity of Cytinus hypocistis (L.) L.
Publication . Silva, Ana Rita; Ayuso, Manuel; García, Pablo A.; Barros, Lillian; Edrada-Ebel, RuAngelie
Cytinus hypocistis (L.) L. is a wild edible parasitic plant on numerous members of the Cistaceae family. Although
its bioactive properties were potentially attributed to its hydrolysable tannin content, its chemical composition is
largely unknown, and the bioactive substances have not yet been identified [1,2]. Thus, this work aimed to
investigate its potential to inhibit human elastase, an important anti-ageing mechanism. The plant material was
collected in Castro Daire, Portugal, from the host species Halimium lasianthum (Lam.) Greuter on the same calendar
date in June for three consecutive years (2018, 2019, and 2020). Metabolomic- based analytical techniques were
applied to disclose the discriminating features of the anti-elastase activity of nine hydroethanolic extracts (three per
year). The discriminating features among samples were identified using a multivariate PLS-DA of the LC-HRMS
data. Dereplication of the discriminating metabolites obtained from the loadings plot revealed that year-one had
the best inhibitory activity with IC50s of 13 to 22 μg/mL and were comprised of high molecular weight
compounds ranging between 500 and 950 Da. However, the second-year collection exhibited a significant decrease
in the biological activity of 120 to 128 μg/mL indicating the occurrence of low molecular weight compounds between
150 and 300 Da. Year three gave IC50s of 74 to 109 μg/mL. This study offers an evidential hypothesis on the effect of
annual environmental changes on the chemical profile of a parasitic plant. Albeit, the relative abundance of the
discriminating features was minimal, it was significant enough to affect the anti-elastase bioactivity of the extracts
Exploring the α-amylase-inhibitory properties of tannin-rich extracts of Cytinus hypocistis on starch digestion
Publication . Correa, Vanesa G.; Garcia-Manieri, Jéssica A.A.; Silva, Ana Rita; Backes, Emanueli; Corrêa, Rúbia C.G.; Barros, Lillian; Bracht, Adelar; Peralta, Rosane M.
Cytinus hypocistis (L.) L. is an edible parasitic plant that grows within the roots of its host. In addition to its use as
famine food in the past, it is also tradidionally used for treating several illnesses such as intestinal problems,
inflammations, tumors, and bleeding. This species is rich in hydrolysable tannins, compounds often associated
with inhibiting starch digestion. Therefore, the present work investigated how effectively C. hypocistis tannin-rich
extracts inhibited enzymes involved in starch digestion and if such effect also occurs in vivo. The latter premise
was approached using the starch tolerance test in mice. Two optimized hydroethanolic extracts were used, a
heat-assisted and an ultrasound-assisted extract, with known hydrolysable tannin content. Both extracts
demonstrated potent inhibition of α-amylase. Inhibitions were of the mixed type with inhibitor constants in the
15 μg/mL range. The inhibition of the intestinal α-glucosidase was at least ten times less effective. The inhibition
of the α-amylase was negatively affected by in vitro gastrointestinal digestion and bovine serum albumin. In vivo,
both extracts inhibited starch digestion at doses between 100 and 400 mg/mL in healthy mice. The highest doses
of the ultrasound and heat extracts diminished the peak glucose levels in the starch tolerance test by 46 and
59.3%, respectively. In streptozotocin diabetic mice, this inhibition occurred only at the dose of 400 mg/mL.
Under this condition, diminution of the peak glucose concentration in the starch tolerance test was equal to
36.7% and 48.8% for the ultrasound and heat extracts, respectively. Maltose digestion was not inhibited by the
C. hypocistis extracts. Qualitatively and quantitatively, thus, the actions of both extracts were similar. The results
allow adding a new biological property to C. hypocistis, namely, the ability to decrease the hyper-glycemic
excursion after a starch-rich meal, propitiating at the same time a diminished caloric intake.
The effect of a tannin enriched topical formulation on mice ears fungal biodiversity
Publication . Silva, Ana Rita; Pereira, Rebeca; Soares, Ana Sofia; Coelho, Ana Cláudia; Ferreira, Isabel C.F.R.; Oliveira, Paula A.; Barros, Lillian
Cytinus hypocistis (L.) L. is a holoparasitic plant on various Cistaceae family members occurring in the
Mediterranean region. This specie has long been used in European popular medicine due to their astringent
and haemostatic properties. Current studies attributed its antioxidant, anti-inflammatory, and antimicrobial
properties to their hydrolysable tannin content. Therefore, the extracts of these plants might be a natural
alternative treatment for many skin conditions.
The main objective of the present work was to study the effect of C. hypocistis enriched topical formulation
(EF) in mice ears fungal isolates using routine mycological techniques [1]. Three EF were prepared with
different concentrations of C. hypocistis extracts: a) 3.1 mg extract (E)/g of cream; b) 6.2 mg E/g of cream;
and c) 12.4 mg E/g of cream. The formulations were carefully mixed to guarantee sample homogeneity. A
negative control (base cream) and the three EF were stored at 4 ºC during the experiments. Thirty samples
were collected in the ears of 30 female mice. The mice were divided in six groups of 5 mice each, to evaluate
the correct dosage and toxicity of the extract for the mice, although these were accessed in another essay.
Two of the groups were of wild type, one was treated with the base cream (G6) and the other with the cream
with highest concentration of extract (G4), and the four others of Papillomavirus models, a positive control
on which was applied the base cream (G5) and rest were treated with three creams with rising concentrations
of the extract (G1, G2 and G3, respectively). The experiment procedure was approved by a Animal Ethics
Committee-DGAV number 0421/000/000/2014, 24/09/2014 (020172).
Before the application, the surface of the ear was rubbed for 1 minute, using a swab and saline solution. The
samples were transferred to Petri dishes with the culture medium Potato Dextrose Agar and the microscopic
identification was carried out using the Lactophenol with Cotton Blue technique for staining the filamentous
fungi and identification of the genera. In the entirety of samples collected on the first collection, 4
filamentous fungal genera were isolated: Penicillium (16.7%) in G1, G4 and G5, Mucor (16.7%) G1, G2 ,
G4 and in G5, Cladosporium (3.33%) in G1, Aspergillus (3.33%) in G6. In total, fungi were isolated in 14
(46.7%) animals. Fungal genera were not identified in three mice from G1 and G5. The remaining samples
exhibiting either other types of microorganisms or no growth at all. After the application of C. hypocistis
cream for 17 days in the ears, only one fungal colony was isolated in a mouse from G4. The genus was not
identified. The results obtained suggest that C. hypocistis reduces the presence of fungal in the ears of mice.
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Funding agency
Fundação para a Ciência e a Tecnologia
Funding programme
POR_NORTE
Funding Award Number
SFRH/BD/145834/2019