背景:Lobostemonfruticosus(L.)H.Buek是Boraginaceae家族的多年生和木质灌木,在南非的开普地区发现。叶子和树枝用于治疗皮肤疾病,如伤口,烧伤,癣,丹毒和湿疹。抗炎,抗菌,已经报道了L.fruticosus的抗病毒和抗增殖活性。然而,有一个空白的研究报告伤口愈合特性的这种植物。
目的:与fruticosus的传统使用保持一致,我们的研究旨在使用体外和体内生物测定来确认植物的伤口愈合潜力。
方法:使用从南非的西开普省收集的样品制备fruticosus的甲醇水提取物(80%v/v),并通过超高效液相色谱与质谱联用(UPLC-MS)进行色谱剖析。进行3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴化物(MTT)细胞毒性测定以筛选提取物的无毒浓度,用于随后在体外划痕测定中使用。人角质形成细胞(HaCaT)和成纤维细胞(BJ-5ta)细胞系均用于体外划痕测定。体内尾鳍截肢试验用于评估fruticosus的伤口愈合潜力。通过监测用各种浓度的植物提取物处理的斑马鱼幼虫的鳍再生。
结果:在fruticosus提取物中初步鉴定出六种主要化合物,即;球形纳米A,球形B,芦丁,rabdosiin,鼠尾草酸和迷迭香酸。还鉴定了潜在毒性的吡咯啶生物碱,并定量证实其存在于119.58ppm(m/m)的低浓度下。在划痕分析中用植物提取物处理HaCaT和BJ-5ta细胞导致细胞迁移增加,这意味着加速伤口闭合。用100μg/mL提取物处理24小时后,据记录,HaCaT和BJ-5ta细胞的伤口闭合率为91.1±5.7%和94.1±1.3%,分别,而未处理的(培养基)对照显示两种细胞系的72.3±3.3%和73.0±4.3%,分别。在24和36小时之间观察到完全的伤口闭合。而未治疗的对照组在观察期结束时(48小时)未达到100%的伤口闭合。在体内,100μg/mL的粗提取物加速了斑马鱼尾鳍再生,达到100.5±3.8%的再生,而未处理的对照在截肢后两天为68.3±6.6%。
结论:该研究肯定了伤口愈合特性,以及使用体外和体内测定法的低毒性。支持传统的药用。应研究针对伤口愈合中涉及的不同机制的其他体外测定以支持当前发现。
BACKGROUND: Lobostemon fruticosus (L.) H.Buek is a perennial and woody shrub of the Boraginaceae family, found in the Cape region of South Africa. The leaves and twigs are used to treat dermatological conditions such as wounds, burns, ringworm, erysipelas and eczema. Anti-inflammatory, antibacterial, antiviral and anti-proliferative activities of L. fruticosus have been reported. However, there is a void in research which reports on the wound healing properties of this plant.
OBJECTIVE: Aligned with the traditional use of L. fruticosus, our study aimed to use in vitro and in vivo bioassays to confirm the wound healing potential of the plant.
METHODS: An aqueous methanol extract (80% v/v) of L. fruticosus was prepared using a sample collected from the Western Cape Province of South Africa and chromatographically profiled by ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay was performed to determine the non-toxic concentrations of the extract for subsequent use in the in vitro scratch assay. Both the human keratinocyte (HaCaT) and fibroblast (BJ-5ta) cell lines were employed in the in vitro scratch assay. The in vivo caudal fin amputation assay was used to assess the wound healing potential of L. fruticosus, by monitoring fin regeneration in zebrafish larvae treated with the plant extract at various concentrations.
RESULTS: Six major compounds were tentatively identified in the L. fruticosus extract namely; globoidnan A, globoidnan B, rutin, rabdosiin, sagerinic acid and rosmarinic acid. The potentially toxic pyrrolizidine alkaloids were also identified and quantitatively confirmed to be present at a low concentration of 119.58 ppm (m/m). Treatment of HaCaT and BJ-5ta cells with the plant extract in the scratch assay resulted in an increase in cell migration, which translates to accelerated wound closure. After 24 hr treatment with 100 μg/mL of extract, wound closure was recorded to be 91.1 ± 5.7% and 94.1 ± 1.3% for the HaCaT and BJ-5ta cells, respectively, while the untreated (medium) controls showed 72.3 ± 3.3% and 73.0 ± 4.3% for the two cell lines, respectively. Complete wound closure was observed between 24 and 36 hr, while the untreated control group did not achieve 100% wound closure by the end of the observation period (48 hr). In vivo, the crude extract at 100 μg/mL accelerated zebrafish caudal fin regeneration achieving 100.5 ± 3.8% regeneration compared to 68.3 ± 6.6% in the untreated control at two days post amputation.
CONCLUSIONS: The study affirms the wound healing properties, as well as low toxicity of L. fruticosus using both in vitro and in vivo assays, which supports the traditional medicinal use. Other in vitro assays that target different mechanisms involved in wound healing should be investigated to support the current findings.