Analysis of Volatile Constituents in Platostoma palustre (Blume) Using Headspace Solid-Phase Microextraction and Simultaneous Distillation-Extraction

Hsian-tsao (Platostoma palustre Blume) is a traditional Taiwanese food. It is admired by many consumers, especially in summer, because of its aroma and taste. This study reports the analysis of the volatile components present in eight varieties of Hsian-tsao using headspace solid-phase microextraction (HS-SPME) and simultaneous distillation-extraction (SDE) coupled with gas chromatography (GC) and gas chromatography-mass spectrometry (GC/MS). HS-SPME is a non-heating method, and the results show relatively true values of the samples during flavor isolation. However, it is a kind of headspace analysis that has the disadvantage of a lower detection ability to relatively higher molecular weight compounds; also, the data are not quantitative, but instead are used for comparison. The SDE method uses distillation 2 h for flavor isolation; therefore, it quantitatively identifies more volatile compounds in the samples while the samples withstand heating. Both methods were used in this study to investigate information about the samples. The results showed that Nongshi No. 1 had the highest total quantity of volatile components using HS-SPME, whereas SDE indicated that Taoyuan Mesona 1301 (TYM1301) had the highest volatile concentration. Using the two extraction methods, 120 volatile components were identified. Fifty-six volatile components were identified using HS-SPME, and the main volatile compounds were α-pinene, β-pinene, and limonene. A total of 108 volatile components were identified using SDE, and the main volatile compounds were α-bisabolol, β-caryophyllene, and caryophyllene oxide. Compared with SDE, HS-SPME sampling extracted a significantly higher amount of monoterpenes and had a poorer detection of less volatile compounds, such as sesquiterpenes, terpene alcohols, and terpene oxide.


Introduction
Hsian-tsao (Platostoma palustre Blume, also known as Mesona procumbens Hemsl. [1]), also called Liangfen Cao or black cincau, belongs to the family Lamiaceae. It is an annual plant that is mainly distributed in tropical and subtropical regions, including Taiwan, southern China, Indonesia, Vietnam, and Burma [2]. Hsian-tsao tea, herbal jelly, and sweet soup with herbal jelly are popular during the summer, and heated herbal jelly is admired by many Taiwanese, especially in winter, because of its

Optimization of the HS-SPME Procedure
The method used was modified from those of Yeh et al. [20]: 1.

2.
Comparisons of the extraction times: The above-mentioned optimal extraction fiber was used in the comparison of the extraction times. The tested extraction times were 10 min, 20 min, 30 min, 40 min, or 50 min, and the extraction temperature was maintained at 25 ± 2 • C. Sample preparation steps were the same as above.

1.
Analysis of the volatile compounds using HS-SPME extraction: a 50/30 µm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber (Supelco, Inc., Bellefonte, PA, USA) was used for aroma extraction. The eight different Hsian-tsao varieties were used as samples. Each sample was homogenized as described above in Section 2.2.1 (1 g was placed in a 7 mL vial (hole cap PTFE/silicone septa)). The SPME fiber was exposed to each sample for 40 min at 25 ± 2 • C; then, each sample was injected into a gas chromatograph injection unit. The injector temperature was maintained at 250 • C and the fiber was held for 10 min. The peak area of a volatile compound or total volatile compounds from the integrator was used to calculate the relative contents.

2.
Analysis of volatile compounds by SDE extraction: 100 g samples of Hsian-tsao were cut with scissors into pieces approximately 1-3 cm in size and were then homogenized for 2 min with 2 L of deionized water and were placed into a 5-L round-bottom flask. The flask was attached to a simultaneous distillation-extraction apparatus and 100 • C steam was used as the heat source and passed through the sample. A 50 mL volume of solvent was prepared by mixing n-pentane/diethyl ether (1:1, v/v) into a pear-shaped flask, then placing it in a 40-45 • C water bath. This distillation circulation continued for 2 h, and the collected solvent extract was added to 200 µL of an internal standard solution of cyclohexyl acetate, and an internal standard was used to obtain the weight concentration of volatile compound in the sample; also, anhydrous sodium sulfate was used to remove the water. Lastly, the distillation column (40 • C, 1 h, 100 cm glass column) was used to volatilize the solvent and the condensed volatile component extracts were collected. 3.
GC analysis of the volatile compounds was conducted using a 7890A GC (Agilent Technologies, Palo Alto, CA, USA) equipped with a DB-1 (60 m × 0 .25 mm i.d. × 0.25 µm film thickness, Agilent Technologies) capillary column and a flame ionization detector. The injector and detector temperatures were maintained at 250 • C and 300 • C, respectively. The oven temperature was held at 40 • C for 1 min and then raised to 150 • C at 5 • C/min and held for 1 min, and then increased from 150 to 200 • C at 10 • C/min and held for 11 min. The carrier gas (nitrogen) flow rate was 1 mL/min. The Kovats indices were calculated for the separated components relative to a C 5 -C 25 n-alkanes mixture [21]. The purpose gas chromatography-flame ionization detector (GC-FID) was used both for retention indices (RI) comparison and quantitation of peak areas. 4.
GC-MS analysis of volatile compounds were identified using an Agilent 7890B GC equipped with DB-1 (60 m × 0.25 mm i.d. × 0.25 µm film thickness) fused silica capillary column coupled to an Agilent model 5977 N MSD mass spectrometer (MS). The GC conditions in the GC-MS analysis were the same as in the GC analysis. The carrier gas (helium) flow rate was 1 mL/min. The electron energy was 70 eV at 230 • C. The constituents were identified by matching their spectra with those recorded in an MS library (Wiley 7N, John Wiley & Sons, Inc. New Jersey, NJ, USA). In addition, the constituents were confirmed by comparing the Kovats indices or GC retention time data with those of authentic standards or data published in the literature. The GC and GC-MS methods used were modified from those of Yeh et al. [20]. 5.
Statistical Analysis: Each sample was extracted in triplicate and the concentration of volatile compounds was determined as the mean value of three repetitions. The data were subjected to a monofactorial variance analysis with Duncan's multiple range method with a level of significance of p < 0.05 (SPSS Base 12.0, SPSS Inc., Chicago, IL, USA).

HS-SPME Extraction Time
The optimal SPME fiber (50/30-μm DVB/CAR/PDMS) was used to extract Nongshi No. 1 at 25 ± 2 °C, and the extraction times from 10 to 50 min were investigated. The total peak area increased from 10-40 min and reached the peak at 40 min ( Figure 2). Silva and Câmara [23] promoted the higher extraction efficiency, corresponding to the higher GC peak areas and the number of identified metabolites. This higher extraction efficiency was achieved using: DVB/CAR/PDMS coating fiber, and 40 °C and 60 min as the extraction temperature and extraction time, respectively. Zhang et al. [24] also obtained optimum extraction conditions, which were using 50/30-μm DVB/CAR/PDMS fiber for 40 min at 90 °C. According to the obtained results, 40 min was selected as the optimal extraction time.
Total peak areas β-pinene Ducki et al. [22] evaluated four different types of SPME fibers (100-µm PDMS, 65-µm PDMS/DVB, 75-µm CAR/PDMS, and 50/30-µm DVB/CAR/PDMS) for the headspace analysis of volatile compounds in cocoa products. The SPME fiber coated with 50/30-µm DVB/CAR/PDMS afforded the highest extraction efficiency. Silva et al. [23] compared the performance of six fibers (PDMS, PDMS/DVB, CW/DVB, PA, CAR/PDMS, and DVB/CAR/PDMS) and found that DVB/CAR/PDMS was the most effective SPME fiber for isolating the volatile metabolites from Mentha × piperita L. fresh leaves based on the total peak areas, reproducibility, and number of extracted metabolites. Yeh et al. [20] reported the volatile components in Phalaenopsis Nobby's Pacific Sunset, and the optimal extraction conditions were obtained using a DVB/CAR/PDMS fiber.
The 50/30-µm DVB/CAR/PDMS was revealed to be the most suitable and was subsequently used in all further experiments.

HS-SPME Extraction Time
The optimal SPME fiber (50/30-µm DVB/CAR/PDMS) was used to extract Nongshi No. 1 at 25 ± 2 • C, and the extraction times from 10 to 50 min were investigated. The total peak area increased from 10-40 min and reached the peak at 40 min ( Figure 2). Silva and Câmara [23] promoted the higher extraction efficiency, corresponding to the higher GC peak areas and the number of identified metabolites. This higher extraction efficiency was achieved using: DVB/CAR/PDMS coating fiber, and 40 • C and 60 min as the extraction temperature and extraction time, respectively. Zhang et al. [24] also obtained optimum extraction conditions, which were using 50/30-µm DVB/CAR/PDMS fiber for 40 min at 90 • C. According to the obtained results, 40 min was selected as the optimal extraction time.

HS-SPME Extraction Time
The optimal SPME fiber (50/30-μm DVB/CAR/PDMS) was used to extract Nongshi No. 1 at 25 ± 2 °C, and the extraction times from 10 to 50 min were investigated. The total peak area increased from 10-40 min and reached the peak at 40 min ( Figure 2). Silva and Câmara [23] promoted the higher extraction efficiency, corresponding to the higher GC peak areas and the number of identified metabolites. This higher extraction efficiency was achieved using: DVB/CAR/PDMS coating fiber, and 40 °C and 60 min as the extraction temperature and extraction time, respectively. Zhang et al. [24] also obtained optimum extraction conditions, which were using 50/30-μm DVB/CAR/PDMS fiber for 40 min at 90 °C. According to the obtained results, 40 min was selected as the optimal extraction time.

Comparisons of the Differences between HS-SPME and SDE
Similar to Table 2, Table 4 shows the eight different Hsian-tsao varieties, along with the 120 components identified using HS-SPME and SDE, of which, 44 were found using both extraction methods, 12 (mainly α-terpinene, δ-3-carene, and cis-α-bergamotene) were identified using HS-SPME but not detected using SDE, and 64 (mainly nonanal, 6-methyl-3,5-heptadien-2-one, and gossonorol) were identified using SDE but not detected using HS-SPME. Table 5 and Figure 4 show that the monoterpene relative content was higher than that of sesquiterpene. Table 6 and Figure 5 show that the SDE samples had a high content of sesquiterpenes, terpene oxide, and terpene alcohols, but a lower content of monoterpenes than the SPME samples. Tersanisni and Berry [33] reported that certain hydrocarbon compounds, such as linalool and α-terpineol, as well as their hydrocarbon interactions, can be interrupted by heat stress, resulting in the induction of volatilization. We detected α-terpineol using SDE but by using HS-SPME. However, both methods identified terpene hydrocarbons as the major components. HS-SPME extracted more terpene hydrocarbons, and the majority was highly volatile monoterpenes with a low molecular weight. SDE extracted mainly sesquiterpenes with higher molecular weights. SDE also identified components that HS-SPME was unable to identify, such as straight-chain acids, aromatic ketones, aromatic esters, terpene aldehydes, terpene ketones, methoxy phenols, and nitrogen-containing compounds. Montserrat et al. [34] analyzed the volatile composition of white salsify (Tragopogon porrifolius L.) and found that SDE used high temperature and a long extraction time, and large quantities of volatile components were lost during the extraction process. Therefore, the SDE method may increase the low volatile compounds with a high molecular weight, such as sesquiterpenes and straight-chain acids. HS-SPME used shorter extraction times, so it was able to extract highly volatile monoterpenes with lower molecular weights. As such, HS-SPME is more appropriate for quality control. This study found that although HS-SPME was more rapid and SDE had a higher temperature and longer extraction time, SDE was able to extract more Hsian-tsao compounds; therefore, both methods can be used to complement each other. Yang et al. [35] compared HS-SPME with traditional methods in the analysis of Melia azedarach and reported that the HS-SPME method is a powerful analytic tool and is complementary to traditional methods for the determination of the volatile compounds in herbs. Comparing both techniques, HS-SPME samples were smaller (1 g) and did not require heating, the data was accurate, and involved less chemical reactions and changes, but the yield of larger molecules were lower, and the identified components were fewer, while SDE needed the use of 100 g of plant material and heating (2 h). The popularity of this method comes from the fact that volatiles with medium to high boiling points are recovered well. The aroma profile can be greatly altered via the formation of artifacts due to heating the sample during isolation. However, Hsian-tsao food needs to be processed using heat; therefore, by combining the HS-SPME and SDE methods of volatile compounds isolation, each isolation technique provides a part of the overall Hsian-tsao profile. Table 5. Percentages of extracted chemical groups of Hsian-tsao analyzed using HS-SPME. All the definitions of the symbols used in Table 2 mean values were also used in Table 5. Notes: All the definitions of the symbols used in Table 3 mean values were also used in Table 4. Notes: All the definitions of the symbols used in Table 3 mean values were also used in Table 4.

Conclusions
This study determined the volatile components present in eight varieties of Hsian-tsao using HS-SPME and SDE methods. A total of 120 volatile components were identified, of which, 56 were verified using HS-SPME and 108 using SDE. HS-SPME extracted more monoterpenes; however, SDE extracted more sesquiterpenes and terpene alcohols, and a terpene oxide, such as β-caryophyllene, α-bisabolol, and caryophyllene oxide. SDE was able to detect more components, but HS-SPME analysis was more convenient. In the future, the two extraction methods can be used in a complementary manner for Hsian-tsao analysis and research.

Conclusions
This study determined the volatile components present in eight varieties of Hsian-tsao using HS-SPME and SDE methods. A total of 120 volatile components were identified, of which, 56 were