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In vitro antioxidant and photoprotective activity of five native Brazilian bamboo species
Industrial Crops & Products 130 (2019) 208–215
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In vitro antioxidant and photoprotective activity of five native Brazilian bamboo species
T
Katarzyna Barbara Wróblewskaa, André Rolim Babyb, Maria Tereza Grombone Guaratinic, ⁎ Paulo Roberto Hrihorowitsch Morenoa, a
Institute of Chemistry, University of São Paulo, Av. Prof. Lineu Prestes 748, 05508-000, São Paulo, SP, Brazil Faculty of Pharmacy, University of São Paulo, São Paulo, Brazil c Institute of Botany, São Paulo, SP, Brazil b
ARTICLE INFO
ABSTRACT
Keywords: Aulonemia Chusquea Merostachys Flavonoids Sun protection factor Photostability
Nature provides an abundant source of antioxidant substances as well as potential compounds for sunscreen products. Asian bamboos, well known and widely cultivated, are already used in cosmetic and pharmaceutical industries thanks to their rich phenolic contents which is related to their antioxidant activity. This study aimed to evaluate the photoprotective and antioxidant activities of five native Brazilian bamboo species. Firstly, hydroalcoholic extracts from culms and leaves of Aulonemia aristulata (Döll) McClure., Chusquea bambusoides Rupr. ex Döll, Chusquea capituliflora Trin. var. pubescens McClure, Chusquea meyeriana Rupr., Merostachys pluriflora Munro ex E.G. Camus were evaluated for their DPPH scavenging activity and total contents of flavonoids and phenolic compounds. Secondly, cosmetic formulations containing the extracts in a combination with three different synthetic ultraviolet (UV) filters (avobenzone, octyldimethyl PABA, and octyl methoxycinnamate) were developed to evaluate their Sun Protection Factor (SPF), critical wavelength (cλ) and photostability by diffuse transmittance analysis to determine the interactions involving the extracts and the anti-UV active ingredients. The bamboos’ antioxidant potential, expressed in IC50, varied between 137.55 and 260 μg/mL. Phenolic contents ranged from 43.64 to 87.81 mg of gallic acid equivalents (GAE) per g of plant material. The extract richest in flavonoids was that from C. bambusoides leaves with 6.44 mg of equivalents of quercetin (EQ) per g of dried leaves. The SPF of the formulations with bamboo extracts varied between 34 and 86 before the irradiation, and the obtained UV absorption profile allowed to classify them as broad spectrum. After irradiation, the SPF values diminished to 14–44, whereas, the area of the absorbed wavelengths remained equal. This study showed that the addition of bamboo extracts to commercial UV filters increased significantly their SPF and photostability.
1. Introduction
Although the skin possesses an elaborate defense system to deal with the UV-induced oxidative stress, humans still need to use additional photoprotective measures to avoid the harmful effects of UV irradiation, such as diminishing sun exposure, wearing protective clothing and using topical sunscreens (Balogh et al., 2011). An optimal sunscreen must be multifunctional being able to reflect, absorb and scatter UV radiation, giving protection throughout the whole UV range and, preferentially, exerting antioxidant activity (Morabito et al., 2011). As the perfect sunscreen substance has not yet been discovered, the need for further research remains the same nowadays. Most sunscreen formulations are composed of several chemicals with each one absorbing at different regions of the UV radiation. However, several of these ingredient mixtures are not photostable, and their unknown photoproducts may be toxic for human skin cells (Butt
Solar radiation was one of the crucial factors for the existence of life on Earth. However, besides its beneficial effects, it also causes deleterious impact on humans, mostly due the exposure to ultraviolet radiation (UV). The shorter UV wavelengths (290–320 nm) are related to the vitamin D and melanin production, as a sunscreen function. The longer wavelengths (320–400 nm) can penetrate to deeper skin layers, generating reactive oxygen and nitrogen species which moderate cell metabolism and functions, and induce genetic changes (Juzeniene and Moan, 2012). In humans, intense overexposure to UVA and UVB radiation provokes erythema (or sunburn), an inflammatory reaction of the skin to UV radiation (Hönigsmann, 2002), oxidative stress, photoaging, immunosuppression and photocarcinogenesis (Polefka et al., 2011).
⁎
Corresponding author. E-mail address: [email protected] (P.R.H. Moreno).
https://doi.org/10.1016/j.indcrop.2018.12.081 Received 4 September 2018; Received in revised form 14 November 2018; Accepted 26 December 2018 0926-6690/ © 2018 Published by Elsevier B.V.
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and Christensen, 2000). For these reasons, the use of natural products has gained interest over the last years, mainly the polyphenols due to their broad UV absorption spectrum, including the UVB and UVA regions. In addition, these compounds can decrease oxidative stress and anti-inflammatory processes, and they can also stimulate DNA-repair and prevent skin damage (Radice et al., 2016). Bamboo is a group of genera of evergreen plants belonging to Poaceae, or grass family. These species are recognized for their multiple biological effects, such as antioxidant, anti-free radical, anti-aging, and antibacterial effects, as well as prevention of cardiovascular diseases (Hu et al., 2000; Kweon et al., 2001; Fujimura et al., 2005; Wang et al., 2012). The biological effects were related to the presence of different phenolic compounds, like flavonoids, cinnamates (Hu et al., 2000), lignophenol derivatives (Akao et al., 2004), anthocyanins, polysaccharides, catechins (Jin et al., 2006) and various volatile compounds (Fu et al., 2002; Takahashi et al., 2010). Although Eastern Asian bamboo species have already been well studied, little is known about the biological activities and chemical composition of the native Brazilian ones. Bamboo species share some common characteristics in phenolic types, accumulating several glycosylated flavones whose aglycones are represented by orientin, homoorientin, vitexin, isovitexin, and tricin (Zhang et al., 2005, Jiao et al. 2007). As natural phenols have been shown to perform topical photoprotective action (Saija et al., 2000; Martorana et al., 2013), the purpose of our study was to verify the potential use of Brazilian bamboo extracts as natural ingredients in sunscreen formulations. Therefore, we evaluated the in vitro antioxidant and photoprotective potential of ethanol extracts from culms and leaves of five native Brazilian bamboo species: Aulonemia aristulata (Döll) McClure., Chusquea bambusoides Rupr. ex Döll, Chusquea capituliflora Trin. var. pubescens McClure, Chusquea meyeriana Rupr., Merostachys pluriflora Munro ex E.G. Camus.
with a sieve of 200 μm. Extracts were prepared in a Soxhlet apparatus with 60% ethanol-water solution, in the proportion of 1.5 L solvent for each 100 g of plant, until exhaustion (until no residue was detected in the extractor solvent). The extracts were concentrated using rotary evaporator under vacuum at 50 °C, dried being lyophilized for 72 h and stored in a freezer at −20 °C. The yield was calculated based on the dry weight basis and expressed as percentage (%). 2.4. Scavenger radical activity Potential antioxidant activity was evaluated using a colorimetric method based on the scavenging of the DPPH radical described by Brand-Williams et al. (1995), adapted for 96-well plates. Bamboo extract samples were dissolved in methanol to obtain final concentrations in microplates: 500, 250, 200, 100, 50 e 25 μg/mL. Quercetin was used as a standard in five different concentrations between 2.5 and 0.25 μg/ mL. To the microplates, were added subsequently 50 μL of the sample/ standard and 150 μL of 200 μmol/L methanol solution of DPPH. Methanol together with DPPH was utilized as a negative control. Sample blanks were prepared by diluting the extracts solutions with methanol, without the reagent. After 30 min of reaction protected from light, the absorbances were measured at λ = 515 nm in a microplate reader (LGC Biotecnologia, LM-LGC). Values of antioxidant activity were expressed by the half maximum inhibitory concentration (IC50, mean ± standard deviation), concentration needed to quench 50% of the DPPH radical, using the linear regression curves of the samples’ radical inhibition. 2.5. Total phenolic contents Total amount of phenols in bamboo extracts was determined by a spectrophotometric method developed by Ainsworth and Gillespie, (2007) using Folin-Ciocalteu reagent. Bamboo extracts were dissolved in methanol to obtain a solution of 1 mg/mL, whereas the final concentration in the plates was 100 μg/mL. Gallic acid in different concentrations (10 – 2.5 μg/mL) was used as reference compound for quantification. The assay was performed by adding to the microplates 20 μL of the sample, 30 μL of distilled water, 50 μL of Folin-Ciocalteu reagent and 100 μL of solution of sodium carbonate (700 mmol/L). Sample blanks were prepared to discount the absorbance of the extracts’ solutions using methanol in place of the reagent. After 2 h of reaction in dark, the absorbance of the samples was measured in spectrophotometer (SpectraMax M4), at λ = 760 nm. The results were expressed in milligrams of gallic acid equivalents per g of plant material (mg GAE/g, mean ± standard deviation).
2. Material and methods 2.1. Chemicals Ethanol and methanol provided by Synth® was used for the extraction process and chemical analyses. Standards - quercetin, gallic acid, and the reagents: 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) and Folin-Ciocalteu used for the assays were obtained from Sigma Aldrich®. Aluminum chloride used for flavonoids quantification was provided by Merck®. Ingredients of photoprotective formulations contained: Aristoflex® AVC and Phenonip® by PharmaSpecial®, butylated hydroxytoluene (BHT) by Vital®, Avobenzone and Propyleneglycol by All Chemistry®, Octyldimethyl PABA by Deg®, Octylmethoxycinnamate and Parrafinum liquidum by Volp®.
2.6. Total flavonoid contents Another spectrophotometric method (Woisky and Salatino, 1998) was used to estimate total flavonoid contents. Test samples in concentration of 5.0 mg/mL (400 μL/mL in the well) were prepared by dissolving dry bamboo extracts in 80% solution of methanol. Quercetin, in five different concentrations between 15 and 2.5 μg/mL in the same solvent solution, was used as quantification reference. For the assay, 20 μL of sample, 210 μL of 80% methanol and 20 μL of 2% aluminum chloride solution were added to the microplates. As blank, some microplate wells received only the sample and 80% methanol. After 30 min in darkness, absorbances were measured at λ = 425 nm in spectrophotometer (SpectraMax M4). The results were expressed in milligrams of quercetin equivalents per g of plant material (mg QE/m, mean ± standard deviation).
2.2. Plant material Culms and leaves of Chusquea bambusoides Rupr. ex Döll and Chusquea capituliflora Trin. var. pubescens McClure were collected at Municipal Natural Springs Park of Paranapiacaba, Santo André (23°46′41″S and 46°18′16″W), whereas those from Aulonemia aristulata (Döll) McClure Chusquea meyeriana Rupr. and Merostachys pluriflora Munro ex E.G. Camus at the Fontes do Ipiranga State Park in São PauloSP, Brazil (23°38′08″-23°40′18″S and 46°36′48″-46°38′00″W). All specimens were identified by Dr. Tarcísio Filgueiras from Botany Institute of São Paulo and deposited at the herbarium in the same institution. Voucher species numbers are: Shirasuna et al. 672 (SP), Shirasuna, RT & Suzuki, R. 1580 (SP), Vinha, D. s/n. 398161 (SP), Grombone, MT s/n 412118 (SP) and Shirasuna, RT 2619 (SP), respectively.
2.7. Preparation of photoprotective formulations
2.3. Crude extract preparation
Sunscreen formulations (20 g) were prepared using m/m proportions. The bamboo extracts were added in concentration of 10%. Aristoflex® AVC (ammonium acryloyldimethyltaurate/VP copolymer), a
Culms and leaves were separated, dried at room temperature, protected from light and grounded in a knives mill (Tecnal TE-580, Brasil) 209
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synthetic polymer, which was an emulsifier and gelling agent was added in 3%. All formulations contained chemical UVA and UVB filters in the following concentrations: 3.0% avobenzone; 8,0% octyldimethyl PABA, and 7.5% octylmethoxycinnamate. Additionally, BHT was used as antioxidant (0.1%), Paraffinum liquidum (mineral oil) as emollient and solvent of the oil phase of the formulation (5%). Propylene glycol was added as humectant (5%) and mixture of 2-phenoxyethanol, methyl 4-hydroxybenzoate, ethyl 4-hydroxybenzoate, propyl 4-hydroxybenzoate and butyl 4-hydroxybenzoate called Phenonip® as preservative (0.75%). Distilled water (q.s.) was a vehicle of the aqueous (gel) phase. In the first step, anti-UV agents were dissolved together by mixing with BHT and mineral oil at 70 °C in a beaker. Dry bamboo extracts were weighed and pulverized in a mortar. One half of total distilled water volume was used to dissolve bamboo extracts in a mortar with propylene glycol and then, the solution obtained was transferred to a beaker with Aristoflex® AVC. The remaining quantity of water was added, and gel was obtained by mixing the water solution with a glass rod. Finally, the UV filter solution was incorporated by parts to the formed gel. The formulation was then homogenized with the preservative in mechanical stirrer (IKA RW 20) at 1000 rpm for 5 min. In total, 11 sunscreen formulations were prepared, 10 of them were test samples prepared with leaf or culm bamboo extracts plus the UV filters, numbered as formulations F1 to F10, and one control formulation (F11), containing only the filters, as presented in Table 1.
c 290 nm
A ( ) d = 0.9
400 nm 290 nm
A ( )d
(2)
The results were presented as mean ± standard deviation. 2.9. Photostability assay Formulations had their functional photostability evaluated using the same procedure as described above with an additional step of irradiating the samples with a fixed dose of artificial solar light (580.08 W/ m2 - 2088 J/m2) in a photostable chamber for one hour at 35 °C. The sample and control absorbances were measured using the same diffuse reflectance spectrophotometer with the identical reading program. The results were also converted to SPF and cλ values and presented as mean ± standard deviation. Nine readings were recorded for each plate. 2.10. Statistical tests To compare the differences among all the samples in the measurements one-way ANOVA and Tukey test were used. Changes in SPF and cλ after irradiation in photoprotective and photostability assessments were detected by t-Student test. All the statistical analyses we performed in Minitab® 17. 3. Results and discussion
2.8. In vitro SPF evaluation
3.1. Crude extract analyses
In vitro photoprotective efficacy of bamboo extracts was estimated by diffuse reflectance spectrophotometer with integrating sphere (Labsphere®UV2000S). For the assay, formulations containing the bamboo extracts and the control were weighed and uniformly spread as a thin film (1.3 mg/cm2) on the surface of a polymethyl-methacrylate plate (PMMA, HD Helioplate® 6 HelioScreen) according to the Cosmetics Europe guidelines (2011), previously known as COLIPA (The European Cosmetic and Perfumery Association). After 40 min of drying, sample absorbances were measured between 290 and 400 nm, with progression rate of 1.0 nm. Nine readings were recorded for each plate. Plates without the film were used as blanks, and two of them were used for the control formulation (F11). Absorbance values were converted to in vitro sun protection factor (SPF) and critical wavelength (cλ) using the Eqs. 1 and 2 (Cosmetics Europe, 2011; Diffey, 2002; Springsteen et al., 1999), where: Eλ is spectral irradiance effectiveness accordingly to CIE (Commission Internationale de l'Eclairage); Sλ is solar spectral irradiance; Tλ is spectral transmittance of the sample; dλ is range of wavelengths and A (λ) is spectral absorbance of the sample.
3.1.1. Extraction yield Since the bamboos are known to possess glycosylated flavonoids as one of the main compounds, 60% ethanol was chosen as a solvent for the extraction process because of its higher polarity. The crude extracts yield varied between 7.6 and 22.2% (m/m), with the leaves affording higher values for all species, as it is usual in plant materials (Table 2). C. capituliflora leaves presented almost the double of extractable material (22.2%) than those from the other species, where the yields ranged from 10 to 14%. In general, culm extracts yields were alike to those obtained for leaves, with the lowest results for M. pluriflora culms. Comparing these values with Asian bamboo species, the native Brazilian bamboos presented similar yields (Wang et al., 2012; Macwan et al., 2010).
SPF =
400 nm E S d 290 nm 400 nm E S T d 290 nm
3.1.2. Antiradical activity Due to their antioxidant effects (Hu et al., 2000), associated with the high polyphenol contents, bamboos are currently proposed as promising anti-UV agents, once there is a correlation between reactive oxygen species (ROS) and UVB exposure (Dinkova-Kostova, 2008). These factors lead to an initial screening for antioxidant properties, expressed as radical scavenger potential, and the evaluation of the total phenol and total flavonoid contents in the Brazilian bamboo extracts. Although, DPPH method does not directly reflect the biological antioxidant activity, it is commonly used, especially for screening purposes, because it is quick, reliable and reproducible. The bamboo extracts presented DPPH scavenging activity (IC50) ranging from 137 to 260 μg/mL, where the most potent extract was C. capituliflora leaves (Table 2). Despite the wide activity range, no significant difference was observed among the culm and leaf samples. In general, all the native Brazilian bamboos presented an antioxidant potential approximately 10 times higher than those observed for the Asian bamboos Phyllostachys pubescens and P. nigra (Park and Jhon, 2010). The antioxidant potential observed for the native Brazilian bamboos were alike to reported for turmeric roots with IC50 = 104.91 μg/mL in the DPPH assay, which are well known for their antioxidant properties (Sukandar et al., 2015). These results indicate a promising antioxidant potential for the bamboo extracts,
(1)
Table 1 List of sunscreen formulations developed for the photoprotective assay sorted by bamboo species and part of the plant used. Formulation
Bamboo species
Part of the plant used
F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11
A. aristulata
Culm Leaves Culm Leaves Culm Leaves Culm Leaves Culm Leaves NA
C. bambusoides C. capituliflora C. meyeriana M. pluriflora Control
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Table 2 Yield (%), antioxidant activity assay (IC50), total phenolic compounds (GAE) and total flavonoid content (QE) tests obtained for native Brazilian bamboo species. Bamboo species Culm Extracts A. aristulata C. bambusoides C. capituliflora C. meyeriana M. pluriflora Leaf Extracts A. aristulata C. bambusoides C. capituliflora C. meyeriana M. pluriflora
Yield [%]
DPPH IC50 [μg/mL]
9.9 12.2 8.6 7.8 7.6
205.68 236.59 244.36 168.18 174.17
± ± ± ± ±
31.22b,c 42.22c 50.30c 21.79a,b 12.88a,b
55.02 43.64 46.31 87.81 54.40
± ± ± ± ±
0.47c,d,e 2.24e 0,95d,e 2.14a 2.30c,d,e
3.16 2.22 4.83 4.60 4.01
± ± ± ± ±
0.38f,g,h,i 0.25i 0.25b,c,d 0.16c,d,e 0.33d,e,f,g
10.1 14.2 22.2 14.2 13.9
260.17 218.16 137.55 205.48 222.52
± ± ± ± ±
42.14c 34.56b,c 15.62a 38.14b,c 20.05b,c
49.54 59.53 57.00 71.03 47.34
± ± ± ± ±
0.74d, 4.83b, 4.49b, 3.61b 0.62d,
3.65 6.44 3.87 4.08 5.93
± ± ± ± ±
0.40d, e, 0.25a 0.31d, e, 0.04d, e, 0.27a, b
GAE [mg/g]
QE [mg/g]
e c, d c, d, e e
f, g, h f, g, h f
IC50 – Inhibition Concentration – concentration needed to inhibit 50% of the radical formation; GAE – Gallic Acid Equivalents; QE – Quercetin Equivalents. Statistic tests were performed separately for each assay. Small letters represent results of one-way ANOVA test with Tukey comparison. Values in the same column that do not share the same letter are statistically different.
found in bamboo extracts that may interact differently with other oil phase ingredients, such as synthetic UV filters. Also, the variations between each formulation properties resulted from distinctive features of each extract.
although the IC50 values were much higher than that from pure quercetin (1.76 μg/mL), the positive control. 3.1.3. Quantification of phenolic compounds and flavonoids As the DPPH scavenging activity might be related with the presence of polyphenols (Dudonné et al., 2009), the contents of total phenolic compounds and flavonoids were measured in the extracts. The total phenolic compounds varied from ˜44 to 88 mg GAE/g of extract as per Table 2. The highest amounts of phenolics were found in the C. meyeriana culm and leaf extract (87.81 and 71.03 mg GAE/g, respectively). All five Brazilian bamboo species showed higher amounts of phenolic than those reported for the Asian species. B. arundinacea containing only 14.6 mg GAE/g (Macwan et al., 2010). Similarly, total phenolic content of Schizostachyum lumampao (Blanco) Merr were 76.7 and 13.5 mg of GAE per 100 g of air-dried sample for the ethanolic and aqueous extracts, respectively (Tongco et al., 2014). Flavonoids are a special group of phenolic compounds which are also involved in the antioxidant properties of plant extracts (Molay et al., 2010). The total amount of flavonoids found in native Brazilian bamboo species, expressed in milligrams equivalents of quercetin (QE), ranged from 2.22 to 6.44 QE per g of extract (Table 2). Leaf extracts were richer in flavonoids than those from culms. The results obtained for other bamboo species varied, for example, in Dendrocalamopsis oldhami (Munro) Keng f. total flavonoid content was higher – 74 mg QE/g of bamboo leaf extract (Lv et al., 2012). On the other hand, aqueous and methanolic extracts from Sasa senanensis (Franch. & Sav.) Rehder gave only 1.18 and 1.85 mg Q/100 g of leaf powder (Khatun et al., 2013), whereas S. lumampao had 70.2 and 17.86 mg QE per 100 g air-dried leaves for the ethanolic and aqueous extracts, respectively (Tongco et al., 2014) which is much less than the Brazilian bamboos. Nevertheless, seasonal variations can be responsible for the differences observed in the chemical composition of the bamboos (Ni et al., 2012).
3.2.2. In vitro photoprotection and photostability assays SPF is the worldwide parameter used to evaluate the efficacy of sunscreens and its general definition is the ratio of the energy of UV radiation required to produce erythema in the protected skin (with sunscreen) to that required to produce erythema in the unprotected skin (without sunscreen), obtained through in vivo assays (Ebrahimzadeh et al., 2014). However, recently some methods using diffuse reflectance spectroscopy (DRS), a non-invasive assay to determine the UV protection, have demonstrated good correlation with in vivo methodologies (Junior et al., 2014). In our study, the photoprotection of sunscreen formulations containing bamboo extracts and well-known UV filters (Avobenzone, Octyldimethyl PABA and Octylmethoxycinnamate) was evaluated by measuring the SPF using DRS before sunlight irradiation and compared with a control containing only the solar agents. All formulations with the bamboo extracts presented SPF values between 34.52 (F3) and 86.15 (F1), showing higher values than the control (F11), SPF 13.00, as can be seen in Table 4. Although the standard deviations for the samples with the extracts were considered high, in some cases more than 50%, statistical analysis showed a significant difference with the control. According to Tukey test, only the C. bambusoides formulations (F3 and F4) were not different from the control. The high standard deviations observed for the formulations containing bamboo extracts might be related to lack of homogeneity of the samples, that might affect the equipment reflectance optic readings. The experiment was performed 3 times, since application of the formulations over the PMMA plates is done manually, and the film characteristics might have been different among the exposed area of analysis, influencing the result variations. In addition, formulations containing only bamboo extracts did not present a considerable SPF (data not shown), but when combined with synthetic filters they were able to increase measured photoprotection. As the SPF alone is an indicator only for a protection against erythemally effective solar UV, largely, confined to the UVB (290–320 nm) and partially short-wavelength UVA (320–340 nm) radiation, it does not provide information regarding protection against long-wavelength UVA1 (340–400 nm) (Hojerová et al., 2011), which can lead to several cumulative damages, such as immunosuppression, skin aging, and various other photodisorders (Velasco et al., 2008). For this reason, critical wavelength (cλ) is another parameter that describes if the photoprotective product achieved broad spectrum of action. It is defined as the wavelength at which the area under the curve gives 90%
3.2. Photoprotection and photostability of bamboo formulations 3.2.1. Formulations All the formulations containing bamboo extracts were emulsified systems. Their individual characteristics are listed in Table 3. The emulsions started to separate small amounts of the oil phase up to 2–3 h after the preparation. Formulations F2, F4, F6 and F8 were defined as more homogeneous due to a uniform aspect. The color of formulations varied from yellowish, light to dark green or brown with a specific herbal odor. The control formulation (F11) was homogenous and with creamy color and a characteristic odor of the gelling base. Difficulties during gel formation can be caused by diverse chemical compounds 211
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Table 3 Formulations received from bamboo extracts and synthetic solar filters. Formulation number
Aspect
Organoleptic properties
Formulation number
Aspect
Organoleptic properties
F1
Partially homogeneous emulsion, brown color and herbal odor
F6
Homogeneous emulsion, green color and herbal odor
F2
Homogeneous emulsion, brown color and herbal odor
F7
Partially homogeneous emulsion, yellowish color and herbal odor
F3
Partially homogeneous emulsion, brownyellowish color and herbal odor
F8
Homogeneous emulsion, green-brown color and herbal odor
F4
Homogeneous emulsion, dark green color and herbal odor
F9
Partially homogeneous emulsion, yellowish color and herbal odor
F5
Partially homogeneous emulsion, yellowish color and herbal odor
F10
Partially homogeneous emulsion, dark orangebrown color and herbal odor
F11 (control)
Homogeneous emulsion, clear yellowish color and specific odor of filters/cream base
Extract type used in formulations: F1 – A. aristulata culms; F2 – A. aristulata leaves; F3 – C. bambusoides culms; F4 –C. bambusoides leaves; F5 – C. capituliflora culms; F6 – C. capituliflora leaves; F7 – C. meyeriana culms; F8 – C. meyeriana leaves; F9 – M. pluriflora culms; F10 – M. pluriflora leaves; F11: water (control).
of the total absorbance of the sample between 290–400 nm. The Food and Drug Administration (FDA) considers 370 nm as the minimal cλ for broad spectrum sunscreens (Polonini et al., 2014). Additionally, an ideal sunscreen must have a minimal SPF value of 15, in accordance with the FDA (Food and Drug Administration, 2007). As can be seen in Table 4, all bamboo formulations can be defined as good efficacy samples (cλ higher than 370 nm and SPF more than 15), whereas the control sample presented broad spectrum of action (cλ higher than 370 nm) but low photoprotective effect (SPF less than 15). Photostability is another essential property describing the effectiveness and safety of sunscreen products, since UV radiation can lead
to reduction of the photoprotective capacity of UV filters and can also generate free radicals (Gonzalez et al., 2007). Avobenzone is a widely used UVA filter that is known to be a rather unstable substance, loosing much of its protective capability after irradiation through tautomerization, fragmentation and photoproduct formation. The photolysis mechanism involves singlet oxygen formation that may induce irreversible modifications in skin proteins, leading to tissue photodegradation and other harmful effects to the skin cells (Abid et al., 2017; Paris et al., 2009). Additionally, it is known that octylmethoxycinnamate in the presence of avobenzone undergoes a photolysis process, rather than the expected E/Z photoisomerization, producing
Table 4 Sun Protection Factor (SPF) and critical wavelength (cλ) of sunscreen formulations containing native Brazilian bamboo extracts, before and after sunlight irradiation. Formulation
SPF (mean ± standard deviation) a
Before irradiation F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 (control)
86.15 61.41 34.52 36.61 78.22 61.59 84.37 51.41 83.19 61.48 13.00
± ± ± ± ± ± ± ± ± ± ±
40.81A,B 37.36A,B,C 10.39C,D 10.68C,D 53.15A,B 35.99A,B,C 40.98A,B 25.84B,C 41.11A,B 32.26A,B,C 2.50D
cλ [nm] b
After irradiation
Before irradiationx
After irradiationx
32.30 ± 15.55E,F,G 22.04 ± 12.16G,H,I 14.33 ± 4.17H,I 35.22 ± 13.48E,F 44.44 ± 37.31E,F 25.93 ± 11.86F,G,H 28.78 ± 20.12E,F,G,H 20.41 ± 13.53G,H,I 41.33 ± 24.91E,F 41.00 ± 27.33E,F 6.00 ± 1.6I
380X 381X 380X 381X 381X 383X 381X 381X 380 X 380X 381X
378X 379X 378X 380X 379X 381X 379X 379X 379X 380X 379X
SPF - Sun Protection Factor; cλ – critical wavelength. Small letters show the results of t-Student test; differences between the values obtained before and after irradiation. Capital letters show the differences between all the samples (one-way ANOVA + Tukey comparison test). Values not sharing the same letter are statistically different. Extract type used in formulations: F1 – A. aristulata culms; F2 – A. aristulata leaves; F3 – C. bambusoides culms; F4 –C. bambusoides leaves; F5 – C. capituliflora culms; F6 – C. capituliflora leaves; F7 – C. meyeriana culms; F8 – C. meyeriana leaves; F9 – M. pluriflora culms; F10 – M. pluriflora leaves; F11: water (control). 212
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Fig. 1. Average UV absorbance profiles for formulations F1-F11 before (A) and after (B) irradiation with artificial sunlight for 1 h. Extract type used in formulations: F1 – A. aristulata culms; F2 – A. aristulata leaves; F3 – C. bambusoides culms; F4 –C. bambusoides leaves; F5 – C. capituliflora culms; F6 – C. capituliflora leaves; F7 – C. meyeriana culms; F8 – C. meyeriana leaves; F9 – M. pluriflora culms; F10 – M. pluriflora leaves; F11: water (control was recorded twice, as F11_1 and F11_2).
mechanisms involved and the compounds responsible for these interactions still need to be elucidated. Several plant extracts have already demonstrated a photoprotective activity by increasing the formulations SPF or other mechanisms. However, acceptable SPF values are only achieved with high amounts of the plant extract, like with the oil fraction of spent coffee grounds and green coffee beans that needed 35% of the oils to give SPF of 51.9 and 82.3 (Marto et al., 2016), or in the presence of inorganic filters, as was observed for formulations containing 1.68% (w/w) Passiflora incarnata L. dry extract with 7.0% (w/w) ethylhexyl methoxycinnamate, 2.0% (w/w) benzophenone-3 and 2.0% (w/w) TiO2 generated an SPF of 20 (Velasco et al., 2008). Comparing with our results, extracts from native Bamboo species at 10% (w/w) concentration could improve the photoprotective activity from formulations using only organic filters, reaching SPF values up to 86, and they also promoted the filters stability as SPF’s remained 2.4 to 7.4 times higher than the control after irradiation.
long lived radicals in the sunscreen film (Sayre et al., 2005). For these reasons, these filters were used in the formulations to test the photostabilization capacity of the Bamboo extracts. The results obtained after irradiation with the artificial sunlight (Table 4) showed that the formulations continued to be broad spectrum sunscreens as the cλ did not change. However, SPF decreased significantly for all the samples, except of F4 (C. bambusoides leaf). The photoprotection efficacy was still good after irradiation (FPS > 15) for most formulations, with exception of F2, F3 and F8 that were not significantly different from the control. The differences observed in the absorbance profiles after irradiation can be seen in Fig. 1. Sunscreens could have antioxidants added in their composition to complement UV filter photoprotection by reducing the damage from the free radicals generated after solar exposure and to stabilize chemical UV filters (Lim et al., 2017; Badea et al., 2015). Natural extracts have been one of the strategies used to broaden the photoprotection spectrum and increase the photostability of sunscreens, mainly those containing polyphenolic compounds due to their ability to absorb the solar spectrum and react with free radicals (Cefali et al., 2016; Korać and Khambholja, 2011). A positive correlation has been found for the phenolic contents, like flavonoids and hydroxycinnamic acids, in an extract and the increase in SPF (Ebrahimzadeh et al., 2014). Flavonoids, besides their UV absorption, may contribute to stabilize the UV filters by multiple mechanisms including oxygen radical scavenging, inhibition of lipid peroxidation and metal ion chelation. It has been demonstrated that rutin stabilization effect on two UV filters cannot be attributed only to its antioxidant activity (Velasco et al., 2008). Additional mechanisms contribute to quercetin effectiveness such as antiradical property, chelation of metal ions acting as catalyst in reactions leading to free radical formation and quenching reactive oxygen species, like the excited triplet states from the organic UV filters butyl methoxydibenzoylmethane and octyl methoxycinnamate (Scalia and Mezzena, 2010). In this sense, the photoprotection and photostability conferred to the formulation by the native Brazilian bamboo species might be related to their phenolic and/or flavonoid contents, but
4. Conclusions In this study, screening of crude extracts from Brazilian bamboo species for potential antioxidant activity, phenolic and flavonoid contents indicated that the phenolic contents are higher than in Asian species and a good radical scavenging activity. These results led to the preparation of sunscreen formulations, with the same plant extracts, in a combination with commercial UV filters. The results showed that the addition of the biological samples increased the SPF and photostability of the synthetic UV filters. According to our observations, due to their rich in phenolic substances composition, the Brazilian bamboo species can provide a promising source of biologically active agents increasing the efficacy of synthetic solar filters. The next step of this research will be to isolate and identify the substances responsible for these properties and their mechanism of action for the most active species.
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In vitro antioxidant and photoprotective activity of five native Brazilian bamboo species
T
Katarzyna Barbara Wróblewskaa, André Rolim Babyb, Maria Tereza Grombone Guaratinic, ⁎ Paulo Roberto Hrihorowitsch Morenoa, a
Institute of Chemistry, University of São Paulo, Av. Prof. Lineu Prestes 748, 05508-000, São Paulo, SP, Brazil Faculty of Pharmacy, University of São Paulo, São Paulo, Brazil c Institute of Botany, São Paulo, SP, Brazil b
ARTICLE INFO
ABSTRACT
Keywords: Aulonemia Chusquea Merostachys Flavonoids Sun protection factor Photostability
Nature provides an abundant source of antioxidant substances as well as potential compounds for sunscreen products. Asian bamboos, well known and widely cultivated, are already used in cosmetic and pharmaceutical industries thanks to their rich phenolic contents which is related to their antioxidant activity. This study aimed to evaluate the photoprotective and antioxidant activities of five native Brazilian bamboo species. Firstly, hydroalcoholic extracts from culms and leaves of Aulonemia aristulata (Döll) McClure., Chusquea bambusoides Rupr. ex Döll, Chusquea capituliflora Trin. var. pubescens McClure, Chusquea meyeriana Rupr., Merostachys pluriflora Munro ex E.G. Camus were evaluated for their DPPH scavenging activity and total contents of flavonoids and phenolic compounds. Secondly, cosmetic formulations containing the extracts in a combination with three different synthetic ultraviolet (UV) filters (avobenzone, octyldimethyl PABA, and octyl methoxycinnamate) were developed to evaluate their Sun Protection Factor (SPF), critical wavelength (cλ) and photostability by diffuse transmittance analysis to determine the interactions involving the extracts and the anti-UV active ingredients. The bamboos’ antioxidant potential, expressed in IC50, varied between 137.55 and 260 μg/mL. Phenolic contents ranged from 43.64 to 87.81 mg of gallic acid equivalents (GAE) per g of plant material. The extract richest in flavonoids was that from C. bambusoides leaves with 6.44 mg of equivalents of quercetin (EQ) per g of dried leaves. The SPF of the formulations with bamboo extracts varied between 34 and 86 before the irradiation, and the obtained UV absorption profile allowed to classify them as broad spectrum. After irradiation, the SPF values diminished to 14–44, whereas, the area of the absorbed wavelengths remained equal. This study showed that the addition of bamboo extracts to commercial UV filters increased significantly their SPF and photostability.
1. Introduction
Although the skin possesses an elaborate defense system to deal with the UV-induced oxidative stress, humans still need to use additional photoprotective measures to avoid the harmful effects of UV irradiation, such as diminishing sun exposure, wearing protective clothing and using topical sunscreens (Balogh et al., 2011). An optimal sunscreen must be multifunctional being able to reflect, absorb and scatter UV radiation, giving protection throughout the whole UV range and, preferentially, exerting antioxidant activity (Morabito et al., 2011). As the perfect sunscreen substance has not yet been discovered, the need for further research remains the same nowadays. Most sunscreen formulations are composed of several chemicals with each one absorbing at different regions of the UV radiation. However, several of these ingredient mixtures are not photostable, and their unknown photoproducts may be toxic for human skin cells (Butt
Solar radiation was one of the crucial factors for the existence of life on Earth. However, besides its beneficial effects, it also causes deleterious impact on humans, mostly due the exposure to ultraviolet radiation (UV). The shorter UV wavelengths (290–320 nm) are related to the vitamin D and melanin production, as a sunscreen function. The longer wavelengths (320–400 nm) can penetrate to deeper skin layers, generating reactive oxygen and nitrogen species which moderate cell metabolism and functions, and induce genetic changes (Juzeniene and Moan, 2012). In humans, intense overexposure to UVA and UVB radiation provokes erythema (or sunburn), an inflammatory reaction of the skin to UV radiation (Hönigsmann, 2002), oxidative stress, photoaging, immunosuppression and photocarcinogenesis (Polefka et al., 2011).
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Corresponding author. E-mail address: [email protected] (P.R.H. Moreno).
https://doi.org/10.1016/j.indcrop.2018.12.081 Received 4 September 2018; Received in revised form 14 November 2018; Accepted 26 December 2018 0926-6690/ © 2018 Published by Elsevier B.V.
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and Christensen, 2000). For these reasons, the use of natural products has gained interest over the last years, mainly the polyphenols due to their broad UV absorption spectrum, including the UVB and UVA regions. In addition, these compounds can decrease oxidative stress and anti-inflammatory processes, and they can also stimulate DNA-repair and prevent skin damage (Radice et al., 2016). Bamboo is a group of genera of evergreen plants belonging to Poaceae, or grass family. These species are recognized for their multiple biological effects, such as antioxidant, anti-free radical, anti-aging, and antibacterial effects, as well as prevention of cardiovascular diseases (Hu et al., 2000; Kweon et al., 2001; Fujimura et al., 2005; Wang et al., 2012). The biological effects were related to the presence of different phenolic compounds, like flavonoids, cinnamates (Hu et al., 2000), lignophenol derivatives (Akao et al., 2004), anthocyanins, polysaccharides, catechins (Jin et al., 2006) and various volatile compounds (Fu et al., 2002; Takahashi et al., 2010). Although Eastern Asian bamboo species have already been well studied, little is known about the biological activities and chemical composition of the native Brazilian ones. Bamboo species share some common characteristics in phenolic types, accumulating several glycosylated flavones whose aglycones are represented by orientin, homoorientin, vitexin, isovitexin, and tricin (Zhang et al., 2005, Jiao et al. 2007). As natural phenols have been shown to perform topical photoprotective action (Saija et al., 2000; Martorana et al., 2013), the purpose of our study was to verify the potential use of Brazilian bamboo extracts as natural ingredients in sunscreen formulations. Therefore, we evaluated the in vitro antioxidant and photoprotective potential of ethanol extracts from culms and leaves of five native Brazilian bamboo species: Aulonemia aristulata (Döll) McClure., Chusquea bambusoides Rupr. ex Döll, Chusquea capituliflora Trin. var. pubescens McClure, Chusquea meyeriana Rupr., Merostachys pluriflora Munro ex E.G. Camus.
with a sieve of 200 μm. Extracts were prepared in a Soxhlet apparatus with 60% ethanol-water solution, in the proportion of 1.5 L solvent for each 100 g of plant, until exhaustion (until no residue was detected in the extractor solvent). The extracts were concentrated using rotary evaporator under vacuum at 50 °C, dried being lyophilized for 72 h and stored in a freezer at −20 °C. The yield was calculated based on the dry weight basis and expressed as percentage (%). 2.4. Scavenger radical activity Potential antioxidant activity was evaluated using a colorimetric method based on the scavenging of the DPPH radical described by Brand-Williams et al. (1995), adapted for 96-well plates. Bamboo extract samples were dissolved in methanol to obtain final concentrations in microplates: 500, 250, 200, 100, 50 e 25 μg/mL. Quercetin was used as a standard in five different concentrations between 2.5 and 0.25 μg/ mL. To the microplates, were added subsequently 50 μL of the sample/ standard and 150 μL of 200 μmol/L methanol solution of DPPH. Methanol together with DPPH was utilized as a negative control. Sample blanks were prepared by diluting the extracts solutions with methanol, without the reagent. After 30 min of reaction protected from light, the absorbances were measured at λ = 515 nm in a microplate reader (LGC Biotecnologia, LM-LGC). Values of antioxidant activity were expressed by the half maximum inhibitory concentration (IC50, mean ± standard deviation), concentration needed to quench 50% of the DPPH radical, using the linear regression curves of the samples’ radical inhibition. 2.5. Total phenolic contents Total amount of phenols in bamboo extracts was determined by a spectrophotometric method developed by Ainsworth and Gillespie, (2007) using Folin-Ciocalteu reagent. Bamboo extracts were dissolved in methanol to obtain a solution of 1 mg/mL, whereas the final concentration in the plates was 100 μg/mL. Gallic acid in different concentrations (10 – 2.5 μg/mL) was used as reference compound for quantification. The assay was performed by adding to the microplates 20 μL of the sample, 30 μL of distilled water, 50 μL of Folin-Ciocalteu reagent and 100 μL of solution of sodium carbonate (700 mmol/L). Sample blanks were prepared to discount the absorbance of the extracts’ solutions using methanol in place of the reagent. After 2 h of reaction in dark, the absorbance of the samples was measured in spectrophotometer (SpectraMax M4), at λ = 760 nm. The results were expressed in milligrams of gallic acid equivalents per g of plant material (mg GAE/g, mean ± standard deviation).
2. Material and methods 2.1. Chemicals Ethanol and methanol provided by Synth® was used for the extraction process and chemical analyses. Standards - quercetin, gallic acid, and the reagents: 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) and Folin-Ciocalteu used for the assays were obtained from Sigma Aldrich®. Aluminum chloride used for flavonoids quantification was provided by Merck®. Ingredients of photoprotective formulations contained: Aristoflex® AVC and Phenonip® by PharmaSpecial®, butylated hydroxytoluene (BHT) by Vital®, Avobenzone and Propyleneglycol by All Chemistry®, Octyldimethyl PABA by Deg®, Octylmethoxycinnamate and Parrafinum liquidum by Volp®.
2.6. Total flavonoid contents Another spectrophotometric method (Woisky and Salatino, 1998) was used to estimate total flavonoid contents. Test samples in concentration of 5.0 mg/mL (400 μL/mL in the well) were prepared by dissolving dry bamboo extracts in 80% solution of methanol. Quercetin, in five different concentrations between 15 and 2.5 μg/mL in the same solvent solution, was used as quantification reference. For the assay, 20 μL of sample, 210 μL of 80% methanol and 20 μL of 2% aluminum chloride solution were added to the microplates. As blank, some microplate wells received only the sample and 80% methanol. After 30 min in darkness, absorbances were measured at λ = 425 nm in spectrophotometer (SpectraMax M4). The results were expressed in milligrams of quercetin equivalents per g of plant material (mg QE/m, mean ± standard deviation).
2.2. Plant material Culms and leaves of Chusquea bambusoides Rupr. ex Döll and Chusquea capituliflora Trin. var. pubescens McClure were collected at Municipal Natural Springs Park of Paranapiacaba, Santo André (23°46′41″S and 46°18′16″W), whereas those from Aulonemia aristulata (Döll) McClure Chusquea meyeriana Rupr. and Merostachys pluriflora Munro ex E.G. Camus at the Fontes do Ipiranga State Park in São PauloSP, Brazil (23°38′08″-23°40′18″S and 46°36′48″-46°38′00″W). All specimens were identified by Dr. Tarcísio Filgueiras from Botany Institute of São Paulo and deposited at the herbarium in the same institution. Voucher species numbers are: Shirasuna et al. 672 (SP), Shirasuna, RT & Suzuki, R. 1580 (SP), Vinha, D. s/n. 398161 (SP), Grombone, MT s/n 412118 (SP) and Shirasuna, RT 2619 (SP), respectively.
2.7. Preparation of photoprotective formulations
2.3. Crude extract preparation
Sunscreen formulations (20 g) were prepared using m/m proportions. The bamboo extracts were added in concentration of 10%. Aristoflex® AVC (ammonium acryloyldimethyltaurate/VP copolymer), a
Culms and leaves were separated, dried at room temperature, protected from light and grounded in a knives mill (Tecnal TE-580, Brasil) 209
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synthetic polymer, which was an emulsifier and gelling agent was added in 3%. All formulations contained chemical UVA and UVB filters in the following concentrations: 3.0% avobenzone; 8,0% octyldimethyl PABA, and 7.5% octylmethoxycinnamate. Additionally, BHT was used as antioxidant (0.1%), Paraffinum liquidum (mineral oil) as emollient and solvent of the oil phase of the formulation (5%). Propylene glycol was added as humectant (5%) and mixture of 2-phenoxyethanol, methyl 4-hydroxybenzoate, ethyl 4-hydroxybenzoate, propyl 4-hydroxybenzoate and butyl 4-hydroxybenzoate called Phenonip® as preservative (0.75%). Distilled water (q.s.) was a vehicle of the aqueous (gel) phase. In the first step, anti-UV agents were dissolved together by mixing with BHT and mineral oil at 70 °C in a beaker. Dry bamboo extracts were weighed and pulverized in a mortar. One half of total distilled water volume was used to dissolve bamboo extracts in a mortar with propylene glycol and then, the solution obtained was transferred to a beaker with Aristoflex® AVC. The remaining quantity of water was added, and gel was obtained by mixing the water solution with a glass rod. Finally, the UV filter solution was incorporated by parts to the formed gel. The formulation was then homogenized with the preservative in mechanical stirrer (IKA RW 20) at 1000 rpm for 5 min. In total, 11 sunscreen formulations were prepared, 10 of them were test samples prepared with leaf or culm bamboo extracts plus the UV filters, numbered as formulations F1 to F10, and one control formulation (F11), containing only the filters, as presented in Table 1.
c 290 nm
A ( ) d = 0.9
400 nm 290 nm
A ( )d
(2)
The results were presented as mean ± standard deviation. 2.9. Photostability assay Formulations had their functional photostability evaluated using the same procedure as described above with an additional step of irradiating the samples with a fixed dose of artificial solar light (580.08 W/ m2 - 2088 J/m2) in a photostable chamber for one hour at 35 °C. The sample and control absorbances were measured using the same diffuse reflectance spectrophotometer with the identical reading program. The results were also converted to SPF and cλ values and presented as mean ± standard deviation. Nine readings were recorded for each plate. 2.10. Statistical tests To compare the differences among all the samples in the measurements one-way ANOVA and Tukey test were used. Changes in SPF and cλ after irradiation in photoprotective and photostability assessments were detected by t-Student test. All the statistical analyses we performed in Minitab® 17. 3. Results and discussion
2.8. In vitro SPF evaluation
3.1. Crude extract analyses
In vitro photoprotective efficacy of bamboo extracts was estimated by diffuse reflectance spectrophotometer with integrating sphere (Labsphere®UV2000S). For the assay, formulations containing the bamboo extracts and the control were weighed and uniformly spread as a thin film (1.3 mg/cm2) on the surface of a polymethyl-methacrylate plate (PMMA, HD Helioplate® 6 HelioScreen) according to the Cosmetics Europe guidelines (2011), previously known as COLIPA (The European Cosmetic and Perfumery Association). After 40 min of drying, sample absorbances were measured between 290 and 400 nm, with progression rate of 1.0 nm. Nine readings were recorded for each plate. Plates without the film were used as blanks, and two of them were used for the control formulation (F11). Absorbance values were converted to in vitro sun protection factor (SPF) and critical wavelength (cλ) using the Eqs. 1 and 2 (Cosmetics Europe, 2011; Diffey, 2002; Springsteen et al., 1999), where: Eλ is spectral irradiance effectiveness accordingly to CIE (Commission Internationale de l'Eclairage); Sλ is solar spectral irradiance; Tλ is spectral transmittance of the sample; dλ is range of wavelengths and A (λ) is spectral absorbance of the sample.
3.1.1. Extraction yield Since the bamboos are known to possess glycosylated flavonoids as one of the main compounds, 60% ethanol was chosen as a solvent for the extraction process because of its higher polarity. The crude extracts yield varied between 7.6 and 22.2% (m/m), with the leaves affording higher values for all species, as it is usual in plant materials (Table 2). C. capituliflora leaves presented almost the double of extractable material (22.2%) than those from the other species, where the yields ranged from 10 to 14%. In general, culm extracts yields were alike to those obtained for leaves, with the lowest results for M. pluriflora culms. Comparing these values with Asian bamboo species, the native Brazilian bamboos presented similar yields (Wang et al., 2012; Macwan et al., 2010).
SPF =
400 nm E S d 290 nm 400 nm E S T d 290 nm
3.1.2. Antiradical activity Due to their antioxidant effects (Hu et al., 2000), associated with the high polyphenol contents, bamboos are currently proposed as promising anti-UV agents, once there is a correlation between reactive oxygen species (ROS) and UVB exposure (Dinkova-Kostova, 2008). These factors lead to an initial screening for antioxidant properties, expressed as radical scavenger potential, and the evaluation of the total phenol and total flavonoid contents in the Brazilian bamboo extracts. Although, DPPH method does not directly reflect the biological antioxidant activity, it is commonly used, especially for screening purposes, because it is quick, reliable and reproducible. The bamboo extracts presented DPPH scavenging activity (IC50) ranging from 137 to 260 μg/mL, where the most potent extract was C. capituliflora leaves (Table 2). Despite the wide activity range, no significant difference was observed among the culm and leaf samples. In general, all the native Brazilian bamboos presented an antioxidant potential approximately 10 times higher than those observed for the Asian bamboos Phyllostachys pubescens and P. nigra (Park and Jhon, 2010). The antioxidant potential observed for the native Brazilian bamboos were alike to reported for turmeric roots with IC50 = 104.91 μg/mL in the DPPH assay, which are well known for their antioxidant properties (Sukandar et al., 2015). These results indicate a promising antioxidant potential for the bamboo extracts,
(1)
Table 1 List of sunscreen formulations developed for the photoprotective assay sorted by bamboo species and part of the plant used. Formulation
Bamboo species
Part of the plant used
F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11
A. aristulata
Culm Leaves Culm Leaves Culm Leaves Culm Leaves Culm Leaves NA
C. bambusoides C. capituliflora C. meyeriana M. pluriflora Control
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Table 2 Yield (%), antioxidant activity assay (IC50), total phenolic compounds (GAE) and total flavonoid content (QE) tests obtained for native Brazilian bamboo species. Bamboo species Culm Extracts A. aristulata C. bambusoides C. capituliflora C. meyeriana M. pluriflora Leaf Extracts A. aristulata C. bambusoides C. capituliflora C. meyeriana M. pluriflora
Yield [%]
DPPH IC50 [μg/mL]
9.9 12.2 8.6 7.8 7.6
205.68 236.59 244.36 168.18 174.17
± ± ± ± ±
31.22b,c 42.22c 50.30c 21.79a,b 12.88a,b
55.02 43.64 46.31 87.81 54.40
± ± ± ± ±
0.47c,d,e 2.24e 0,95d,e 2.14a 2.30c,d,e
3.16 2.22 4.83 4.60 4.01
± ± ± ± ±
0.38f,g,h,i 0.25i 0.25b,c,d 0.16c,d,e 0.33d,e,f,g
10.1 14.2 22.2 14.2 13.9
260.17 218.16 137.55 205.48 222.52
± ± ± ± ±
42.14c 34.56b,c 15.62a 38.14b,c 20.05b,c
49.54 59.53 57.00 71.03 47.34
± ± ± ± ±
0.74d, 4.83b, 4.49b, 3.61b 0.62d,
3.65 6.44 3.87 4.08 5.93
± ± ± ± ±
0.40d, e, 0.25a 0.31d, e, 0.04d, e, 0.27a, b
GAE [mg/g]
QE [mg/g]
e c, d c, d, e e
f, g, h f, g, h f
IC50 – Inhibition Concentration – concentration needed to inhibit 50% of the radical formation; GAE – Gallic Acid Equivalents; QE – Quercetin Equivalents. Statistic tests were performed separately for each assay. Small letters represent results of one-way ANOVA test with Tukey comparison. Values in the same column that do not share the same letter are statistically different.
found in bamboo extracts that may interact differently with other oil phase ingredients, such as synthetic UV filters. Also, the variations between each formulation properties resulted from distinctive features of each extract.
although the IC50 values were much higher than that from pure quercetin (1.76 μg/mL), the positive control. 3.1.3. Quantification of phenolic compounds and flavonoids As the DPPH scavenging activity might be related with the presence of polyphenols (Dudonné et al., 2009), the contents of total phenolic compounds and flavonoids were measured in the extracts. The total phenolic compounds varied from ˜44 to 88 mg GAE/g of extract as per Table 2. The highest amounts of phenolics were found in the C. meyeriana culm and leaf extract (87.81 and 71.03 mg GAE/g, respectively). All five Brazilian bamboo species showed higher amounts of phenolic than those reported for the Asian species. B. arundinacea containing only 14.6 mg GAE/g (Macwan et al., 2010). Similarly, total phenolic content of Schizostachyum lumampao (Blanco) Merr were 76.7 and 13.5 mg of GAE per 100 g of air-dried sample for the ethanolic and aqueous extracts, respectively (Tongco et al., 2014). Flavonoids are a special group of phenolic compounds which are also involved in the antioxidant properties of plant extracts (Molay et al., 2010). The total amount of flavonoids found in native Brazilian bamboo species, expressed in milligrams equivalents of quercetin (QE), ranged from 2.22 to 6.44 QE per g of extract (Table 2). Leaf extracts were richer in flavonoids than those from culms. The results obtained for other bamboo species varied, for example, in Dendrocalamopsis oldhami (Munro) Keng f. total flavonoid content was higher – 74 mg QE/g of bamboo leaf extract (Lv et al., 2012). On the other hand, aqueous and methanolic extracts from Sasa senanensis (Franch. & Sav.) Rehder gave only 1.18 and 1.85 mg Q/100 g of leaf powder (Khatun et al., 2013), whereas S. lumampao had 70.2 and 17.86 mg QE per 100 g air-dried leaves for the ethanolic and aqueous extracts, respectively (Tongco et al., 2014) which is much less than the Brazilian bamboos. Nevertheless, seasonal variations can be responsible for the differences observed in the chemical composition of the bamboos (Ni et al., 2012).
3.2.2. In vitro photoprotection and photostability assays SPF is the worldwide parameter used to evaluate the efficacy of sunscreens and its general definition is the ratio of the energy of UV radiation required to produce erythema in the protected skin (with sunscreen) to that required to produce erythema in the unprotected skin (without sunscreen), obtained through in vivo assays (Ebrahimzadeh et al., 2014). However, recently some methods using diffuse reflectance spectroscopy (DRS), a non-invasive assay to determine the UV protection, have demonstrated good correlation with in vivo methodologies (Junior et al., 2014). In our study, the photoprotection of sunscreen formulations containing bamboo extracts and well-known UV filters (Avobenzone, Octyldimethyl PABA and Octylmethoxycinnamate) was evaluated by measuring the SPF using DRS before sunlight irradiation and compared with a control containing only the solar agents. All formulations with the bamboo extracts presented SPF values between 34.52 (F3) and 86.15 (F1), showing higher values than the control (F11), SPF 13.00, as can be seen in Table 4. Although the standard deviations for the samples with the extracts were considered high, in some cases more than 50%, statistical analysis showed a significant difference with the control. According to Tukey test, only the C. bambusoides formulations (F3 and F4) were not different from the control. The high standard deviations observed for the formulations containing bamboo extracts might be related to lack of homogeneity of the samples, that might affect the equipment reflectance optic readings. The experiment was performed 3 times, since application of the formulations over the PMMA plates is done manually, and the film characteristics might have been different among the exposed area of analysis, influencing the result variations. In addition, formulations containing only bamboo extracts did not present a considerable SPF (data not shown), but when combined with synthetic filters they were able to increase measured photoprotection. As the SPF alone is an indicator only for a protection against erythemally effective solar UV, largely, confined to the UVB (290–320 nm) and partially short-wavelength UVA (320–340 nm) radiation, it does not provide information regarding protection against long-wavelength UVA1 (340–400 nm) (Hojerová et al., 2011), which can lead to several cumulative damages, such as immunosuppression, skin aging, and various other photodisorders (Velasco et al., 2008). For this reason, critical wavelength (cλ) is another parameter that describes if the photoprotective product achieved broad spectrum of action. It is defined as the wavelength at which the area under the curve gives 90%
3.2. Photoprotection and photostability of bamboo formulations 3.2.1. Formulations All the formulations containing bamboo extracts were emulsified systems. Their individual characteristics are listed in Table 3. The emulsions started to separate small amounts of the oil phase up to 2–3 h after the preparation. Formulations F2, F4, F6 and F8 were defined as more homogeneous due to a uniform aspect. The color of formulations varied from yellowish, light to dark green or brown with a specific herbal odor. The control formulation (F11) was homogenous and with creamy color and a characteristic odor of the gelling base. Difficulties during gel formation can be caused by diverse chemical compounds 211
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Table 3 Formulations received from bamboo extracts and synthetic solar filters. Formulation number
Aspect
Organoleptic properties
Formulation number
Aspect
Organoleptic properties
F1
Partially homogeneous emulsion, brown color and herbal odor
F6
Homogeneous emulsion, green color and herbal odor
F2
Homogeneous emulsion, brown color and herbal odor
F7
Partially homogeneous emulsion, yellowish color and herbal odor
F3
Partially homogeneous emulsion, brownyellowish color and herbal odor
F8
Homogeneous emulsion, green-brown color and herbal odor
F4
Homogeneous emulsion, dark green color and herbal odor
F9
Partially homogeneous emulsion, yellowish color and herbal odor
F5
Partially homogeneous emulsion, yellowish color and herbal odor
F10
Partially homogeneous emulsion, dark orangebrown color and herbal odor
F11 (control)
Homogeneous emulsion, clear yellowish color and specific odor of filters/cream base
Extract type used in formulations: F1 – A. aristulata culms; F2 – A. aristulata leaves; F3 – C. bambusoides culms; F4 –C. bambusoides leaves; F5 – C. capituliflora culms; F6 – C. capituliflora leaves; F7 – C. meyeriana culms; F8 – C. meyeriana leaves; F9 – M. pluriflora culms; F10 – M. pluriflora leaves; F11: water (control).
of the total absorbance of the sample between 290–400 nm. The Food and Drug Administration (FDA) considers 370 nm as the minimal cλ for broad spectrum sunscreens (Polonini et al., 2014). Additionally, an ideal sunscreen must have a minimal SPF value of 15, in accordance with the FDA (Food and Drug Administration, 2007). As can be seen in Table 4, all bamboo formulations can be defined as good efficacy samples (cλ higher than 370 nm and SPF more than 15), whereas the control sample presented broad spectrum of action (cλ higher than 370 nm) but low photoprotective effect (SPF less than 15). Photostability is another essential property describing the effectiveness and safety of sunscreen products, since UV radiation can lead
to reduction of the photoprotective capacity of UV filters and can also generate free radicals (Gonzalez et al., 2007). Avobenzone is a widely used UVA filter that is known to be a rather unstable substance, loosing much of its protective capability after irradiation through tautomerization, fragmentation and photoproduct formation. The photolysis mechanism involves singlet oxygen formation that may induce irreversible modifications in skin proteins, leading to tissue photodegradation and other harmful effects to the skin cells (Abid et al., 2017; Paris et al., 2009). Additionally, it is known that octylmethoxycinnamate in the presence of avobenzone undergoes a photolysis process, rather than the expected E/Z photoisomerization, producing
Table 4 Sun Protection Factor (SPF) and critical wavelength (cλ) of sunscreen formulations containing native Brazilian bamboo extracts, before and after sunlight irradiation. Formulation
SPF (mean ± standard deviation) a
Before irradiation F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 (control)
86.15 61.41 34.52 36.61 78.22 61.59 84.37 51.41 83.19 61.48 13.00
± ± ± ± ± ± ± ± ± ± ±
40.81A,B 37.36A,B,C 10.39C,D 10.68C,D 53.15A,B 35.99A,B,C 40.98A,B 25.84B,C 41.11A,B 32.26A,B,C 2.50D
cλ [nm] b
After irradiation
Before irradiationx
After irradiationx
32.30 ± 15.55E,F,G 22.04 ± 12.16G,H,I 14.33 ± 4.17H,I 35.22 ± 13.48E,F 44.44 ± 37.31E,F 25.93 ± 11.86F,G,H 28.78 ± 20.12E,F,G,H 20.41 ± 13.53G,H,I 41.33 ± 24.91E,F 41.00 ± 27.33E,F 6.00 ± 1.6I
380X 381X 380X 381X 381X 383X 381X 381X 380 X 380X 381X
378X 379X 378X 380X 379X 381X 379X 379X 379X 380X 379X
SPF - Sun Protection Factor; cλ – critical wavelength. Small letters show the results of t-Student test; differences between the values obtained before and after irradiation. Capital letters show the differences between all the samples (one-way ANOVA + Tukey comparison test). Values not sharing the same letter are statistically different. Extract type used in formulations: F1 – A. aristulata culms; F2 – A. aristulata leaves; F3 – C. bambusoides culms; F4 –C. bambusoides leaves; F5 – C. capituliflora culms; F6 – C. capituliflora leaves; F7 – C. meyeriana culms; F8 – C. meyeriana leaves; F9 – M. pluriflora culms; F10 – M. pluriflora leaves; F11: water (control). 212
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Fig. 1. Average UV absorbance profiles for formulations F1-F11 before (A) and after (B) irradiation with artificial sunlight for 1 h. Extract type used in formulations: F1 – A. aristulata culms; F2 – A. aristulata leaves; F3 – C. bambusoides culms; F4 –C. bambusoides leaves; F5 – C. capituliflora culms; F6 – C. capituliflora leaves; F7 – C. meyeriana culms; F8 – C. meyeriana leaves; F9 – M. pluriflora culms; F10 – M. pluriflora leaves; F11: water (control was recorded twice, as F11_1 and F11_2).
mechanisms involved and the compounds responsible for these interactions still need to be elucidated. Several plant extracts have already demonstrated a photoprotective activity by increasing the formulations SPF or other mechanisms. However, acceptable SPF values are only achieved with high amounts of the plant extract, like with the oil fraction of spent coffee grounds and green coffee beans that needed 35% of the oils to give SPF of 51.9 and 82.3 (Marto et al., 2016), or in the presence of inorganic filters, as was observed for formulations containing 1.68% (w/w) Passiflora incarnata L. dry extract with 7.0% (w/w) ethylhexyl methoxycinnamate, 2.0% (w/w) benzophenone-3 and 2.0% (w/w) TiO2 generated an SPF of 20 (Velasco et al., 2008). Comparing with our results, extracts from native Bamboo species at 10% (w/w) concentration could improve the photoprotective activity from formulations using only organic filters, reaching SPF values up to 86, and they also promoted the filters stability as SPF’s remained 2.4 to 7.4 times higher than the control after irradiation.
long lived radicals in the sunscreen film (Sayre et al., 2005). For these reasons, these filters were used in the formulations to test the photostabilization capacity of the Bamboo extracts. The results obtained after irradiation with the artificial sunlight (Table 4) showed that the formulations continued to be broad spectrum sunscreens as the cλ did not change. However, SPF decreased significantly for all the samples, except of F4 (C. bambusoides leaf). The photoprotection efficacy was still good after irradiation (FPS > 15) for most formulations, with exception of F2, F3 and F8 that were not significantly different from the control. The differences observed in the absorbance profiles after irradiation can be seen in Fig. 1. Sunscreens could have antioxidants added in their composition to complement UV filter photoprotection by reducing the damage from the free radicals generated after solar exposure and to stabilize chemical UV filters (Lim et al., 2017; Badea et al., 2015). Natural extracts have been one of the strategies used to broaden the photoprotection spectrum and increase the photostability of sunscreens, mainly those containing polyphenolic compounds due to their ability to absorb the solar spectrum and react with free radicals (Cefali et al., 2016; Korać and Khambholja, 2011). A positive correlation has been found for the phenolic contents, like flavonoids and hydroxycinnamic acids, in an extract and the increase in SPF (Ebrahimzadeh et al., 2014). Flavonoids, besides their UV absorption, may contribute to stabilize the UV filters by multiple mechanisms including oxygen radical scavenging, inhibition of lipid peroxidation and metal ion chelation. It has been demonstrated that rutin stabilization effect on two UV filters cannot be attributed only to its antioxidant activity (Velasco et al., 2008). Additional mechanisms contribute to quercetin effectiveness such as antiradical property, chelation of metal ions acting as catalyst in reactions leading to free radical formation and quenching reactive oxygen species, like the excited triplet states from the organic UV filters butyl methoxydibenzoylmethane and octyl methoxycinnamate (Scalia and Mezzena, 2010). In this sense, the photoprotection and photostability conferred to the formulation by the native Brazilian bamboo species might be related to their phenolic and/or flavonoid contents, but
4. Conclusions In this study, screening of crude extracts from Brazilian bamboo species for potential antioxidant activity, phenolic and flavonoid contents indicated that the phenolic contents are higher than in Asian species and a good radical scavenging activity. These results led to the preparation of sunscreen formulations, with the same plant extracts, in a combination with commercial UV filters. The results showed that the addition of the biological samples increased the SPF and photostability of the synthetic UV filters. According to our observations, due to their rich in phenolic substances composition, the Brazilian bamboo species can provide a promising source of biologically active agents increasing the efficacy of synthetic solar filters. The next step of this research will be to isolate and identify the substances responsible for these properties and their mechanism of action for the most active species.
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