Analysis of Volatile Compounds from Different Parts of Houttuynia cordata Thunb.

Houttuynia cordata Thunb. is a medicinal and edible plant that has been commonly used in traditional Chinese medicine since ancient times. This study used headspace solid-phase microextraction (HS-SPME) and direct injection, combined with gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS), to identify the volatile compounds in H. cordata. Extraction from different parts of the plant using different extraction techniques for the identification of volatile compounds were determined. A total of 93 volatile components were analyzed in the leaves, stems, rhizomes, and whole plant samples of H. cordata. The leaves contained more (Z)-3-hexenal, β-myrcene, (Z)-β-ocimene, and (4E,6E)-allo-ocimene; the stems contained more geranyl acetate and nerolidol; and rhizomes contained more α-pinene, β-pinene, limonene, 2-undecanone, and decanoyl acetaldehyde. Among them, the essential oil extracted by HS-SPME could produce more monoterpenes, while direct injection could obtain higher contents of aliphatic ketones, terpene esters, sesquiterpenes, and was more conducive to the extraction of 2-undecanone and decanoyl acetaldehyde.


Introduction
Houttuynia cordata Thunb., belonging to Saururaceae, is a medicinal and edible perennial herb native to China, Japan, and Taiwan [1]. H. cordata is rich in nutrients and contains a variety of vitamins, amino acids, and trace elements, such as zinc, potassium, and copper [2]. The physiologically active substances of H. cordata include essential oils, steroids, and flavonoids [3], which have many pharmacological properties, including antibacterial, antiviral, anti-inflammatory, antioxidant, and anticancer effects. Many studies have been conducted on its active components and pharmacological properties [4][5][6][7][8].
Essential oils, secondary metabolites of plants, are industrially important natural products [9]. Essential oils were utilized in pharmaceutical and other related medical, and the amount of published evidence on aromatherapy and essential oils has gradually increased [10,11]. The essential oil components of H. cordata include decanoyl acetaldehyde, 2-undecanone, β-myrcene, decanal, and trans-caryophyllene [1]. Because the essential oil compounds of H. cordata will affect its pharmacological effects, analyses of the volatile components are also used to determine plant quality [12].
To date, there have been many studies on the volatile components of H. cordata. Kosuge [13] used steam distillation to extract essential oils from H. cordata and isolated

Analysis of Volatile Compounds from Different Parts of H. cordata
The leaves, stems, rhizomes, and whole plants of fresh H. cordata were analyzed for differences in volatile compounds (Figures 1-3). Fresh plants and essential oils were analyzed using HS-SPME and direct injection. A total of 91 volatile components were identified in the H. cordata samples. The yield of essential oils was 0.09% (leaves), 0.02% (stems), 0.04% (rhizomes), and 0.04% (whole plants). Chen et al. [20] analyzed Angelica acutiloba essential oil and found that the highest content of essential oils was in the leaves. The main compounds from different parts were not the same, and the overall components and contents were different. In addition, the year-to-year yields of essential oil were slightly different, which may be due to differences in cultivation and climatic conditions [21].
2-undecanone, β-myrcene, decanal, and trans-caryophyllene [1]. Because the essential oil compounds of H. cordata will affect its pharmacological effects, analyses of the volatile components are also used to determine plant quality [12].
To date, there have been many studies on the volatile components of H. cordata. Kosuge [13] used steam distillation to extract essential oils from H. cordata and isolated decanoyl acetaldehyde, which has an antibacterial effect and is known to cause the unique stinking smell of H. cordata [14]. However, this component is easily oxidized into 2-undecanone during distillation and storage [15]. Both are important volatile components of H. cordata [14,16]. Yang et al. [17] analyzed 25 volatile compounds in H. cordata by GC-MS, including α-pinene, camphene, β-pinene, β-myrcene, (+)-limonene, γ-terpinene, decanal, linalool, β-caryophyllene, and 2-undecanone. Asakawa et al. [18] analyzed volatile compounds in different parts of the H. cordata plant. The study indicated that the main component of all parts analyzed was 4-tricancanone, and β-myrcene was the main monoterpene in the flowers, leaves, and stems, while the main monoterpene in the rhizomes and roots was β-pinene, and 1-decanal was the main polyketide in leaves and stems. Xu et al. [19] analyzed monoterpenes in the essential oils of three H. cordata accessions, and the results showed that the number and content of monoterpenes were different in different plant parts and different accessions.
In this study, extraction from different parts of the H. cordata plant, and different extraction methods on the volatile components. The results of this study can be used as a reference for the extraction and utilization of H. cordata in the future.
Asakawa et al. [18] analyzed the volatile components of different parts of H. cordata and showed that the main component of each part was 4-tricancanone. The main monoterpene in rhizomes and roots was β-pinene, while in flowers, leaves, and stems, it was β-myrcene. 1-decanal is the main polyketide compound in leaves and stems. Haghighi et al. [27] studied the effects of ecotypes and different plant parts (leaves, flowers, and fruits) on essential oil from Vitex pseudo-negundo. The results showed that there were significant differences in the yield and chemical characteristics of the essential oils in different plant parts. Zribi et al. [28] analyzed the volatile components and essential oils of Tunisian Borago officinalis L. and showed that the main components of different parts of this plant differed; octanal was the main component in the flowers, while in leaves, it was nonanal.

Comparison of Different Extractions
Comparing HS-SPME of fresh plants, HS-SPME of essential oil, and direct injection of essential oil, HS-SPME from fresh plants produced the smallest number of volatile compounds, followed by HS-SPME of essential oil, while direct injection of essential oil produced the most. Analysis of fresh plants by HS-SPME identified aliphatic alcohols and aliphatic aldehydes of low molecular weight, while analysis of essential oils by HS-SPME identified the highest content of monoterpenes. Direct injection of essential oils could identify more aliphatic ketones, sesquiterpenes, and terpene esters ( Table 2).
Farag and Wessjohann [29] compared the volatile compound profiles of Glycyrrhiza glabra L. roots extracted by SPME and steam distillation. The results showed that SPME could easily extract several small molecular weight monoterpenes, while more compounds could be identified in the essential oils extracted by steam distillation, including the volatiles generated by chemical reactions during the heating process. Peng et al. [30] analyzed the volatile components of kumquat (Fortunella margarita Swingle) and showed that HS-SPME/GC could identify a higher proportion of monoterpenes but fewer sesquiterpenes than that by DI/GC. Gao et al. [31] compared different extractions to volatile components of Pu-erh ripe tea and observed that HS-SPME was beneficial for the extraction of highly volatile compounds, such as low molecular weight alcohols, aldehydes, ketones, and hydrocarbons. Yang et al. [32] compared HS-SPME with conventional extraction in the analysis of Melia azedarach, and Kung et al. [33] analyzed Platostoma palustre (Blume) and pointed out that HS-SPME is a powerful analytical tool that can complement traditional methods. Overall, the results of these three methods showed all have high monoterpene content. Direct injection can be used to analyze more classes of volatile compounds, especially the larger molecular weight components, including important components of H. cordata such as 2-undecanone, and decanoyl acetaldehyde.
H. cordata has long been used as an edible vegetable and in traditional medicine [2]. Due to its pharmacological properties, it has been gradually applied in many fields, such as medicine, health food, preservatives and cosmetics, with great potential for development. After the extraction was complete, the fiber was inserted into the inlet of the GC or GC-MS and desorbed for 20 min. This experiment was repeated in triplicate.

Extraction of Essential Oil from H. cordata
A fresh H. cordata sample (600 g) was washed with clean water, and then 1800 mL of distilled water was added to homogenize (TATUNG, TJC-2200) for 30 s. The homogenate was then placed in a 5 L round-bottom flask for steam distillation. The extraction time was 3 h, and the extract was stored at 4 • C until analysis. The experiment was repeated in triplicates. The yields of essential oils from different parts of H. cordata were 0.09% (leaves), 0.02% (stems), 0.04% (rhizomes), and 0.04% (whole plants).

Gas Chromatography-Flame Ionization Detector (GC-FID)
GC was performed with an Agilent Model 7890A GC (Santa Clara, CA, USA), with a 60 m × 0.25 mm id Agilent DB-1 fused-silica capillary non-polar column with a film thickness of 0.25 µm; the HS-SPME injection mode was splitless, and the injection mode of direct injection was split. The GC heating conditions were as follows: the initial temperature was maintained at 40 • C for 1 min, then raised to 150 • C at 5 • C/min, maintained for 1 min, then raised to 200 • C at 10 • C/min and maintained for 11 min. The inlet temperature was 250 • C, the detector temperature was 300 • C, and a flame ionization detector (FID) was used for detection. The carrier gas was nitrogen at a flow rate of 1 mL/min.

Gas Chromatography-Mass Spectrometry (GC-MS)
GC-MS was conducted with an Agilent Model 5977A quadrupole mass spectrometer (Mass Selective Detector, MSD) coupled to an Agilent Model 7890B GC (Palo Alto, CA, USA). The operating conditions and column were the same as in Section 3.2.3. The carrier gas was helium, the ion source temperature of the MSD was 230 • C, and the electron energy was 70 eV. The transfer line was set at 250 • C. The mass range was 30-350 m/z. The quadrupole temperature was 150 • C. The mass spectra data were compared and judged using the Wiley 7N mass spectrum library.

Analysis of Essential Oil from H. cordata
For GC, 1 µL of essential oil was injected, while 0.5 µL was injected for GC-MS. For HS-SPME, 0.1 mL of the essential oil was added to a 4 mL cylindrical glass bottle (Supelco Co., No. 27136) with a Teflon rubber pad. Additionally, 50/30 µm DVB/CAR/PDMS fiber was used for extraction for 5 min, and the extraction temperature was room temperature. The fiber was then inserted into the inlet of the GC or GC-MS. All experiments were performed in triplicates.

Retention Index (RI) Comparison
The GC retention index of the volatile components in this experiment was based on a mixture of C 5 -C 25 n-alkane standards (Sigma-Aldrich, St. Louis, MO, USA) and the GC retention time was used as a reference under the same conditions. The RI was calculated according to the method described by Curvers et al. in reference [34].

Relative Percentage Calculation
After volatile components were identified, the percentage composition was calculated using the peak area normalization measurements. The formula is as follows: volatile component peak area total peak areas ×100% In addition to the volatile compounds of the sample, HS-SPME will also adsorb the impurity of the bottle or any silicon-containing coating. The total percentage in the above tables did not reach 100%, due to deducted from these impurities.

Conclusions
The volatile compounds of H. cordata leaves, stems, rhizomes, and whole plants were compared. The leaves of H. cordata had the highest essential oil content, but the rhizomes had higher 2-undecanone and decanoyl acetaldehyde content. Therefore, using rhizomes as raw materials is beneficial for extracting key components of H. cordata. Additionally, HS-SPME and direct injection of essential oil are highly complementary. Together, they cover the full range of volatilities and trace components and provide relatively complete data on the volatile components of H. cordata. Data Availability Statement: Data sharing not applicable. No new data were created or analyzed in this study. Data sharing is not applicable to this article.