Natural Product Research Formerly Natural Product Letters
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Anti-inflammatory activity of Brunfelsia uniflora root extract: phytochemical characterization and pharmacologic potential of this under-investigated species Patrícia Weimer , Lenise Maria Spies , Ronete Haubert , João Alfredo Santos de Lima , Rage Weidner Maluf , Rochele Cassanta Rossi & Edna Sayuri Suyenaga To cite this article: Patrícia Weimer , Lenise Maria Spies , Ronete Haubert , João Alfredo Santos de Lima , Rage Weidner Maluf , Rochele Cassanta Rossi & Edna Sayuri Suyenaga (2020): Anti-inflammatory activity of Brunfelsia�uniflora root extract: phytochemical characterization and pharmacologic potential of this under-investigated species, Natural Product Research, DOI: 10.1080/14786419.2020.1827403 To link to this article: https://doi.org/10.1080/14786419.2020.1827403
Published online: 01 Oct 2020.
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NATURAL PRODUCT RESEARCH https://doi.org/10.1080/14786419.2020.1827403
SHORT COMMUNICATION
Anti-inflammatory activity of Brunfelsia uniflora root extract: phytochemical characterization and pharmacologic potential of this underinvestigated species Patrıcia Weimera,b , Lenise Maria Spiesc, Ronete Haubertc, Jo~ao Alfredo Santos de Limad, Rage Weidner Malufc, Rochele Cassanta Rossia,e and Edna Sayuri Suyenagaa,c Faculdade de Farmacia, Universidade do Vale do Rio dos Sinos (UNISINOS), S~ao Leopoldo, Brazil; Programa de Pos-Graduac¸~ao em Ci^encias Farmac^euticas, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Brazil; cFaculdade de Farmacia, Universidade Feevale, Novo Hamburgo, Brazil; d Departamento Administrativo, Universidade do Vale do Rio dos Sinos (UNISINOS), S~ao Leopoldo, Brazil;ePrograma de Pos-Graduac¸~ao em Nutric¸~ao e Alimentos, Universidade do Vale do Rio dos Sinos (UNISINOS), S~ao Leopoldo, Brazil a
b
ABSTRACT
Brunfelsia uniflora (Pohl) D. Don roots have been widely used in folk medicine for treating inflammatory conditions. However, few studies have elucidated compounds that justify their traditional use. This study was conducted to characterize the phytochemical profile and evaluate the in vitro antioxidant capacity, and in vivo anti-inflammatory activity of extracts obtained from B. uniflora roots by comparing an herbal remedy (HR) with the crude hydroalcoholic extract (CHE). In the phytochemical analysis, scopoletin was identified as the marker compound. In quantitative analyses, CHE showed better results than HR. Furthermore, CHE had an effective anti-inflammatory activity. Animals treated with CHE (200 mg/kg) showed an 89.1% and a 73.8% reduction in edema volume after 1 hour of edema induction compared with those treated with negative control and positive control (indomethacin), respectively. These results show that B. uniflora root extracts have promising antioxidant and anti-inflammatory activities, thus corroborating their application in ethnomedicine.
ARTICLE HISTORY
Received 27 May 2020 Accepted 5 September 2020 KEYWORDS
Solanaceae; scopoletin; inflammation; liquid chromatography; radical scavenger
CONTACT Patrıcia Weimer [email protected] Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2020.1827403. ß 2020 Informa UK Limited, trading as Taylor & Francis Group
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1. Introduction The species Brunfelsia uniflora (Pohl) D. Don belongs to the family Solanaceae and it is commonly known as “Manaca, Mercury-vegetable, and Spring flower” in Brazil. It has been used as a medicinal plant for various applications such as an anesthetic, a laxative, and a hypothermic agent. Nevertheless, it is mostly used in inflammatory conditions, such as arthritis, owing to its anti-inflammatory and hypothermic activities (Plowman 1977; Elisabetsky and Souza 2010). Inflammatory processes are associated with different diseases and are initiated by resident immune cells in response to an injury, which could be chemical, physical, or biological in nature, causing physiological changes at the cellular and tissue levels. One way in which the body combats lesions is by producing substances such as reactive species, free radicals, and inflammatory mediators that play vital roles in selfdefense against pathogenic microorganisms and harmful substances. However, in excess, the activity of these substances may damage various protein and lipid structures (Gregus 2012). The use of extracts and vegetable derivatives in these cases is based on their antioxidant capacity and presence of bioactive substances, which promote the stabilization of free radicals and suppress inflammatory mediators production (Byrns and Penning 2011; Gregus 2012). Among the bioactive metabolites present in medicinal plants, the phenolic compounds and flavonoids have been notably used in the treatment of inflammatory diseases because of their antioxidant capacity attributable to phenolic hydroxylates. These phenolic hydroxylates stabilize free radicals and reactive oxygen species formed during inflammatory processes, generating new phenoxyl radicals, which are less harmful and are stabilized through intramolecular hydrogen bonds and electron displacement (Filipovic et al. 2015). Flavonoids also inhibit the production of inflammatory mediators (prostaglandins and leukotrienes) generated after the induction of the arachidonic acid cascade by injury (Adebayo et al. 2015). Despite the widespread use of B. uniflora roots in Brazilian folk medicine, only few studies have evaluated its phytochemical and pharmacological profiles, and these studies assessed the leaves. The aim of this study was to characterize the
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phytochemical profile, evaluate the in vitro antioxidant capacity, and in vivo antiinflammatory activity of B. uniflora root extracts by comparing herbal remedy (HR, prepared by the local population) and a crude hydroalcoholic extract (CHE, prepared under standard conditions).
2. Results and discussion In phytochemical screening, HR, CHE, and aqueous extract (AE - control) samples showed positive results for phenolic compounds, flavonoids, coumarins, alkaloids, and steroid nucleus (compounds with cyclopentanoperhydrophenanthrene ring). Some studies have identified the alkaloids manacine and manacein in the roots of B. uniflora (Mors and Ribeiro 1957; Harri and Mato 2008). The genus of the Solanaceae family, including the genus Brunfelsia, are characterized by the presence of alkaloids, saponins, coumarins, and compound with steroid nucleus (Plowman 1977). In this study, phytochemical screening revealed positive results for steroid nucleus, saponins, and coumarins, confirming the chemotaxonomic markers of the genus. Data from thin layer chromatography (TLC) are shown in Supplementary Material (Table S1). The results only confirmed the presence of scopoletin in the samples. In the second eluent system of TLC, one compound present in HR and CHE samples had the same retention factor (Rf) as quercetin; however, its spot color differed under UV light, and was not stained by AlCl3 solution. Additionally, this unknown substance showed intense blue fluorescence, suggesting the presence of coumarin group, which was confirmed in the third eluent system. Rf in TLC is related to the lipophilicity of molecules; hence, in this case, substances with the same Rf value have similar lipophilicity. Therefore, the lipophilicity of the unknown compound was similar to that of quercetin. Lipophilicity can be evaluated using the physicochemical descriptor LogP. Negative values or values close to zero indicate hydrophilic molecular structures, whereas higher and positive values correspond to the lipophilic characteristic of molecules. Some studies have reported the presence of the coumarins scopoletin and aesculetin in B. uniflora roots (Mors and Ribeiro 1957; Harri and Mato 2008). While investigating the theoretical LogP values, it was verified that scopoletin has the same LogP value (1.5) as quercetin (NCBI 2017). Owing to the similar lipophilicity, the presence of scopoletin was investigated, using a third TLC system specific to coumarin compounds. Although ferulic and caffeic acids do not belong to the class of coumarins, they also demonstrate blue fluorescence; therefore, they were included in this analysis (Wagner and Bladt 1995). The same Rf value and blue fluorescence indicated the presence of scopoletin in HR and CHE. Confirmation and quantification of scopoletin were performed by HPLC. HR and CHE samples had similar retention time, of 3.96 minutes (Supplementary material, Figure S1). CHE had 139.86 ± 0.63 lg/g of scopoletin, and HR had 43.02 ± 1.62 lg/g. The results were statistically different (student’s t-test, p < 0.001). Considering the importance of phenolic and flavonoid compounds, and their identification in phytochemical screening, these compounds were quantified in terms of gallic acid (GAE) and quercetin (QE) equivalents, respectively. The phenolic content was 460.9 ± 9.7 mg GAE/g in CHE and 272.4 ± 3.8 mg GAE/g in HR, while flavonoid content
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was 386.6 ± 2.5 mg QE/g in CHE and 227.9 ± 2.1 mg QE/g in HR. No reference values were found for the contents of phenolic compounds and flavonoids in B. uniflora roots extracts. In addition, the results obtained for the content of phenolic compounds and flavonoids were significantly higher in the CHE sample than HR (student’s ttest, p < 0.001). The differences in phenolic, flavonoid, and scopoletin contents, between HR and CHE, were attributed to the distinct extraction conditions. While preparing HR the root: solvent (w/v) ratio, extraction time, and particle size of the plant material were not controlled. Such uncontrolled parameters directly interfer with extractive process efficiency, especially for more rigid plant parts such as roots, which have a more compact histological structure that hampers the solvent penetration (Falkenberg et al. 2010). In contrast, fragmentation of the plant material while preparing CHE increased the contact surface between plant tissues and solvent, thus facilitating further extraction of the substances of interest. In addition, owing to the lipophilicity of these substances, the use of a solvent with higher alcohol content (70% v/v) favored the extraction, thus justifying why CHE samples had the highest concentrations of phenolic compounds, flavonoids, and scopoletin. Several damages in inflammatory processes are associated with the exacerbated production of free radicals by host defense mechanisms. The inability of endogenous reducing systems (superoxide dismutase and glutathione peroxidase enzymes) to stabilize the exacerbated concentration of free radicals results in an unbalance that induces the oxidative stress (Filipovic et al. 2015). To resolve this, exogenous antioxidants must be used to avoid irreversible damage e.g., the loss of tissue functionality (Morales-Del-Rio et al. 2015). The antioxidant capacities of CHE, HR, and scopoletin were evaluated by in vitro ABTSþ and DPPH scavenging assays (Supplementary material, Figure S2). The antioxidant capacity of a sample evaluated by these techniques was defined as the lowest concentration capable of inhibiting 50% of the respective radical (IC50 value). The results of ABTSþ and DPPH assays demonstrated that CHE had 2-fold higher antioxidant capacity than HR (Figure S2). As determined by the ABTSþ assay (Figure S2-A), CHE and HR had IC50 values of 1678.00 ± 11.26 mg/mL and 3441.00 ± 36.05 mg/mL, respectively. As determined by the DPPH assay (Figure S2-B), CHE and HR had IC50 values of 37698.00 ± 3437.00 mg/mL and 68452.00 ± 5155.00 mg/ mL, respectively. These results are in agreement with the higher phenolic, flavonoids, and scopoletin contents in CHE sample than HR sample. However, when comparing the antioxidant capacity of HR and CHE with the isolated standards of scopoletin and Trolox (positive control), both samples presented lower results. Additionally, no statistical differences were observed between scopoletin and Trolox in the ABTSþ assay (p ¼ 0.9273). Some extracts and substances exhibit antioxidant activity by reducing metal ions or chelating ions. Generally, free radicals react with different molecular structures and substances, including metal ions. For example, the oxidation of FeII in the heme group of hemoglobin to FeIII leads to the formation of methemoglobin that decreases oxygen transport. Iron is also associated with different biological functions such as enzymatic reactions, and synthesis of neurotransmitters. Recently, the relationship between
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metabolic imbalance of this metal and neurodegenerative diseases has been evaluated (Beard 2001; Gregus 2012; Belaidi and Bush 2016). The ability of CHE and HR to reduce ions FeIII to FeII was evaluated by the FRAP assay, and the results were expressed as ferrous sulfate equivalents (FSE). The results obtained were 4.59 ± 0.09 mmol FSE/g for CHE and 2.49 ± 0.099 mmol FSE/g for HR (student’s t-test, p < 0.05). This suggested that the antioxidant activity of CHE was superior to that of HR in the antioxidant assays (ABTSþ, DPPH, and FRAP) owing to its higher scopoletin, phenolic, and flavonoid contents. Considering that both CHE and HR extracts had a similar phytochemical profile, the same marker, and prioritizing the reduction of animal use, the in vivo anti-inflammatory activity was evaluated only for CHE sample, as it was obtained via a standardized extraction method. The anti-inflammatory activity of the CHE of B. uniflora was confirmed by the carrageenan-induced rat paw edema assay. Both treatment groups (100 mg/kg and 200 mg/kg), demonstrated superior activity than the negative control group (saline solution), with a 69.9% and 89.1% reduction in edema volume, respectively, 1 h after edema induction (Supplementary Material Table S2). Furthermore, compared with the administration of indomethacin (positive control), the administration of 200 mg of CHE/kg showed a 73.8% and 30.9% reduction of edema volume 1 h and 4 h after induction, respectively. In contrast, the animals treated with 100 mg/kg of CHE showed superior edema reduction than the positive control just 1 h after induction. The in vivo anti-inflammatory activity of CHE could be directly related to the antioxidant potential of this extract and to the presence of scopoletin. Since the extract compounds displayed a radical-scavenging capacity and could stabilizing the free radicals generated during the inflammatory process. The identified marker, scopoletin (7hydroxy-6-methoxycoumarin), has been identified in vast plant species. For elucidating the mechanisms underlying the anti-inflammatory activity of this coumarin, in vitro studies were performed using murine macrophages (RAW 264.7) stimulated by lipopolysaccharide. These studies showed a dose-dependent suppression of the cyclooxygenase 2 enzyme (COX-2, enzyme activated in inflammatory processes) by scopoletin, and pro-inflammatory cytokines inhibition (PGE2, IL-6, IL-b, and TNF-a). However, it had no effect on the constitutive enzyme COX-1 (Kim et al. 2004). In addition, Dou et al. (2013) also confirmed the suppression capacity of scopoletin in IL-6 release. Scopoletin has been shown to inhibit 5-lipoxygenase enzyme in vitro (Mogana et al. 2013; Rzodkiewicz et al. 2015). And by in vivo rat model of visceral pain induced by acetic acid, scopoletin exhibited antinociceptive effects (Meotti et al. 2006; Rzodkiewicz et al. 2015).
3. Experimental section The experimental section is detailed in the supplementary material.
4. Conclusions The results revealed the phytochemical profiles, in vitro antioxidant activity, and in vivo anti-inflammatory activity of herbal remedy (HR) and crude hydroalcoholic
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extract (CHE) of B. uniflora roots; the constituents identified in the extracts justified their application in folk medicine for inflammatory conditions. Additionally, the high contents of phenolic compounds and flavonoid in the CHE sample made its antioxidant activity better than that of the HR. The differences between HR and CHE samples in the quantitative analyses resulted from the extraction process, in which CHE was obtained through standard pharmacopeial conditions and HR was obtained by the local population without standardization regarding the alcoholic concentration of the extracting solvent, root: solvent proportion, etc. Extracts obtained under standardized conditions can be reproduced either in future experiments or in interlaboratory analysis. The data suggest that the marker compound, scopoletin, exhibits its anti-inflammatory activity by suppressing enzymes and releasing inflammatory mediators. Finally, the investigation of in vivo anti-inflammatory activity showed promising results based on the reduction in the volume of edema, observed in the early stages of analysis. This study gives new perspectives for the study of the pharmacological potential of B. uniflora and its roots.
Disclosure statement No potential conflict of interest was reported by the authors.
Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
ORCID Patrıcia Weimer
http://orcid.org/0000-0002-2024-8228
References Adebayo SA, Dzoyem JP, Shai LJ, Eloff JN. 2015. The anti-inflammatory and antioxidant activity of 25 plant species used traditionally to treat pain in southern African. BMC Complement Altern Med. 15(1):1–10. Beard JL. 2001. Iron biology in immune function, muscle metabolism and neuronal functioning. J Nutr. 131(May):586S–580S. Belaidi AA, Bush AI. 2016. Iron neurochemistry in Alzheimer’s disease and Parkinson’s disease: targets for therapeutics. J Neurochem. 139:179–197. Byrns MC, Penning TM. 2011. Environmental toxicology: carcinogens an heavy metals. In: Brunton LL, editor. Goodman and Gilman’s the pharmacological basis of therapeutics. 12th ed. New York: McGraw Hill; p. 1853–1877. Dou Y, Tong B, Wei Z, Li Y, Xia Y, Dai Y. 2013. Scopoletin suppresses IL-6 production from fibroblast-like synoviocytes of adjuvant arthritis rats induced by IL-1b b stimulation. Int Immunopharmacol. 17(4):1037–1043. http://dx.doi.org/10.1016/j.intimp.2013.10.011. Elisabetsky E, Souza GC. 2010. Etnofarmacologia como ferramenta na busca de subst^ancias ati~es CMO, editor. Farmacogn da planta ao Medicam. 6th ed. Porto Alegre, vas. In: Simo polis: Editora da UFRGS, Editora da UFSC; p. 107–122. Floriano
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~es CMO. 2010. Introduc¸~ao a analise fitoquımica. In: Falkenberg M. d B, dos Santos RI, Simo ~es CMO, editor. Farmacogn da planta ao Medicam. 6th ed. Porto Alegre, Floriano polis: Simo Editora da UFRGS, Editora da UFSC; p. 229–246. - orovic J, Markovic JD, Lucic B, Amic D. 2015. QSAR of the free radical Filipovic M, Markovic Z, D scavenging potency of selected hydroxybenzoic acids and simple phenolics. Comptes Rendus Chim. 18(5):492–498. Gregus Z. 2012. Mecanismos de toxicidade. In: Klaassen CD, Watkins III JB, editors. Fundam em Toxicol Casarett e Doul. 2nd ed. Porto Alegre: AMGH Editora Ltda; p. 21–46. ticas. 2nd ed. Nova Odessa: Harri L, Mato FJA. 2008. Plantas Medicinais no Brasil: nativas e exo Instituto Plantarum. Kim HJ, Jang SI, Kim YJ, Chung HT, Yun YG, Kang TH, Jeong OS, Kim YC. 2004. Scopoletin suppresses pro-inflammatory cytokines and PGE2 from LPS-stimulated cell line, RAW 264.7 cells. Fitoterapia. 75(3–4):261–266. Meotti FC, Ardenghi JV, Pretto JB, Souza MM, Moura J. dAvila, Junior AC, Soldi C, Pizzolatti MG, Santos ARS. 2006. Antinociceptive properties of coumarins, steroid and dihydrostyryl-2pyrones from Polygala sabulosa (Polygalaceae) in mice. J Pharm Pharmacol. 58(1):107–112. Mogana R, Teng-Jin K, Wiart C. 2013. Anti-inflammatory, Anticholinesterase, and Antioxidant Potential of Scopoletin Isolated from Canarium patentinervium Miq. (Burseraceae Kunth). Evidence-based. Complement Altern Med. 2013:1–6. Morales-Del-Rio JA, Gutierrez-Lomelı M, Robles-Garcıa MA, Aguilar JA, Lugo-Cervantes E, Guerrero-Medina PJ, Ruiz-Cruz S, Cinco-Moroyoqui FJ, Wong-Corral FJ, Del-Toro-Sanchez CL. 2015. Anti-inflammatory activity and changes in antioxidant properties of leaf and stem extracts from Vitex mollis kunth during in vitro digestion. Evidence-Based Complement Altern Med. 2015:1–8. Mors WB, Ribeiro O. 1957. Occurrence of scopoletin in the genus brunfelsia. J Org Chem. 22(8): 978–979. NCBI NC for BI. 2017. PubChem. Rockv NCBI [Internet]. [accessed 2017 Oct 10]. https://pubchem. ncbi.nlm.nih.gov/. Plowman T. 1977. Brunfelsia in ethnomedicine. Bot Mus Lealf Harv Univ. 25(10):289–320. Rzodkiewicz P, Gasinska E, Maslinski S, Bujalska-Zadrozny M. 2015. Antinociceptive properties of esculetin in non-inflammatory and inflammatory models of pain in rats. Clin Exp Pharmacol Physiol. 42(2):213–219. Wagner H, Bladt S. 1995. Coumarin drugs. In: Wagner H, Bladt S, editors. Plant drug analysis: a thin layer chromatography atlas. 2nd ed. New York: Springer-Verlag Berlin Heindelberg; p. 125–146.
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Anti-inflammatory activity of Brunfelsia uniflora root extract: phytochemical characterization and pharmacologic potential of this under-investigated species Patrícia Weimer , Lenise Maria Spies , Ronete Haubert , João Alfredo Santos de Lima , Rage Weidner Maluf , Rochele Cassanta Rossi & Edna Sayuri Suyenaga To cite this article: Patrícia Weimer , Lenise Maria Spies , Ronete Haubert , João Alfredo Santos de Lima , Rage Weidner Maluf , Rochele Cassanta Rossi & Edna Sayuri Suyenaga (2020): Anti-inflammatory activity of Brunfelsia�uniflora root extract: phytochemical characterization and pharmacologic potential of this under-investigated species, Natural Product Research, DOI: 10.1080/14786419.2020.1827403 To link to this article: https://doi.org/10.1080/14786419.2020.1827403
Published online: 01 Oct 2020.
Submit your article to this journal
Article views: 6
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=gnpl20
NATURAL PRODUCT RESEARCH https://doi.org/10.1080/14786419.2020.1827403
SHORT COMMUNICATION
Anti-inflammatory activity of Brunfelsia uniflora root extract: phytochemical characterization and pharmacologic potential of this underinvestigated species Patrıcia Weimera,b , Lenise Maria Spiesc, Ronete Haubertc, Jo~ao Alfredo Santos de Limad, Rage Weidner Malufc, Rochele Cassanta Rossia,e and Edna Sayuri Suyenagaa,c Faculdade de Farmacia, Universidade do Vale do Rio dos Sinos (UNISINOS), S~ao Leopoldo, Brazil; Programa de Pos-Graduac¸~ao em Ci^encias Farmac^euticas, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Brazil; cFaculdade de Farmacia, Universidade Feevale, Novo Hamburgo, Brazil; d Departamento Administrativo, Universidade do Vale do Rio dos Sinos (UNISINOS), S~ao Leopoldo, Brazil;ePrograma de Pos-Graduac¸~ao em Nutric¸~ao e Alimentos, Universidade do Vale do Rio dos Sinos (UNISINOS), S~ao Leopoldo, Brazil a
b
ABSTRACT
Brunfelsia uniflora (Pohl) D. Don roots have been widely used in folk medicine for treating inflammatory conditions. However, few studies have elucidated compounds that justify their traditional use. This study was conducted to characterize the phytochemical profile and evaluate the in vitro antioxidant capacity, and in vivo anti-inflammatory activity of extracts obtained from B. uniflora roots by comparing an herbal remedy (HR) with the crude hydroalcoholic extract (CHE). In the phytochemical analysis, scopoletin was identified as the marker compound. In quantitative analyses, CHE showed better results than HR. Furthermore, CHE had an effective anti-inflammatory activity. Animals treated with CHE (200 mg/kg) showed an 89.1% and a 73.8% reduction in edema volume after 1 hour of edema induction compared with those treated with negative control and positive control (indomethacin), respectively. These results show that B. uniflora root extracts have promising antioxidant and anti-inflammatory activities, thus corroborating their application in ethnomedicine.
ARTICLE HISTORY
Received 27 May 2020 Accepted 5 September 2020 KEYWORDS
Solanaceae; scopoletin; inflammation; liquid chromatography; radical scavenger
CONTACT Patrıcia Weimer [email protected] Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2020.1827403. ß 2020 Informa UK Limited, trading as Taylor & Francis Group
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1. Introduction The species Brunfelsia uniflora (Pohl) D. Don belongs to the family Solanaceae and it is commonly known as “Manaca, Mercury-vegetable, and Spring flower” in Brazil. It has been used as a medicinal plant for various applications such as an anesthetic, a laxative, and a hypothermic agent. Nevertheless, it is mostly used in inflammatory conditions, such as arthritis, owing to its anti-inflammatory and hypothermic activities (Plowman 1977; Elisabetsky and Souza 2010). Inflammatory processes are associated with different diseases and are initiated by resident immune cells in response to an injury, which could be chemical, physical, or biological in nature, causing physiological changes at the cellular and tissue levels. One way in which the body combats lesions is by producing substances such as reactive species, free radicals, and inflammatory mediators that play vital roles in selfdefense against pathogenic microorganisms and harmful substances. However, in excess, the activity of these substances may damage various protein and lipid structures (Gregus 2012). The use of extracts and vegetable derivatives in these cases is based on their antioxidant capacity and presence of bioactive substances, which promote the stabilization of free radicals and suppress inflammatory mediators production (Byrns and Penning 2011; Gregus 2012). Among the bioactive metabolites present in medicinal plants, the phenolic compounds and flavonoids have been notably used in the treatment of inflammatory diseases because of their antioxidant capacity attributable to phenolic hydroxylates. These phenolic hydroxylates stabilize free radicals and reactive oxygen species formed during inflammatory processes, generating new phenoxyl radicals, which are less harmful and are stabilized through intramolecular hydrogen bonds and electron displacement (Filipovic et al. 2015). Flavonoids also inhibit the production of inflammatory mediators (prostaglandins and leukotrienes) generated after the induction of the arachidonic acid cascade by injury (Adebayo et al. 2015). Despite the widespread use of B. uniflora roots in Brazilian folk medicine, only few studies have evaluated its phytochemical and pharmacological profiles, and these studies assessed the leaves. The aim of this study was to characterize the
NATURAL PRODUCT RESEARCH
3
phytochemical profile, evaluate the in vitro antioxidant capacity, and in vivo antiinflammatory activity of B. uniflora root extracts by comparing herbal remedy (HR, prepared by the local population) and a crude hydroalcoholic extract (CHE, prepared under standard conditions).
2. Results and discussion In phytochemical screening, HR, CHE, and aqueous extract (AE - control) samples showed positive results for phenolic compounds, flavonoids, coumarins, alkaloids, and steroid nucleus (compounds with cyclopentanoperhydrophenanthrene ring). Some studies have identified the alkaloids manacine and manacein in the roots of B. uniflora (Mors and Ribeiro 1957; Harri and Mato 2008). The genus of the Solanaceae family, including the genus Brunfelsia, are characterized by the presence of alkaloids, saponins, coumarins, and compound with steroid nucleus (Plowman 1977). In this study, phytochemical screening revealed positive results for steroid nucleus, saponins, and coumarins, confirming the chemotaxonomic markers of the genus. Data from thin layer chromatography (TLC) are shown in Supplementary Material (Table S1). The results only confirmed the presence of scopoletin in the samples. In the second eluent system of TLC, one compound present in HR and CHE samples had the same retention factor (Rf) as quercetin; however, its spot color differed under UV light, and was not stained by AlCl3 solution. Additionally, this unknown substance showed intense blue fluorescence, suggesting the presence of coumarin group, which was confirmed in the third eluent system. Rf in TLC is related to the lipophilicity of molecules; hence, in this case, substances with the same Rf value have similar lipophilicity. Therefore, the lipophilicity of the unknown compound was similar to that of quercetin. Lipophilicity can be evaluated using the physicochemical descriptor LogP. Negative values or values close to zero indicate hydrophilic molecular structures, whereas higher and positive values correspond to the lipophilic characteristic of molecules. Some studies have reported the presence of the coumarins scopoletin and aesculetin in B. uniflora roots (Mors and Ribeiro 1957; Harri and Mato 2008). While investigating the theoretical LogP values, it was verified that scopoletin has the same LogP value (1.5) as quercetin (NCBI 2017). Owing to the similar lipophilicity, the presence of scopoletin was investigated, using a third TLC system specific to coumarin compounds. Although ferulic and caffeic acids do not belong to the class of coumarins, they also demonstrate blue fluorescence; therefore, they were included in this analysis (Wagner and Bladt 1995). The same Rf value and blue fluorescence indicated the presence of scopoletin in HR and CHE. Confirmation and quantification of scopoletin were performed by HPLC. HR and CHE samples had similar retention time, of 3.96 minutes (Supplementary material, Figure S1). CHE had 139.86 ± 0.63 lg/g of scopoletin, and HR had 43.02 ± 1.62 lg/g. The results were statistically different (student’s t-test, p < 0.001). Considering the importance of phenolic and flavonoid compounds, and their identification in phytochemical screening, these compounds were quantified in terms of gallic acid (GAE) and quercetin (QE) equivalents, respectively. The phenolic content was 460.9 ± 9.7 mg GAE/g in CHE and 272.4 ± 3.8 mg GAE/g in HR, while flavonoid content
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was 386.6 ± 2.5 mg QE/g in CHE and 227.9 ± 2.1 mg QE/g in HR. No reference values were found for the contents of phenolic compounds and flavonoids in B. uniflora roots extracts. In addition, the results obtained for the content of phenolic compounds and flavonoids were significantly higher in the CHE sample than HR (student’s ttest, p < 0.001). The differences in phenolic, flavonoid, and scopoletin contents, between HR and CHE, were attributed to the distinct extraction conditions. While preparing HR the root: solvent (w/v) ratio, extraction time, and particle size of the plant material were not controlled. Such uncontrolled parameters directly interfer with extractive process efficiency, especially for more rigid plant parts such as roots, which have a more compact histological structure that hampers the solvent penetration (Falkenberg et al. 2010). In contrast, fragmentation of the plant material while preparing CHE increased the contact surface between plant tissues and solvent, thus facilitating further extraction of the substances of interest. In addition, owing to the lipophilicity of these substances, the use of a solvent with higher alcohol content (70% v/v) favored the extraction, thus justifying why CHE samples had the highest concentrations of phenolic compounds, flavonoids, and scopoletin. Several damages in inflammatory processes are associated with the exacerbated production of free radicals by host defense mechanisms. The inability of endogenous reducing systems (superoxide dismutase and glutathione peroxidase enzymes) to stabilize the exacerbated concentration of free radicals results in an unbalance that induces the oxidative stress (Filipovic et al. 2015). To resolve this, exogenous antioxidants must be used to avoid irreversible damage e.g., the loss of tissue functionality (Morales-Del-Rio et al. 2015). The antioxidant capacities of CHE, HR, and scopoletin were evaluated by in vitro ABTSþ and DPPH scavenging assays (Supplementary material, Figure S2). The antioxidant capacity of a sample evaluated by these techniques was defined as the lowest concentration capable of inhibiting 50% of the respective radical (IC50 value). The results of ABTSþ and DPPH assays demonstrated that CHE had 2-fold higher antioxidant capacity than HR (Figure S2). As determined by the ABTSþ assay (Figure S2-A), CHE and HR had IC50 values of 1678.00 ± 11.26 mg/mL and 3441.00 ± 36.05 mg/mL, respectively. As determined by the DPPH assay (Figure S2-B), CHE and HR had IC50 values of 37698.00 ± 3437.00 mg/mL and 68452.00 ± 5155.00 mg/ mL, respectively. These results are in agreement with the higher phenolic, flavonoids, and scopoletin contents in CHE sample than HR sample. However, when comparing the antioxidant capacity of HR and CHE with the isolated standards of scopoletin and Trolox (positive control), both samples presented lower results. Additionally, no statistical differences were observed between scopoletin and Trolox in the ABTSþ assay (p ¼ 0.9273). Some extracts and substances exhibit antioxidant activity by reducing metal ions or chelating ions. Generally, free radicals react with different molecular structures and substances, including metal ions. For example, the oxidation of FeII in the heme group of hemoglobin to FeIII leads to the formation of methemoglobin that decreases oxygen transport. Iron is also associated with different biological functions such as enzymatic reactions, and synthesis of neurotransmitters. Recently, the relationship between
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metabolic imbalance of this metal and neurodegenerative diseases has been evaluated (Beard 2001; Gregus 2012; Belaidi and Bush 2016). The ability of CHE and HR to reduce ions FeIII to FeII was evaluated by the FRAP assay, and the results were expressed as ferrous sulfate equivalents (FSE). The results obtained were 4.59 ± 0.09 mmol FSE/g for CHE and 2.49 ± 0.099 mmol FSE/g for HR (student’s t-test, p < 0.05). This suggested that the antioxidant activity of CHE was superior to that of HR in the antioxidant assays (ABTSþ, DPPH, and FRAP) owing to its higher scopoletin, phenolic, and flavonoid contents. Considering that both CHE and HR extracts had a similar phytochemical profile, the same marker, and prioritizing the reduction of animal use, the in vivo anti-inflammatory activity was evaluated only for CHE sample, as it was obtained via a standardized extraction method. The anti-inflammatory activity of the CHE of B. uniflora was confirmed by the carrageenan-induced rat paw edema assay. Both treatment groups (100 mg/kg and 200 mg/kg), demonstrated superior activity than the negative control group (saline solution), with a 69.9% and 89.1% reduction in edema volume, respectively, 1 h after edema induction (Supplementary Material Table S2). Furthermore, compared with the administration of indomethacin (positive control), the administration of 200 mg of CHE/kg showed a 73.8% and 30.9% reduction of edema volume 1 h and 4 h after induction, respectively. In contrast, the animals treated with 100 mg/kg of CHE showed superior edema reduction than the positive control just 1 h after induction. The in vivo anti-inflammatory activity of CHE could be directly related to the antioxidant potential of this extract and to the presence of scopoletin. Since the extract compounds displayed a radical-scavenging capacity and could stabilizing the free radicals generated during the inflammatory process. The identified marker, scopoletin (7hydroxy-6-methoxycoumarin), has been identified in vast plant species. For elucidating the mechanisms underlying the anti-inflammatory activity of this coumarin, in vitro studies were performed using murine macrophages (RAW 264.7) stimulated by lipopolysaccharide. These studies showed a dose-dependent suppression of the cyclooxygenase 2 enzyme (COX-2, enzyme activated in inflammatory processes) by scopoletin, and pro-inflammatory cytokines inhibition (PGE2, IL-6, IL-b, and TNF-a). However, it had no effect on the constitutive enzyme COX-1 (Kim et al. 2004). In addition, Dou et al. (2013) also confirmed the suppression capacity of scopoletin in IL-6 release. Scopoletin has been shown to inhibit 5-lipoxygenase enzyme in vitro (Mogana et al. 2013; Rzodkiewicz et al. 2015). And by in vivo rat model of visceral pain induced by acetic acid, scopoletin exhibited antinociceptive effects (Meotti et al. 2006; Rzodkiewicz et al. 2015).
3. Experimental section The experimental section is detailed in the supplementary material.
4. Conclusions The results revealed the phytochemical profiles, in vitro antioxidant activity, and in vivo anti-inflammatory activity of herbal remedy (HR) and crude hydroalcoholic
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extract (CHE) of B. uniflora roots; the constituents identified in the extracts justified their application in folk medicine for inflammatory conditions. Additionally, the high contents of phenolic compounds and flavonoid in the CHE sample made its antioxidant activity better than that of the HR. The differences between HR and CHE samples in the quantitative analyses resulted from the extraction process, in which CHE was obtained through standard pharmacopeial conditions and HR was obtained by the local population without standardization regarding the alcoholic concentration of the extracting solvent, root: solvent proportion, etc. Extracts obtained under standardized conditions can be reproduced either in future experiments or in interlaboratory analysis. The data suggest that the marker compound, scopoletin, exhibits its anti-inflammatory activity by suppressing enzymes and releasing inflammatory mediators. Finally, the investigation of in vivo anti-inflammatory activity showed promising results based on the reduction in the volume of edema, observed in the early stages of analysis. This study gives new perspectives for the study of the pharmacological potential of B. uniflora and its roots.
Disclosure statement No potential conflict of interest was reported by the authors.
Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
ORCID Patrıcia Weimer
http://orcid.org/0000-0002-2024-8228
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