Characterization of the Volatile Constituents of Plai (Zingiber purpureum) by Gas Chromatography–Mass Spectrometry

Zingiber purpureum Roscoe, known as plai in Thailand, is a perennial plant of the Zingiberaceae family and has traditionally been used in Southeast Asian countries to treat inflammation, pain, and asthma. In this study, we performed the characterization of the volatile constituents in ethyl acetate extracts of plai. Ethyl acetate extracts derived from the rhizomes of plai were subjected to gas chromatography–mass spectrometry, and the key peaks in the total ion current chromatograms were annotated or identified. In total, twenty-one compounds were identified using isolation procedures or standards, and nine compounds were annotated by comparing their Kovats retention index (RI) and electron ionization (EI) mass spectra with those in the literature. Most of the identifications were inconsistent with the tentative annotations found via library search and suggested that some peaks were incorrectly assigned in previous studies. Thus, to avoid further misannotations and contribute to the research on dereplication, the RI value, EI mass spectral data, and NMR spectroscopy data of the isolated compounds are reported.


Introduction
The goal of metabolomics research on medicinal plants is to comprehensively and accurately identify all low-molecular-weight metabolites [1], thus providing an effective approach to evaluating the quality of medicinal plants [2].These studies are mainly based on targeted and untargeted analyses [1].In untargeted analyses, annotation and identification are critical to converting metabolomics data into meaningful biological knowledge [3].However, especially in studies on medicinal plants, the process of annotation and identification of metabolites remains a major bottleneck due to the limited data in libraries and lack of standards [1,4].
In 2007, four different levels of metabolite identification were defined by the chemical analysis working group of the Metabolomics Standards Initiative (MSI) [5,6], namely, the identified compounds (level 1), putatively annotated compounds (level 2), putatively characterized compound classes (level 3), and unknown compounds (level 4).Recently, level 0, which includes compounds identified via isolation and full stereochemical characterization, was established as a new confidence level of metabolite identification [6,7].In many studies, authentic standards were not used; therefore, annotations (levels 2 and 3) and not identifications (levels 0 and 1) were achieved [8].
Zingiber purpureum Roscoe (syn Z. cassumunar Roxb.) is a perennial plant in the Zingiberaceae known as plai (phlai) in Thailand and bangle or bengle in Indonesia [9][10][11].Over the past two decades, Z. montanum (J.Koenig) Link ex A.Dietr. has been accepted as the scientific name for plai (cassumunar ginger), whereas Bai et al. recently proposed that Z. purpureum Roscoe is the correct name for this plant [12].This herb is widely used as a remedy or component of herbal recipes in Asian countries [9].In Thailand, it is used as the main component in massage oil to relieve muscle pain and is consumed to relieve asthma [9,13].In the Thai Herbal Pharmacopoeia, plai is listed as an anti-inflammatory, counter-irritant, and mosquito-repellent herb [14].In fact, products using plai oil are currently made and distributed to alleviate muscle pain [15].In Indonesia, bangle has been used to relieve colic in children [13], to treat abdominal obesity in postpartum women [16], and as a vermifuge and an analeptic for the uterus [17].Scientific studies have revealed the bioactivities of the extracts or fractions of this plant behind these traditional uses, such as antioxidant, anti-inflammatory, antifungal, antimicrobial, anti-asthma, neuroprotective, anticancer, antiaging, and skin whitening effects [18].
In this study, to confirm or revise the peak annotation of the volatile constituents in plai extracts, we performed their characterization.In particular, the compounds corresponding to key peaks were isolated, and their structures were elucidated based on NMR spectroscopic data.Finally, Kovats retention index (RI) value, electron ionization (EI) mass spectral data, and NMR data of the isolated compounds are reported to contribute to further studies on the chemical constituents of plai.

Results and Discussion
Previous studies have shown that essential oils obtained through the hydrodistillation of plai contained phenylbutenoid monomers [26], and the ethyl acetate fraction of the 70% ethanol extract of plai contained phenylbutenoid monomers and dimers [33].Therefore, herein, the dried rhizomes of two plai samples purchased in Thailand and Indonesia were extracted using ethyl acetate, and then the extracts were subjected to GC-MS (Figure 1).Thirty major peaks were detected in the GC-MS total ion current (TIC) chromatograms of the extracts, and their tentative annotation was performed via library search using the Wiley 9 database (Table 1 and Figure 2).purpureum Roscoe is the correct name for this plant [12].This herb is widely used as a remedy or component of herbal recipes in Asian countries [9].In Thailand, it is used as the main component in massage oil to relieve muscle pain and is consumed to relieve asthma [9,13].In the Thai Herbal Pharmacopoeia, plai is listed as an anti-inflammatory, counter-irritant, and mosquito-repellent herb [14].In fact, products using plai oil are currently made and distributed to alleviate muscle pain [15].In Indonesia, bangle has been used to relieve colic in children [13], to treat abdominal obesity in postpartum women [16], and as a vermifuge and an analeptic for the uterus [17].Scientific studies have revealed the bioactivities of the extracts or fractions of this plant behind these traditional uses, such as antioxidant, anti-inflammatory, antifungal, antimicrobial, anti-asthma, neuroprotective, anticancer, antiaging, and skin whitening effects [18].Plai contains several types of secondary metabolites.Among them, phenylbutenoids, curcuminoids, and essential oil constituents are the major bioactive compounds [18].In particular, phenylbutenoids are major characteristic compounds of this herb and show various biological activities, including anti-asthma, anticancer, anti-inflammatory, chondroprotective, and melanogenic effects [18,19].Owing to their volatility, gas chromatography-mass spectrometry (GC-MS) is often employed for the analysis of the extracts and oils of plai [15,[20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39].Although these studies contributed to the elucidation of chemical constituents in plai, some peaks are still unidentified, and some discrepancies in peak annotations are found in the literature.
In this study, to confirm or revise the peak annotation of the volatile constituents in plai extracts, we performed their characterization.In particular, the compounds corresponding to key peaks were isolated, and their structures were elucidated based on NMR spectroscopic data.Finally, Kovats retention index (RI) value, electron ionization (EI) mass spectral data, and NMR data of the isolated compounds are reported to contribute to further studies on the chemical constituents of plai.

Results and Discussion
Previous studies have shown that essential oils obtained through the hydrodistillation of plai contained phenylbutenoid monomers [26], and the ethyl acetate fraction of the 70% ethanol extract of plai contained phenylbutenoid monomers and dimers [33].Therefore, herein, the dried rhizomes of two plai samples purchased in Thailand and Indonesia were extracted using ethyl acetate, and then the extracts were subjected to GC-MS (Figure 1).Thirty major peaks were detected in the GC-MS total ion current (TIC) chromatograms of the extracts, and their tentative annotation was performed via library search using the Wiley 9 database (Table 1 and Figure 2).Next, verification of the tentative annotations was conducted.The annotation of peaks 1, 2, 4-8, and 13 was verified by comparing the EI mass spectrum and RI value with those reported [40].Peaks 3 and 11 were identified as sabinene (3) and terpinen-4-ol (11), respectively, using standards.In addition, peak 12 was assigned to 3,4-dimethoxybenzaldehyde (12) via isolation [41].Therefore, the annotation and identification of these peaks was consistent with the annotation found via library search.
The EI mass spectrum of peak 16 showed the molecular ion peak at 218 (m/z) and the base ion peak at 136 (m/z).It was annotated as δ-cuparenol (16a) via a library search with 80% similarity, and Risnawati et al. could not identify this peak [32].In this study, a search in the Adams database revealed that xanthorrhizol showed these features [40], which is consistent with other studies [49,50].Accordingly, using the standard, peak 16 was finally identified as xanthorrhizol (16), which is reported as a component of Z. purpureum Roscoe for the first time.
In total, twenty-one compounds, including fifteen phenylbutenoids and one phenylbutanoid, were identified, and nine compounds were annotated.The results indicate that some peaks were incorrectly assigned in previous investigations [20][21][22][23][24][25][27][28][29][30][31]34].To avoid further misannotations and to contribute to the research on dereplication, the RI value, EI mass spectral data, and NMR data of the isolated compounds are summarized in Table 1 and in the Materials and Methods section.Some of these data, such as the RI value of compounds 23-30 and the EI mass spectral data of compound 21, are reported for the first time.

Plant Materials
Dried rhizomes of Zingiber purpureum Roscoe (Zingiberaceae) were purchased from the crude drug market in Surakarta, Indonesia, in August 2017 and Bangkok, Thailand, in July 2018.These crude drugs were authenticated by one of the authors (A.S.).A voucher specimen (RIN-170102 and 180107) has been deposited at the Museum of Materia Medica, Ritsumeikan University (Kusatsu, Shiga, Japan).

GC-MS Analysis
The pulverized samples were extracted with 1 mL of ethyl acetate per 10 mg sample for 24 h at room temperature.After extraction, the samples were filtered through a 0.45 µm Millipore filter unit (Advantec, Tokyo, Japan) and subjected to GC-MS by injecting 1 µL of sample in the splitless mode.The injector temperature was set at 270 • C, and the carrier gas (helium) was set at a constant flow rate of 1 mL/min.Metabolites were separated using a DB-5MS capillary column (30 m × 0.25 mm i.d., film thickness 0.25 µm, Agilent Technologies, Santa Clara, CA, USA).The GC oven temperature was initially set at 50 • C and held for 3 min, increased to 300 • C at a rate of 10 • C/min, and then maintained at 300 • C for 12 min.Mass spectrometry was performed in the EI mode with an electron energy of 70 eV.The temperature of the ion source and interface was set at 270 • C.
Tentative annotations were performed via library search using the Wiley 9 database.Identifications and annotations were performed according to the confidence levels of metabolite identification defined by the chemical analysis working group of MSI.Briefly, identifications with levels 0 and 1 were isolation and standard, respectively.The structures of isolated compounds were established based on NMR spectroscopic data [41,[46][47][48][51][52][53][54][55].Annotations with level 2 were RI value and EI mass spectral data matched with those in the literature [40].

Conclusions
In this study, the characterization of the volatile constituents in ethyl acetate extracts prepared from the rhizomes of plai was performed.In the GC-MS TIC chromatograms, thirty major peaks were detected, and their corresponding compounds were annotated or identified.Eventually, twenty-one compounds, including fifteen phenylbutenoids and one phenylbutanoid, were identified by means of isolation procedures or using standard compounds, and nine compounds were annotated on the basis of RI value and EI mass spectral data.Most of the identifications were inconsistent with tentative annotations obtained via library search, indicating the presence of incorrect peak assignments in previous studies.To avoid further misannotations and to contribute to studies on dereplication, the RI value, EI mass spectral data, and NMR data of the isolated compounds are reported.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules29061216/s1,Table S1: Relative contents (%) of major 30 compounds in ethyl acetate extracts derived from the rhizomes of plai purchased in Thailand and Indonesia; Figure S1: EI-MS spectra of isolated compounds.

Figure 1 .
Figure 1.Gas chromatography-mass spectrometry total ion current chromatograms of ethyl acetate extracts of plai purchased in Thailand (A) and Indonesia (B).The numbers in the figure refer to the structures in Figure 2.

Figure 1 .
Figure 1.Gas chromatography-mass spectrometry total ion current chromatograms of ethyl acetate extracts of plai purchased in Thailand (A) and Indonesia (B).The numbers in the figure refer to the structures in Figure 2.

Figure 2 .
Figure 2. Chemical structures of annotated or identified compounds.

Figure 2 .
Figure 2. Chemical structures of annotated or identified compounds.

Table 1 .
Annotation or identification of volatile constituents in the ethyl acetate extracts of plai samples.