Elsevier

Fitoterapia

Volume 143, June 2020, 104554
Fitoterapia

Evaluation of Cypholophus macrocephalus sap as a treatment for infected cutaneous ulcers in Papua New Guinea

https://doi.org/10.1016/j.fitote.2020.104554Get rights and content

Abstract

Cypholophus macrocephalus sap is used to treat bacterially infected cutaneous leg ulcers in Papua New Guinea. High resolution LC-MS analysis of the sap revealed it to be rich in sulphated flavonoids. We assessed the effects of the sap on the differentiation and pro-inflammatory anti-microbial responses of M1 macrophages using IL-6 and TNF-α ELISAs and found significant increases in M1 macrophage IL-6 expression with concentrations as low as 243 ng/ml sap. Neutrophil IL-6 and TNF-α expression was also significantly increased but to a lesser degree. Matrix metalloproteinases (MMPs) 1, 2, 8 and 9 which are known to contribute to the toxic nature of wound exudates were inhibited by the sap at 24 μg/ml. The sap was tested with several bacterial species known to colonize cutaneous ulcers in Papua New Guinea but proved not to be active. Cypholophus sap stimulates pro-inflammatory, anti-microbial M1 macrophage and neutrophil responses at very low concentrations, whilst also inhibiting MMPs. The combination of an enhanced innate immune response and inhibition of MMPs in ulcer exudate, may contribute to the eradication of bacteria and healing of these infected ulcers. The sap concentrations used in these assays are readily achievable in an in vivo context.

Introduction

The country of Papua New Guinea, which forms the eastern half of New Guinea and its outlying islands, has been ranked second in a world ranking based on a biocultural index that takes into account a combination of biological and human cultural diversity [11]. Despite this, to date, of New Guinea's 1100 indigenous groups only 117 have been the subject of an ethnobotanical study [3]. To the east of the island of New Guinea lies the island of New Britain, one of the longest populated tropical rainforest islands in the Pacific. Archaeological evidence from Yombon in Whitman Range that forms part of the central backbone of the island suggests human habitation there dates back at least 35,000 years [17]. We have been carrying out ethnopharmacological research in the Kandrian inland area that extends into the foothills of the Whiteman Range, since 1998. Our focus has been on plants used to treat cutaneous leg ulcers, which are a highly common bacterial infection that until recently has received relatively little attention in tropical disease research [[18], [19], [20]]. The focus of this research is to identify promising plant saps that could be used as first-line treatments for cutaneous ulcers, in remote areas that lack access to basic health facilities.

Cutaneous skin ulcers of the lower leg are a common ailment in island Melanesia, and although relatively little studied in terms of their microbiology and pathology, they nonetheless present a significant area of unmet clinical need. Treatment options in Papua New Guinea include topical antiseptics, such as chlorhexidine; and antibiotics, such as amoxicillin; but for communities living in remote rainforest areas, access to these treatments may be limited or non-existent. For the inland Kaulong whose living conditions range from more established villages to small hamlets, and even temporary camps in rainforest clearings, access to medicine can be rather limited [18]. Even if medical help is sought in an aid post, the very process of traversing muddy forest paths to these aid posts is likely to further exacerbate the condition by submerging the infected wounds in rainforest mud. For this reason, wound healing plant saps that can be easily found in the local environment may prove to be useful first line treatments for cutaneous ulcers.

Until recently, little was known about the microbiology of cutaneous ulcers in Papua New Guinea. The classification of different types of cutaneous ulcers is still an area of ongoing research. Tropical ulcers which develop from small skin breaks, tend to be large and undermined, and have conventionally been thought of as infections involving spirochetes and fusiform bacteria, such as Fusobacterium ulcerans [1,12]. Early research using culture-based methods identified β-haemolytic Streptococci, Staphylococcus aureus, Corynebacterium diphtheriae and Corynebacterium haemolyticum from infected skin lesions from children in the highlands of Papua New Guinea [13]. More recently, researchers applied metagenomics to cutaneous ulcers in Lihir Island, Papua New Guinea, revealing a multitude of bacterial pathogens, such as Haemophilus ducreyi, Treponema pallidum subspecies pertenue, Streptococcus dysgalactiae and Arcanobacterium haemolyticum [16].

Aside from the bacteriology of the ulcers, the host immuno-inflammatory response of the wound environment is likely to have a strong bearing on clinical outcomes with cutaneous ulcers in Papua New Guinea. In non-healing wounds prevalent in the western world, for example, diabetic ulcers and pressure sores, persistent or dysregulated inflammation is known to be key to chronicity [21,27]. Although chronic inflammation can contribute to wound persistence in the type of ulcers found in the west, an increase in pro-inflammatory responses from innate immune cells, such as macrophages and neutrophils, which are directly involved in defending against microbial pathogens could prove beneficial, if induced early on in the pathogenesis.

Here, we examine the wound healing potential of the sap of Cypholophus macrocephalus (Blume) Wedd., a small shrub in the Urticaceae family with a distribution ranging from Malesia to New Guinea across to the pacific islands. The sap is applied directly to cutaneous ulcers by the Kaulong speaking population of Papua New Guinea. The work presented here is to the best of our knowledge, the first phytochemical and bioactivity study of this species.

Section snippets

Ethnobotanical data collection

Approval for the ethnobotanical survey was provided by the Papua New Guinea National Research Institute, the provincial government in West New Britain and the local community itself. The ethnobotanical survey commenced in August 1998 in the village of Umbi (Kandrian Inland, West New Britain, Papua New Guinea). Initially, a putative list of cutaneous ulcer plant medicines was obtained by simply walking though rainforest with key respondents while discussing plant uses. The putative plant

Cypholophus macrocephalus is applied topically to treat cutaneous ulcers by the inland Kaulong population of Papua New Guinea

Ethnobotanical research was initiated with the inland Kaulong community in 1998, with follow up work completed in 2017. Careful questioning of key ethnobotanical respondents over this large time period provided data from six respondents consistently supporting the use of the sap of this species as a topical treatment for cutaneous ulcers. The plant use is further supported by a previous report of Cypholophus stem being used to treat ulcers in Waigeo Island in Papua Province Indonesia [22]. In

Discussion

Sulphated flavonoids have been previously identified from certain plant families, such as Arecaceae, Asteraceae, Bixaceae, Dilleniaceae, Frankeniaceae, Gramineae, Guttiferae, Juncaceae, Malvaceae, Tamaricaceae, Umbelliferae and Verbenaceae [23]. Quercetin-O-sulphates (including single and multi-sulphate esters) have been frequently observed in the Asteraceae [6]. Although epi/catechin-O-sulphate has been detected as a metabolite in the human body after consumption of dietary flavonoids,

Funding information

Funding for this study came from a grant from the Christensen Fund for Melanesia, USA, 2016-8040.

Declaration of Competing Interest

The authors declare no competing interests.

Acknowledgements

We are highly grateful for ethnobotanical and plant collection support provided in Umbi Village in the Kandrian Inland region of West New Britain and to the West New Britain government for assistance in the field. We would also like to thank Catherine Fulton and Anthony Tsolaki for assistance with microbiology.

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