Research paper
Topical analgesic, anti-inflammatory and antioxidant properties of Oxybaphus nyctagineus: Phytochemical characterization of active fractions

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Abstract

Ethnopharmacological relevance

Oxybaphus nyctagineus (Michx.) Sweet has traditionally been used by several Native American tribes predominantly as a topical anti-inflammatory and analgesic agent.

Aim of the study

To evaluate the antioxidant, analgesic and anti-inflammatory activity of the extracts prepared from the aerial parts of Oxybaphus nyctagineus and to characterize the major chemical constituents of the bioactive extracts.

Materials and methods

Crude polar and apolar extracts (PCE and ACE) of the herb of Oxybaphus nyctagineus were prepared and tested in the models of the CFA-induced hyperalgesia in rat knee and carrageenan-induced paw edema in rat. To identify the active compounds, subfractions were prepared by column chromatography and subjected in vitro assays, such as antioxidant assays (DPPH, peroxynitrite (ONOO) scavenging), and the LPS-induced IL-1β release test in human monocytes. Preparative HPLC was employed for the isolation of active substances, while phytochemical analysis was performed by mean of LC–MS/MS and NMR.

Results

The topically administered PCE and ACE of Oxybaphus nyctagineus demonstrated a significant analgesic and anti-inflammatory effect in the inflammation animal models. The subfraction A4 of ACE and the subfraction P5 of PCE considerably inhibited the LPS-induced IL-1β release in human monocytes, while the strongest activity was localized in the subfraction P5 in the antioxidant assays. The HPLC–MS/MS and NMR analysis revealed that 6-methoxyflavonol diglycosides, namely patuletin-3-O-robinobioside (1), 6-methoxykaempferol-3-O-robinobioside (2), spinacetin-3-O-robinobioside (3), and hydroxy-polyenoic fatty acids, namely corchorifatty acid B (4), 9-hydroxy-10E,12Z,15Z-octadecatrienoic acid (9-HOT acid) (5), and 9-hydroxy-10E,12Z-octadecadienoic acid (9-HOD acid) (6) were present in PCE, and in ACE as major compounds.

Conclusion

The results of this study established a pharmacological evidence for the traditional use of Oxybaphus nyctagineus as an anti-inflammatory agent used topically, and provided data on its phytochemical composition for the first time.

Introduction

Oxybaphus nyctagineus (Michx.) Sweet (syn.: Allionia nyctaginea, Mirabilis nyctaginea) commonly known as heart-leaf four-o’clock or heart-leaf umbrella-wort belongs to the family Nyctaginaceae. The common names of this perennial plant refer to the physiology of blooming and the morphology of the flowers. The small, pink flowers of wild four-o’clock open in the late afternoon (around 4 p.m.) from May to September, which are covered by large papery bracts forming ‘umbrellas’ (Cruden, 1973, Cardina et al., 2009). The wild four o’clock is native to North America (Spellenberg, 2004), but by now, due to its ornamental value (Carrol, 2006), spreading strategy and high tolerance (Doll, 2002), it is widely distributed as an invasive species in several European countries, e.g.: in Austria (Melzer, 1971), in Romania (Culita, 2007), in Poland (Ceynowa-Gieldon, 1988), and in Hungary (Solymosi, 2008).

Native American tribes utilized different parts of Oxybaphus nyctagineus in diverse ways to treat wide scale of diseases. Among the Ponca it was called Makan-wasek (‘strong medicine’) and its chewed root was used as a remedy for wounds (Gilmore, 1919). The Pawnees knew this plant as Kahtstakat (‘yellow medicine’) and applied the dried root to cure sore mouth in babies, while women drank as decoction after childbirth to relieve abdominal swelling (Gilmore, 1919, Foster and Duke, 1999). The Sioux drank the same decoction to reduce fever (Gilmore, 1919, Cardina et al., 2009). For the external treatment of fracture and swelling of the limbs, a mixture from the dried herb and soft grease was prepared by the Sioux (Densmore, 1918, Gilmore, 1919). The Ojibwe Indians also used this plant (called ‘goko’ coadji’ bik’, i.e., pig root) to reduce sprains and swellings (Smith, 1932). The Navajo Indians applied its root and aerial part externally as ‘fire medicine’ for burns, scalds, swellings and sores in form of dusting powder, poultice and ointment (Wyman and Harris, 1941, Elmore, 1944). Interestingly, the Acoma and Laguna Indians smoked its leaves as tobacco (Swank, 1932), while the Sioux combined and boiled it with Echinacea angustifolia and taken as a vermifuge (Gilmore, 1919). Internally, the leaf or root tea was used for bladder ailments (Foster and Duke, 1999). Despite the above summarized extensive and diverse therapeutic uses of wild four-o’clock, to the best of our knowledge, no data regarding neither the phytochemical composition, nor the bioactivity of this species has been reported in the literature to date.

Considering that Oxybaphus nyctagineus was traditionally used to treat fractures and sprains, to reduce swellings, fever, inflammation and for wound healing, the basic purpose of our research was to investigate the antioxidant, analgesic and anti-inflammatory activity of the crude extracts of Oxybaphus nyctagineus. In parallel, we aimed to characterize the major chemical constituents of the bioactive fractions in order to verify its beneficial ethnopharmacological effects. In addition, the significant spreading of Oxybaphus nyctagineus as a weed in Hungary strengthened the relevance of our study.

Section snippets

Chemicals and reagents

Analytical grade of dimethylsulfoxide, methanol, dioxane, and trifluoroacetic acid was obtained from Merck (Darmstadt, Germany). Thiopental was purchased from Sandoz (Kundl, Austria). Diclofenac gel (topical formulation of diclofenac) with active substance content of 10 mg/g was purchased from Ratiopharm (product no. L09783, Ulm, Germany). All other solvents, reagents and reference compounds were of analytical grade and purchased from Sigma-Aldrich (St Louis, MO, USA). Purified water (18 mΩ) was

Effects of PCE and ACE on carrageenan-induced paw edema in rats

The results are summarized in Fig. 1. Maximum edema formation to carrageenan was observed at 2–3 h after carrageenan injection in the control group. Pretreatment with PCE, ACE (2.5 mg/paw each), and diclofenac (1 mg/paw) attenuated the edema formation with 38%, 39% and 73% maximum inhibition, respectively. Maximum inhibitions of edema formation by Oxybaphus nyctagineus extracts and diclofenac gel were maintained at 30–60 min post-carrageenan administration. An antiphlogistic effect of Oxybaphus

Discussion and conclusion

Oxybaphus nyctagineus has been traditionally used by Native American tribes for the treatment of several inflammatory diseases; however the scientific basis for its folklore use is lacking in the literature. In order to prove the anti-inflammatory and analgesic activities of Oxybaphus nyctagineus, polar and apolar crude extracts (PCE and ACE) of the aerial parts were prepared and submitted to in vivo tests, such as the carrageenan-induced hind paw edema model in rats and the CFA-induced

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  • 1

    These authors have contributed equally to this work.

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