Improved method to obtain essential oil, asarinin and sesamin from Asarum heterotropoides var. mandshuricum using microwave-assisted steam distillation followed by solvent extraction and antifungal activity of essential oil against Fusarium spp

Highlights

• Microwave steam distillation followed by solvent extraction was used.

• Essential oil was obtained from Asarum heterotropoides var. mandshuricum.

• Asarinin and sesamin were successfully extracted from deoiled roots and rhizomes.

• The proposed technique showed an advantage in yield and time than other techniques.

• Isolated essential oil exerted better antifungal activity against Fusarium spp.

Abstract

An efficient microwave-assisted steam distillation followed by a solvent extraction method (MSDE) was established to isolate the essential oil, asarinin and sesamin from the roots and rhizomes of Asarum heterotropoides var. mandshuricum (AHVM). In the MSDE technology applied in this work, moistened roots and rhizomes of AHVM powders are applied to this system. After extracting the essential oil by steam distillation, the powder residue of the roots and rhizomes of AHVM is relatively dry. Hence, this method can save energy consumption, and the loss of asarinin and sesamin can be reduced during the subsequent extraction process and improve essential oil yield. The developed method extracted the essential oil to reach the maximum extraction amount (15.80 ± 0.79 mL/kg) after microwave irradiation for 30 min and maintained the yield balance. Through comparison, MSDE was chosen as the best approach, and under the optimum conditions (90 % aqueous ethanol, liquid–solid ratio of 19.0 mL/g and microwave irradiation power of 332 W) obtained by first order kinetic models and response surface analysis with Box–Behnken design, satisfactory yields of asarinin (1.81 ± 0.72 mg/g) and sesamin (0.74 ± 0.09 mg/g) were achieved. In accordance with the yields of asarinin and sesamin, our research optimized the process parameters. According to the comprehensive comparison of these parameters, the proposed process described in this study has greater advantages than traditional methods. Minimum inhibitory concentrations (MIC_S) of the essential oil, which obtain from the roots and rhizomes of AHVM of five kinds of Fusariums spp. (F. avenaceum, F. trichothecioides, F. sporotrioides, F. sambucinum and F. culmorum) were determination to be 1.25 mg/mL, 1.5 mg/mL, 1.5 mg/mL, > 2 mg/mL and > 2 mg/mL, respectively. The results of the antifungal test showed that the essential oil from the roots and rhizomes of AHVM showed remarkable antifungal activities, in which, F. avenaceum was the most sensitive to the essential oil, with the MIC₅₀ values of 0.625 mg/mL.

Introduction

Microwave extraction technology has been widely used in the extraction of plant essential oils (EOs) and non-volatile components. It is mainly summarized into two types of methods: one is solvent-free microwave method, this method is suitable for fresh raw materials, or the plant itself contains fresh raw materials. The water is distilled to obtain the EO. However, although this method can obtain EOs, it cannot simultaneously obtain non-volatile target components. Another method is to mix the plant material and water in the same container for microwave distillation. The steps to obtain the EO, asarinin and sesamin by traditional experimental methods are as follows: (1) the root and rhizome of AHVM are evenly mixed with water at a defined proportion; (2) the EO in AHVM is extracted by hydrodistillation; 3) water is removed from the residual soliquoid of AHVM by distillation (a drying temperature of 80 ± 5 °C for about 4 h was used in this experiment.); (4) the compounds asarinin and sesamin are extracted with ethanol; (5) organic solvents are separated from the AHVM oils; and (6) rotary evaporation is utilized to remove ethanol From the extract and to obtain an extract containing asarinin and sesamin. This method can obtain the EO, and the water-soluble component in the system, but the fat-soluble ingredients are not available using this method and this method causes the material to be wetted with a large amount of water and requires a drying treatment.

Conventional extraction methods such as conventional steam distillation (SD), conventional hydrodistillation (HD) and microwave-assisted hydrodistillation (MHD) methods have some disadvantages such as can not take into account the extraction of volatile components (EOs) and the extraction of non-volatile components (asarinin, sesamin) and lower efficiency, we lack some effective means. Alternatively, microwave-assisted steam distillation involves placing the premoistened materials in a microwave reactor without adding any solvent while steam distillation is performed at normal pressure. Since the water in the bottom flask and premoistened plant materials in the upper flask are irradiated with microwaves, the pressure in the system increases, causing the oil cells to rupture; this results in the release of EOs accompanied with steam distillation from the premoistened plant materials. Generally, microwave steam distillation has been extensively used in the extraction of EO from aromatic and medicinal plants (Chemat et al., 2006), and there is no excess water participation in the entire reaction system, which is of great benefit for subsequent processing. Nevertheless, the microwave-assisted steam distillation followed by solvent extraction MSDE method novelty developed in this paper can take into consideration the extraction of volatile components (EOs) and the extraction of non-volatile components (asarinin, sesamin).

AHVM is a well-known cold temperate vivacious herbaceous plant belonging to the Aristolochiaceae family (Hwang et al., 2003). This plant is primarily located northeast and north of China, Japan and Korea (Dan et al., 2010; Zeng et al., 2004) and is diffusely planted in the three northeastern provinces of China (Pharmacopoeia of the People's Republic of China (Volume I), 2015). Farmers can grow this plant up to 7500 kg/hm² (Chen et al., 2018a). The herbaceous plant is recorded in ancient herbalists that can be used as herbal medicinal products for a long historical source (Oh et al., 2010). The herb has been used in the remedy of various diseases, such as toothache, headache, cough, aphthous stomatitis, gingivitis, and rheumatic arthritis (Huang et al., 2014; Jing et al., 2017a, b; Oh et al., 2010). Some phytochemical studies of AHVM have revealed the presence of EOs (Chen et al., 2018b) and furofuran-type lignans, sesamin and asarinin (Huang et al., 2014). Moreover, the EO from the roots and rhizomes of AHVM has traditionally been used as an antibacterial agent (Haque et al., 2016), an antifungal agent (Dan et al., 2010; Liu et al., 2007) and an insecticide (Han et al., 2012; Ji et al., 2013).

Sesamin and asarinin are two C-7’ epimeric lignans (Fig. 1) (Li et al., 2005). Asarinin is used as the index component of the roots and rhizomes of AHVM in the Chinese Pharmacopoeia (Pharmacopoeia of the People's Republic of China (Volume I), 2015). Asarinin increased intracellular dopamine levels (Park et al., 2017), had an anti-rejection effect and prolonged allograft heart survival (Gu et al., 2015; Zhang et al., 2006), It is also known as a precursor of phytoestrogens and enterolactone and exhibits antiestrogenic activity (Jin and Hattori, 2011). Sesamin produces neuroprotective effects (Cheng et al., 2006; Guo et al., 2015), improves cognitive functions (Ito et al., 2018), and promotes recovery from mental fatigue (Imai et al., 2018). Sesamin acts as an antioxidant (Hemalatha and Ghafoorunissa, 2007) and ameliorates oxidative stress (Hsieh et al., 2011). Sesamin is also believed to improve cardiac function (Su et al., 2014) and exhibits anti-inflammatory and antifibrogenic actions (Periasamy et al., 2014), and may protect against cardiac remodeling (Fan et al., 2017). Therefore, the EO and asarinin and sesamin of AHVM are valuable components and deserve increasing attention. Because of asarinin and sesamin solubility in water, a prolonged heating time and the complicated separation process, the yields of target analytes received by such means are very low and result in a loss of asarinin and sesamin. Alternatively, microwave-assisted steam distillation followed by solvent extraction (MSDE) can acquire the EO quickly due to combining microwave irradiation heating with distillation. However, its application to the extraction of nonvolatile target analytes from the deoiled plant materials has not yet been reported. The use of MSDE technology described in this research can lower the asarinin and sesamin waste in subsequent operational processes because the system does not introduce excess water; furthermore, since it does not require drying, the energy consumption of the operational process can be reduced effectively.

The genus Fusarium is a kind of filamentous fungi and taxonomic status belongs to family Nectriaceae (Burgess, 1981). Fusarium species are known to cause a huge range of diseases on an extraordinary range of wheat, corn, cereal and other organic substrates and are common pathogens of crops (Jedidi et al., 2018). They reduce yield and produce mycotoxins during infection of crops tissues (Parikh et al., 2018). In addition, some previous reports have shown that pollutants infected by Fusarium can secrete mycotoxins under certain conditions. This is a toxic metabolite with a certain degree of mutagenic and carcinogenic effects. Mycotoxins cannot be destroyed by cooking because of the are thermo stable (Parikh et al., 2018; Dananjaya et al., 2017). They can continuously accumulate in the human body and are difficult to remove from the organism. Due to the serious harm of Fusarium spp. to human health and ecological health, people have chemically synthesized a variety of fungicides agents to reduce the harm caused by Fusarium spp.. The usual antimicrobial chemicals (benzimidazoles, aromatic hydrocarbons and sterol biosynthesis inhibitors) used in agriculture and forestry for plants and Chinese herbal medicine disease control are associated with series of problems (Singh and Sahota, 2018; Heneberg et al., 2018; Slana and Dolenc, 2013). Currently, there are reports that because of the carcinogenic and teratogenic properties and residual toxicity of chemical synthetic agents, people have more disputes about its safety. (de Souza Cândido et al., 2014). For these reasons, people began to doubt the safety of fungicides agents that are chemically synthesized or chemically added. Therefore, people and society have an increasing demand for natural non-toxic and side-effect biocides. Hence, because of the advantage of EOs which is the natural antimicrobial activity, could contribute to reducing the risk of chemical fungicide handling to control decay in crops, plants and Chinese herbal medicine. Meaningfully, there are no recent reports on the antifungal activity of EO, which from the roots and rhizomes of AHVM against Fusarium spp.. This article uses various antifungal parameters such as: antifungal diameter, MIC, MBC and time killing curve to preliminarily discuss and study the antifungal effect of the EO on Fusarium spp. we used in this antifungal activity experimental process are as follows: F. avenaceum, F. trichothecioides, F. sporotrioides, F. sambucinum and F. culmorum.

The aim of this work was to innovate an easy, effective way to distill and extract EO from the roots and rhizomes of AHVM and for the high-efficiency simultaneous extraction of asarinin and sesamin from the residue. The parameters that affect the courses were scientifically optimized, and the quantity obtained by four different extraction methods (MSD, MHD, SD and HD) of roots and rhizomes of AHVM EO were evaluated using the gravimetric method. Asarinin and sesamin extracted by MSDE, MHDE, HDE and SDE were detected by high performance liquid chromatography. The obtained EO by four different extraction methods (MSD, MHD, SD and HD) were also characterized by gas chromatography-mass spectrometry (GC-MS). Evaluating the antifungal activity of the EOs from the roots and rhizomes of AHVM on these five Fusarium species.