Chemical composition, in vitro antioxidant activity and α-glucosidase inhibitory effects of the essential oil and methanolic extract of Elsholtzia densa Benth.

Abstract Elsholtzia densa Benth. is a traditional aromatic herb used in the pharmaceutical and flavouring industries. To analyse and compare the chemical composition, the oils and nonvolatile compounds in E. densa and Mosla chinensis Maxim. were extracted via hydrodistillation, solvent extraction or ultrasound-assisted extraction. Seventy-three volatile compounds in the volatile oil (0.35 ± 0.06%) obtained by E. densa via hydrodistillation were investigated by gas chromatography–mass spectrometer and compared based on different parameters. Also, the antioxidant activity and α-glucosidase inhibitory effects of the five sub-fractions of the methanolic extract were studied and the ethyl acetate sub-fraction (EC50 = 7.9 μg/mL) and petroleum ether sub-fraction (EC50 = 0.0955 mg/mL) showed the strongest activity, respectively. This study has provided a scientific basis for scientific collection, effective development, use of E. densa Benth., and suggested that it can be used as a potential source of antioxidants in food and a potential candidate for the management of type 2 diabetes mellitus.


Introduction
Since ancient times, herbs and spices have been used as additives to improve the flavour and organoleptic properties of food. A total of 40 Elsholtzia species are widely distributed in eastern Asia. Most species are used as traditional pharmaceutical herbs as well as applied in the perfume and flavouring industries (Gong 1997).
Elsholtzia densa Benth. is often used as a substitute for Mosla chinensis Maxim. This herb is also considered a promising source of effective compounds to improve the microbiological safety of food. Elsholtzia species are commonly used in traditional medicine to treat colds, fever, dysentery, digestion disorders and heat stroke, among others (Liu et al. 2007). However, it was little reported about the antioxidant activity and α-glucosidase inhibitory effects of E. densa.
The aerial parts are usually used for the extraction of essential oil from E. densa via hydrodistillation. (Sun et al. 1995). However, less focus has been given to the chemical composition of roots and the nonvolatile compounds of E. densa and other extraction methods. In this paper, the chemical components of different parts of E. densa were compared. And the chemical components derived from different extraction methods were compared to screen for a suitable method that would yield more chemical compounds in a shorter period of time. The differences between E. densa and M. chinensis were studied to determine the common effective components of the two species (Liu et al. 2015). Furthermore, the potential of antioxidant activity and α-glucosidase inhibitory effects of E. densa were studied.

Comparison of the chemical composition of different plant parts
Hydrodistillation yielded 0.30, 0.35 and < 0.10% of a pale yellow oil from the E. densa whole plants, aerial parts and roots, respectively. These essential oils and nonvolatile water extracts were investigated by GC/MS. A total of 73 volatile and 20 nonvolatile components were identified. The retention index of the compounds and their relative percentages are listed in Tables S1 and S2. The main volatile component of the aerial parts is 3-octanone (8.68%), whereas that of the roots is geranyl benzoate (14.75%). These two compounds are usually used as functional antioxidants, antiradicals and antimicrobials in food (Sacchetti et al. 2005). The main nonvolatile component in the aerial parts and roots is toluene (34.34 and 55.01%, respectively). Toluene is often used to sterilise microbial cultures and to lyse bacterial cells in bacterial enzyme assays (Inoue & Horikoshi 1989). Compared with the chemical components in the aerial parts, seven volatile and four nonvolatile compounds were found in common with the root components. Moreover, 24 volatile and 7 nonvolatile compounds were unique to the roots and were not identified in the aerial parts.
This comparison shows that aside from essential oils, nonvolatile water extracts are important sources of antioxidants and antimicrobials. The roots of E. densa contain various compounds, and further research on their applications is necessary.

Comparison of the chemical composition of extracts from different methods
E. densa oils obtained via HD, SE and UAE were investigated by GC/MS (Mann et al. 2013); a total of 40, 19 and 25 compounds, respectively, were identified. The results are listed in Table S3. The main components of the extracts from HD, SE and UAE are α-caryophyllene (14.34%), tetrapentacontane (31.16%) and hexatriacontane (12.91%), respectively. α-Caryophyllene and tetrapentacontane were found to demonstrate antioxidant activity, whereas tetrapentacontane and hexatriacontane have antimicrobial activity (Geethalakshmi & Sarada 2013). The components extracted via SE and UAE are similar and are relatively different from those extracted via HD. High-polarity compounds were extracted by water in HD, whereas low-polarity compounds were extracted by ethyl acetate in SE and UAE. More compounds were detected in the HD extract because of the large proportion of high-polarity compounds; the high heating temperatures made the compounds dissolve more efficiently.
Hydrodistillation avoids the use of organic solvents, which is a green and efficient method that can retain the organoleptic characteristics of the starting materials. Compared with SE, UAE obtained more compounds and also shortened the extraction time, which confirmed it to be a novel and efficient extraction method.

Comparison of the chemical compositions of E. densa and M. chinensis
The oils and nonvolatile compounds extracted by HD from E. densa and M. chinensis were investigated with GC/MS; the results are listed in Tables S4 and S5. A total of 40 and 43 volatile compounds, as well as 15 and 9 nonvolatile compounds, were identified in E. densa and M. chinensis, respectively. The main volatile components of E. densa and M. chinensis are α-caryophyllene (14.34%) and 4-methylene-1-(1-methylethyl)-bicyclo[3.1.0]hexane (15.51%), respectively. Meanwhile, the main nonvolatile component is allyl n-octyl ether (13.31%) in E. densa and toluene (32.24%) in M. Chinensis. A total of 11 volatile compounds and 1 common nonvolatile compound were common to E. densa and M. chinensis. These results confirm that E. densa contains some similar compounds that can substitute those of M. chinensis. Meanwhile, the identification of specific compounds in E. densa indicates the presence of potential novel active compounds. Further study is required to establish the applications of these compounds in E. densa.

Antioxidant activity of the extract
The antioxidant activity of the five sub-fractions of methanolic extract has been evaluated by DPPH radical-scavenging test. Free radical-scavenging properties of the five sub-fractions of methanolic extract are presented in Table 1. Regarding the EC 50 values, all the extracts and the commercial standard (Vitamin C) depleted the initial DPPH concentration by 50% within 0.5 h. The lower EC 50 value represents the higher antioxidant activity of the sample. The EC 50 value of Vitamin C is 2.98 μg/mL and the free radical-scavenging activities of all subfractions were in the order: Ethyl acetate sub-fraction > Butanol sub-fraction > Chloroform sub-fraction > Petroleum ether sub-fraction.

α-glucosidase inhibition assay
The α-glucosidase inhibitory effects of the five sub-fractions of methanolic extract were tested successively and the EC 50 values are presented in Table 1. The lower EC 50 value represents the higher α-glucosidase inhibitory effect of the sample. The EC 50 value of the acarbose is 35.12 mg/mL and the α-glucosidase inhibitory effects of all sub-fractions were in the order: Petroleum ether sub-fraction > Chloroform sub-fraction > Ethyl acetate subfraction > Butanol sub-fraction > Water sub-fraction. According to the results, the petroleum sub-fraction showed much stronger inhibitory effect than the positive control, which may suggest the active substances would more likely belongs to the low-poverty sub-fraction.

Conclusions
This study has provided a scientific basis for scientific collection, effective development and use of E. densa Benth. as well as the formulation of quality standards. Our results suggested that E. densa can be used as a potential source of antioxidants in food and a candidate for the management of type 2 diabetes mellitus.

Supplementary material
Experimental details are available online, alongside Tables S1-S5 and Figures S1-S2.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by the National Scientific Foundation of China [grant number 81171957].