Identification of key odorants in honeysuckle by headspace-solid phase microextraction and solvent-assisted flavour evaporation with gas chromatography-mass spectrometry and gas chromatograph-olfactometry in combination with chemometrics

Keran Su; Xin Zhang; Shao Quan Liu; LiHui Jia; Benjamin Lassabliere; Kim Huey Ee; Aileen Pua; Rui Min Vivian Goh; Jingcan Sun; Bin Yu; XiaoXue Hu

doi:10.1371/journal.pone.0237881

. 2020 Aug 20;15(8):e0237881. doi: 10.1371/journal.pone.0237881

Identification of key odorants in honeysuckle by headspace-solid phase microextraction and solvent-assisted flavour evaporation with gas chromatography-mass spectrometry and gas chromatograph-olfactometry in combination with chemometrics

Keran Su ¹, Xin Zhang ², Shao Quan Liu ^1,^*, LiHui Jia ², Benjamin Lassabliere ³, Kim Huey Ee ³, Aileen Pua ¹, Rui Min Vivian Goh ¹, Jingcan Sun ³, Bin Yu ^3,^*, XiaoXue Hu ^4,^*

Editor: Tommaso Lomonaco⁵

PMCID: PMC7440650 PMID: 32817641

Abstract

At present, the identification of honeysuckle aroma depends on experienced tasters, which results in inconsistencies due to human error. The key odorants have the potential to distinguish the different species and evaluate the quality of honeysuckle. Hence, in this study, a more scientific approach was applied to distinguish various honeysuckles. The volatile compounds of different species and parts of honeysuckle were separately extracted by headspace-solid phase microextraction (HS-SPME) and solvent assisted flavor evaporation (SAFE). Compounds with greater volatility such as aldehydes, limonene, γ-terpinene, and terpinolene were preferentially extracted by HS-SPME. As a complementary extraction method to HS-SPME, SAFE was found to recover comparatively more polar compounds such as eugenol, decanoic acid, and vanillin. Subsequently, key odorants with the highest flavour dilution (FD) factors were detected by aroma extract dilution analysis (AEDA). These were benzaldehyde, 4-ethylphenol, decanoic acid, vanillin, 3-methyl-2-butenal, and β-ionone in honeysuckle flowers and γ-octalactone, 4-ethyl phenol, and vanillin in honeysuckle stem. Finally, principal component analysis (PCA) was conducted to analyze not only the key odorants of species and parts of honeysuckle but also their different origins. The results of PCA suggested that the species of honeysuckle contributed much more to variations in aroma rather than their origins. In conclusion, the application of the key odorants combined with PCA was demonstrated as a valid approach to differentiate species, origins, and parts of honeysuckle.

Introduction

Honeysuckle is a kind of herbal plant with many species and widely used and grown in northern hemisphere countries [1]. It has been consumed as traditional medicine and tea beverage for thousands of years [2]. In addition to the treatment of pneumonia and respiratory infections, and the improvement of consumers’ health, honeysuckle possesses anti-inflammatory, antioxidant, and antiviral properties [3,4]. For this reason, it has been gradually added into foods and beverages to improve product value and to provide adjuvant therapy [5,6]. Recently, the aroma of herbal plants in food products has also increasingly gained popularity and consumer acceptance around the world [7]. Key aroma profiles helps to distinguish the different species and determines the quality of honeysuckle. At present, the quality of honeysuckle is evaluated by professional tasters that causes inconsistent and inaccurate results [8]. Therefore, a systematic study of the volatile compositions of different species, parts and origins of honeysuckle was conducted in this study.

The selection of an appropriate extraction method is a prerequisite to study volatile compounds in honeysuckle. The complex plant matrix requires the development of various sample preparation approaches to address the issue of sample complexity [9]. Some solvent extraction methods operate under high temperatures and use toxic organic solvents that are prone to thermal reactions and cause adverse effects to environment. In comparison, headspace-solid phase microextraction (HS-SPME) is a relatively low-temperature, solvent-free, and rapid approach [10]. For this reason, it is a popular method applied in the food matrix of natural materials [11,12]. HS-SPME is suitable for the isolation of highly volatile compounds. In comparison to HS-SPME, solvent-assisted flavour evaporation (SAFE) is able to extract less volatile and a greater range of polar compounds from complex matrices [13]. By applying high vacuum and low-temperature conditions, SAFE avoids aroma modification during the extraction of heat-liable aroma compounds from natural materials [14]. Besides, SAFE reduces the number of side reactions that may occur during aroma isolation, such as enzymatic degradation [15]. Hence, these two extraction methods are suitable for isolating volatile compounds from natural materials.

The potency of the aroma is often not proportionate to its peak area detected by chromatography-mass spectrometry (GC-MS) because many high abundance compounds may have a high odor threshold and thus do not serve as key odorants in the overall matrix [16]. Hence, it was difficult to distinguish between the aromas of different honeysuckle species using the volatile characteristics alone. As well known, aroma extract dilution analysis (AEDA) has been widely used to fill this gap by suggesting the contribution of each compound through the flavour dilution (FD)-factor [17]. The key odorants detected by AEDA may then be used to better describe the characteristic aroma of honeysuckle, and has the added potential to distinguish different species of honeysuckle.

Therefore, the objective of this study was to extract the volatile compounds in various honeysuckles using HS-SPME in conjunction with SAFE, both coupled to GC-MS/FID. Furthermore, their key odorants were identified using AEDA. Lastly, principal component analysis (PCA) was performed to show the differences in the aromatic profiles of honeysuckle samples from different species, origins, and parts of the plants.

Materials and methods

Sample preparation

Two main species of honeysuckle flowers are Lonicera japonica Flos (Jinyinhua) and Lonicera Flos (Shanyinhua) [18]. The stems of honeysuckle are also commonly used as medicine [6]. All of the flowers and stems of honeysuckle belong to the family of Caprifoliaceae [19] and contain similar chemical constituents [6], but they cannot be used interchangeably. With the help of Hubei Provincial Hospital of Traditional Chinese Medicine, six common honeysuckle samples with permit number 20160077 were purposefully selected for this study. These are flowers of honeysuckle: Lonicera japonica Flos (LJF, Pingyi County, Shandong province, China) and Lonicera Flos (LF, Longshan County, Hunan province, China), stems of Lonicera japonica: Lonicera japonica Caulis (LJC, Pingyi County, Shandong province, China), and stems of Lonicera: Lonicera Caulis (LC, Longshan County, Hunan province, China). In addition, LJF from Fengqiu County, Henan province, China and LF from Enshi County, Hubei province, China were also complemented for PCA to compare the differences between origins. All samples were packed in heat-seal vacuum aluminium foil bags for every 100 g portion sand stored in a 5°C refrigerator. The aluminium foil bag can isolate the external humidity, and all samples were used within one week after repacking.

HS-SPME procedure

The HS-SPME procedure was conducted as described in our previous publication [20]. Two different sample preparation methods, namely dry and brewed, were applied in this study. In the dry method, 0.200 g of LJF (Shandong or Henan), LF (Hunan or Hubei), LC, or LJC was weighed (d = 0.001 g and max 620 g, Mettler Toledo, America) and added directly in a glass headspace vial with a PTFE-coated silicone septum (Agilent, California, USA). The brewed honeysuckle was prepared by adding 20.000 g of honeysuckle in 200.000 g of 80°C Ultrapure water (Human Corporation Arioso Power Series, Seoul, South Korea). The optimal brewing conditions were determined to be 80°C and 30 min (results not shown here). After brewing, the mixture was filtered through a metal sieve and chilled in an ice bath for 10 min. The cooled filtrate (2.000 g) was then transferred into a headspace vial. A Supelco 85 μm Carboxen/Polydimethylsiloxane (CAR/PDMS) fibre (Pennsylvania, USA) was used to extract volatiles from headspace vial at designated extraction temperatures (40, 60, and 80°C) and extraction times (5, 30, and 60 min). Finally, the fibre was inserted in the GC injector for the subsequent GC analysis.

SAFE procedure

Brewed honeysuckle was prepared similarly as described above but in this case 60.000 g of sample was added in 600.0 g of 80°C Ultrapure water was weighed by the balance (d = 0.1 g and max 8200). After brewing at 80°C for 30 min, the mixture was filtered through a metal sieve and chilled in an ice bath for 10 min. The chilled filtrate (300 mL) was then transferred into a 500 mL beaker. An internal standard solution (0.060 g) was added to the filtrate and stirred for 5 min. The internal standard solution was prepared as follows: 0.300 g butyl butyryl lactate (VWR, Pennyslvania, USA), which was found not to coelute with other volatile compounds in honeysuckle in both MS and FID chromatograms, was added in 250 mL volumetric flask, then dilute to volume with ethanol (VWR, Pennyslvania, USA). The formula for calculation of the concentration of each compound was shown in Eq (1). The extraction conditions of SAFE was adapted from our previous work, with minor modifications [21]. The extraction conditions of SAFE (Glasbläserei Bahr, Germany) were 5 × 10⁻⁶ mbar, the pressure was maintained by Edwards PFPE RV3 rotary vane pump (West Sussex, UK) and Edwards Diffstak diffusion pump (West Sussex, UK), and the water bath temperature was 40°C.

\frac{P e a k a r e a o f i n t e r n a l s t a n d a r d}{c o n c e n t r a t i o n o f i n t e r n a l s t a n d a r d} = \frac{P e a k a r e a o f e a c h c o m p o u n d}{c o n c e n t r a t i o n o f c o r r e s p o n d i n g c o m p o u n d}

(1)

GC-MS/FID analysis

Analytes quantitation and identification analysis were conducted by Agilent 7890B GC equipped with a flame ionisation detector (FID) and Agilent 5977B mass selective detector (MSD) (California, USA). CTC CombiPAL autosampler (Zwingen, Switzerland) was employed to insert samples into GC injector. GC column was Agilent HP-INNOWax column (60 m × 250 μm × 0.25 μm) (Woodbridge, USA). The GC conditions used were injector temperature 250°C; splitless mode; helium carrier gas; column flow rate 1.2 ml/min; FID temperature was 300°C; EI mode was 70 eV. The temperature gradient in the GC oven was 50°C for 5 minutes, increased to 240°C at rate of 5°C/min, and held at 240°C for 40 minutes. In-house and NIST 14 MS libraries (Connecticut, USA) were applied to identify the eluted compounds by matching the mass spectra, and further confirmed with the linear retention indices (LRI) of in-house standard compounds. LRI values of the HP-INNOWax column were calculated by a mixture of Supelco C7-40 alkanes (VWR, Pennsylvania, USA). Alkane standard and pure standards were run under the same conditions with the sample. All experiments were carried out in triplicate, and the results were reported as mean values of the peak area of each compound extracted by HS-SPME and mean values of concentration of each compound extracted by SAFE.

AEDA

For the AEDA analysis, the SAFE concentrates of LJF, LF and LJC were diluted stepwise with dichloromethane (VWR; Pennyslvania, USA) at 5ⁿ (n = 0, 1, 2, 3, and so on). Each dilution was sniffed and evaluated via GC-O by four experienced flavourists (2 male and 2 female; aged 26–50). Gas chromatograph-olfactometry (GC-O) conditions were referred from our previous study [22]. All GC-O panellists had experience in conducting GC-O analyses before this study. The odor of each compound was described by these panellists. The strength of each compound was represented by their flavour dilution factor (FD-factor), which was the highest dilution at which nothing could be smelled at the GC-O port. Compounds corresponding to each odor were identified by injection of pure standards under the same GC-O condition.,

Data processing and statistical analysis

Student’s t-test was performed to test the significance level of the difference between samples at two different conditions. Data of key aroma compounds obtained from AEDA was processed by PCA in the R programming language. The data was processed and transformed with data.table and magrittr packages and visualized them with ggplot2, ggbiplot, and patchwork packages.

Results and discussion

The selection of HS-SPME extraction conditions

It is critical to select the extraction conditions (temperature and time) for natural materials. That is because high temperature and long-time extraction could destroy their volatile compounds, while lower temperatures and shorter extraction times could lead to poor sensitivity and recovery [23,24]. Therefore, different extraction temperatures (40, 60, and 80°C) and extraction times (5, 30, and 60 min) were applied to study the effects of extraction conditions on the release of volatile compounds from honeysuckle (both dry and brewed).

As reported in the literature [25–28], hexanal, cis-3-hexenol, acetic acid, benzyl alcohol, and β-ionone were important compounds in honeysuckle. Hence, they were chosen as indicators for the effect of extraction temperatures on both dry and brewed LJF. In Fig 1(A), the peak area of hexanal, acetic acid, and β-ionone increased with the increase of temperature but decreased after 60°C. Other aldehydes (e.g. 2-butenal, heptanal, trans-2-hexenal, and benzaldehyde) exhibited a similar trend. For cis-3-hexenol and benzyl alcohol, an increase in temperature resulted in the higher peak areas, which was observed for other alcohols such as geraniol, octanol, and 2-phenylethyl alcohol. Hence, alcohols were easier to be detected at the higher temperatures in dry honeysuckle.

The peak areas of the selected compounds after brewing are shown in Fig 1(B). Hexanal and other aldehydes such as 2-butenal, heptanal, trans-2-hexenal, and benzaldehyde showed an inverse trend compared to dry LJF. Hence, aldehydes were a little bit harder to be detected at a higher temperature in brewed honeysuckle. When the temperature was gradually increased from 40°C to 80°C, the peak areas of the most alcohol compounds showed high significant increase, except cis-3-hexenol, which reached the greatest peak area at 60°C. cis-3-Hexenol appeared to stop rising at 60°C, accompanied by increments in geraniol and β-ionone in samples after brewing, which is similar to the previous study [29], which may be the result of thermally-related reactions. The peak areas of acetic acid and β-ionone had the same trends as the peak areas increase when the extraction temperature was above 60°C. Compared to dry honeysuckle extraction, acetic acid was more difficult to be isolated in brewed honeysuckle.

Fig 2 shows the peak areas of major volatile compounds in dry and brewed LJF at various extraction times at 80°C. In Fig 2(A), hexanal, cis-3-hexenol, and acetic acid were found to have the highest peak areas at 30 min, while benzyl alcohol and β-ionone recorded their respective highest peak areas at 60 min in dry LJF. For brewed LJF (Fig 2(B)), an extraction time of 30 min resulted in the greatest peak areas for cis-3-hexenol, acetic acid, and β-ionone. The peak area of benzyl alcohol was the highest with a 60 min extraction time, similar to dry LJF. Hexanal exhibited a unique trend. When the extraction time increased from 5 min to 30 min, the peak area of hexanal significantly decreased (p<0.001), and as the extraction time increased from 30 min to 60 min, the peak area of hexanal significantly increased (p<0.001). This phenomenon may be due to the hydration of hexanal and it could be fully extracted at low temperature [21].

The trends of each compound in dry LJC and LJC after brewing were similar to dry LJF and LJF after brewing. Hence, the results of the LJC were not shown in this study. Most of the volatile compounds in dry and brewed honeysuckle generally increased with the increase of temperature and had the greatest peak area at 30 min. Therefore, 80°C and 30 min were chosen as the extraction conditions both in dry and brewed samples for further analysis.

Volatile profiles of different species and parts of honeysuckle by HS-SPME

Following the selection of HS-SPME extraction conditions, dry and brewed LJF, LF, LJC, and LC were analysed by GC-MS/FID under HS-SPME conditions of 80°C and 30 min. One hundred and two compounds were tentatively identified using HS-SPME-GC-MS and shown in Fig 3. Higher volatile compounds were preferentially found, such as some aldehydes, limonene, γ-terpinene and terpinolene. The results are shown in S1 Table. The most abundant compounds in dry LJF were 2-butenal, hexanal, hexanol, cis-3-hexenol, acetic acid, benzaldehyde, octanol, benzyl alcohol, and 2-phenylethyl alcohol, etc., and these compounds have also been reported by Wang et al. [30]. The main compounds in brewed LJF were almost the same as those in dry LJF. However, the abundances of the same compounds were different in brewed and dry LJF. For example, the peak areas of aldehydes and acetic acid in brewed LJF were lower while alcohol compounds were greater when compared to dry LJF. The abundance differences were likely due to the different sample matrix. The changes of volatile compounds of LF were similar to LJF. Although hexadecenoic acid and octadecanoic acid were reported to be the quantitatively highest compounds in Lonicera Flos [31], they were not detected in our study. This might be due to the different extraction methods, such as microwave, hydro distillation, and ultrasound, it used. These methods cause thermal reactions or decomposition during extraction. Some terpenes (e.g. α-copaene, α-cubebene, γ-muurolene, and α-muurolene) were detected in dry LJC and LC but not in brewed stem and flower samples. Until now, there have been few studies on volatile compounds of honeysuckle, but they only focused on one species such as LJF or LF [25,30]. Few studies have been conducted to differentiate the volatile compounds in honeysuckle flower of two species, or in the different parts of a single species [6,32]. Hence, volatile compounds in various honeysuckles and different status may provide different aroma spectrum when they were added in food products.

Fig 3 — The numbers of each peak correspond to the compounds in S1 Table.

Volatile profiles of different species and parts of honeysuckle extracted by SAFE

LJF, LF, LJC, and LC were isolated at 40°C water bath by SAFE. S2 Table listed one hundred and eight compounds across all honeysuckle species, such as hexanal, 1-octen-3-ol, benzaldehyde, and eugenol, and these compounds were reported by literature [32]. SAFE was a complementary extraction method to HS-SPME due to its success in extracting higher polar compounds, higher molecular weight, and less volatile compounds such as eugenol, decanoic acid, and vanillin. SAFE is a modified distillation technique conducted under low-temperature and high-vacuum conditions, which aims to reduce the degradation of heat-sensitive volatiles and generation of artifacts as compared to other extraction methods like simultaneous distillation extraction (SDE) and hydro-distillation. When the extraction is performed using SDE, many linalool derivatives such as linalool oxides (furanoid and pyranoid) [25] as well as esters (bornyl actate, methyl hexadecanoate and methyl linolenate) [33] were detected. All these volatiles might be the result of thermal reactions during the SDE extraction process. Volatile compounds with lower boiling points such as pentanal, hexanal, trans-2-pentenal, and heptanal could not be detected by hydro-distillation [34] due to volatilisation during extraction. Therefore, the volatile compounds isolated by SAFE minimises the modification of volatiles in honeysuckle to obtain a more representative aroma extract.

Key odorants of honeysuckle

Thirty-six odorants were identified in LJF, LF, and LJC (Table 1). These odorants with the highest FD-factors had sweet, floral, fruity, burnt, phenolic, and vanilla-like odor qualities. Benzaldehyde (bitter almond, burnt sugar; FD-factor: 625), 4-ethyl phenol (phenolic, smoky; FD-factor: 625), decanoic acid (sour, citrus; FD-factor: 625) and vanillin (vanilla, sweet; FD-factor: 625) were detected at the highest FD-factors in LJF. In LF, 3-methyl-2-butenal (fruity, sweet; FD-factor: 625) and β-ionone (seaweed, floral; FD-factor: 625) were the most potent odorants by FD-factor. The highest FD-factors in LJC was 125, and these odorants were γ-octalactone (sweet, waxy; FD-factor: 125), 4-ethyl phenol (phenolic, smoky; FD-factor: 125) and vanillin (vanilla, sweet; FD-factor: 125).

Table 1. Key odorants (flavour dilution factor ≥ 1) with their respective flavour dilution factors in SAFE extracts of Lonicera japonica Flos, Lonicera Flos and Lonicera japonica Caulis.

Olfactometric detection was conducted by four flavourists.

No.	Compound	Expt. LRI^†	Ref. LRI^‡	Odor quality^§	Flavour dilution factor^∥			Identification^⊥
No.	Compound	Expt. LRI^†	Ref. LRI^‡	Odor quality^§	Lonicera japonica flos	Lonicera flos	Lonicera japonica Caulis	Identification^⊥
1	2-Butenal	1051	1047	floral	25	125	-	MS, LRI, O
2	trans-2-Pentenal	1137	1127	fruity, green	-	125	-	MS, LRI, O
3	Butanol	1141	1142	sweet, whiskey	-	-	25	MS, LRI, O
4	1-Penten-3-ol	1155	1159	horseradish, green	-	125	-	MS, LRI, O
5	3-Methyl-2-butenal	1202	1215	fruity, sweet	-	625	-	MS, LRI, O
6	trans-2-Hexenal	1224	1216	green	-	5	-	MS, LRI, O
7	3-Methyl pyridine	1292	1292	green, earthy	25	5	5	MS, LRI, O
8	trans-2-Heptenal	1330	1323	Vegetable, green	-	25	-	MS, LRI, O
9	cis-3-Hexenol	1380	1382	green, cut grass	5	5	-	MS, LRI, O
10	trans-2-Octenal	1433	1429	waxy, green	-	125	-	MS, LRI, O
11	Heptanol	1448	1453	herbal, green	5	5	-	MS, LRI, O
12	Furfural	1469	1461	sweet, woody	-	-	25	MS, LRI, O
13	trans,trans-2,4-Heptadienal	1501	1495	vegetable, green	-	5	-	MS, LRI, O
14	1-(2-Furanyl)-ethanone	1512	1499	sweet, caramel	-	25	-	MS, LRI, O
15	Benzaldehyde	1537	1520	bitter almond, burnt sugar	625	5	-	MS, LRI, O
16	Linalool	1537	1547	floral, green	-	25	-	MS, LRI, O
17	Octanol	1549	1557	waxy, green	5	-	5	MS, LRI, O
18	trans,cis-2,6-Nonadienal	1584	1584	green, cucumber	-	-	5	MS, LRI, O
19	γ-Butanolactone	1637	1632	caramel, sweet	5	-	5	MS, LRI, O
20	1-Nonanol	1646	1660	floral, rose	-	5	-	MS, LRI, O
21	α-Terpineol	1696	1697	woody, floral	25	25	5	MS, LRI, O
22	γ-Hexanolactone	1723	1694	sweet, herbal	125	125	25	MS, LRI, O
23	1-Phenethyl alcohol	1801	1801	fresh, sweet	5	-	5	MS, LRI, O
24	Hexanoic acid	1840	1846	sour, cheese, fatty	5	5	1	MS, LRI, O
25	Guaiacol	1861	1861	phenolic, smoky	5	-	25	MS, LRI, O
26	Benzyl alcohol	1879	1870	sweet, floral	5	25	-	MS, LRI, O
27	γ-Octalactone	1930	1910	sweet, waxy	-	-	125	MS, LRI, O
28	2-Phenethyl alcohol	1914	1906	honey, rose	5	25	5	MS, LRI, O
29	β-Ionone	1971	1971	seaweed, floral	125	625	25	MS, LRI, O
30	Phenol	2004	2000	phenolic, plastic, rubbery	25	-	5	MS, LRI, O
31	Pantolactone	2029	2029	candy	5	125	25	MS, LRI, O
32	γ-Nonalactone	2041	2024	coconut, creamy	-	-	5	MS, LRI, O
33	4-Ethyl phenol	2179	2187	phenolic, smoky	625	125	125	MS, LRI, O
34	2-Methoxy-4-vinyl phenol	2197	2212	woody	5	25	25	MS, LRI, O
35	Decanoic acid	2262	2276	sour, citrus	625	-	-	MS, LRI, O
36	Vanillin	2578	2568	vanilla, sweet	625	125	125	MS, LRI, O

Open in a new tab

'-' Means compounds were not detected.

^†Expt. LRI: linear retention index on an HP-Innowax column relative to C7-C40 alkane standards.

^‡Ref. LRI: Reference retention index values from literature: NIST 14 MS library.

^§Odor quality of compounds described by flavourists.

^∥Flavour dilution-factor refers to the highest dilution at which the compound can be detected by at least three flavourists.

^⊥Identification methods: MS = Comparison with mass spectrum of the compound in the NIST library version 2.2; LRI = Comparison of retention index with that of the compound in the NIST library version 2.2; O = Comparison of the retention time and odor quality of the eluted compound with standard.

Some odorants were found to be unique to a particular honeysuckle species. For example, decanoic acid (sour, citrus; FD-factor: 625) showed the highest FD-factors in LJF, but it was not detected in LJC and LF. 1-Penten-3-ol (horseradish, green; FD-factor: 125), 3-methyl-2-butenal (fruity, sweet; FD-factor: 625), trans-2-octenal (waxy, green; FD-factor: 125) had high FD-factors in LF, but were not found to be key odorants in LJF and LJC. On the other hand, γ-octalactone (sweet, waxy; FD-factor: 125) had the highest FD-factor in LJC but it was not detected in LJF and LF. Besides that, trans-2-hexenal (green; FD-factor: 5), trans-2-heptenal (vegetable, green; FD-factor: 25), trans, trans-2,4-heptadienal (vegetable, green; FD-factor: 5), 1-(2-furanyl)-ethanone (sweet, caramel; FD-factor: 25), linalool (floral, green; FD-factor: 25) and 1-nonanol (floral, rose; FD-factor: 5) were only detected in LF, while butanol (sweet, whiskey; FD-factor: 25), furfural (sweet, woody; FD-factor: 25) and trans, trans-2,6-nonadienal (green, cucumber; FD-factor: 5) and γ-nonalactone (coconut, creamy; FD-factor: 5) were unique in LJC. In particular, pantolactone, which has a candy-like odor, could be confidently identified by GC-O with FD-factors of 5, 125, and 25 in LJF, LF, and LJC respectively, despite appearing as a trace compound in the GC-MS/FID analysis. The results suggested that the key odorants among LJF, LF, and LJC were quite different.

Principal component analysis

Volatile studies often result in large, complicated datasets [35]. PCA is often used as a statistical tool to improve the efficiency of data analysis. In this study, PCA was conducted to pinpoint the main areas of variation among different species, parts and origins of honeysuckle. PCA could also suggest specific volatile features that contributed most significantly to the differences observed between honeysuckle samples. In the present study, the key odorants that were identified by GC-O and contributed to the aroma of honeysuckle were selected for comparison by PCA instead of the total volatile composition by GC-MS/FID. This allowed for a more streamlined analysis of various honeysuckles by eliminating volatile compounds that were not confirmed to be significant aroma contributors in honeysuckle. The data collected from dry and brewed LJF (Shandong and Henan province, China), LF (Hunan and Hubei province, China), LJC, and LC extracted by HS-SPME and SAFE are shown in Fig 4.

Fig 4(A) shows the biplot of key odorants in various honeysuckle extracted by HS-SPME-GC/MS without brewing. In the plot, PC 1 and 2 accounted for 45.6% and 27.0% of variations, respectively. Heptanol (11), 1-nonanol (20), and furfural (12) contributed greatly to the separation of LF from other species and parts of honeysuckle. Both LF samples extracted by HS-SPME contained more abundant amounts of these compounds regardless of their origins, although 1-nonanol (20) had a relatively greater peak area in LF Hunan. Similarly, γ-nonanolactone (32), 2-methoxy-4-vinyl phenol (34), and vanillin (36) were found to determine the separation of LJC on the positive area of PC 1 and negative area of PC 2 in the plot. In Fig 4(A), the plot shows that the two LJF samples were clustered together, despite having different origins. A similar observation was seen in the two LF samples. Hence, the origin likely had a smaller impact on the differences in volatile compounds than the species. On the other hand, LJC and LC were separated from each other in the plot.

The comparison of key odorants in honeysuckle after brewing extracted by HS-SPME is shown in Fig 4(B). PC 1 and 2 accounted for 33.6% and 26.3% of the variations, respectively. In the plot, compounds, such as α-terpineol (21), furfural (12), 1-penten-3-ol (4) and octanol (17), determined the separation of LF from other species and parts of honeysuckle. α-Terpineol (21), furfural (12) and 1-penten-3-ol (4) were only detected in LF. Octanal (17) had a greater peak area in LF compared to other species. Similarly, hexanoic acid (24), γ-nonanolactone (32) and 2-methoxy-4-vinyl phenol (34) determined the plot on the positive area of PC 1 and PC 2. Hexanoic acid (24) shown a greater peak area in LJC. Similarly, to the dry samples, the separation was observed for different species, but not for different origins.

The results of key odorants of honeysuckle extracted by SAFE are shown in Fig 4(C). PC 1 was 34.5%, and PC 2 was 29.1%. In the plot, 1-(2-furanyl)-ethenone (14), linalool (16), 1-nonanol (20), and 3-methyl-2-butenal (5) determined the separation of LF on the positive area of PC 1 and the negative area of PC 2. 3-Methyl-2-butenal (5), 1-(2-furanyl)-ethenone (14), linalool (16), and 1-nonanol (20) were only found in LF. Compounds, such as trans, cis-2,6-Nonadienal (18),) γ-nonalactone (32), 4-ethyl phenol (33), 2-methoxy-4-vinyl phenol (34) and vanillin (36), were found to determine LJC on the negative area of PC 1 and PC 2. trans, cis-2,6-Nonadienal (18) was only found in LJC, while the other four compounds had greater peak areas in LJC. Again, clustering was observed for LJF and LF of different origins. γ-Nonalactone (32) and 2-methoxy-4-vinyl phenol (34) determined the separation of LJC, and 1-nonanol (20) and furfural (12) determined the separation of LF from the other various honeysuckle extracted by HS-SPME and SAFE.

Conclusion

In this research, it was found that HS-SPME was able to extract volatile aldehydes more efficiently than SAFE, with larger peak areas obtained in the dry flowers of honeysuckle than in the brewed samples. With the help of SAFE, volatile compounds were minimally modified, and the compounds of lower volatility (e.g., eugenol, decanoic acid, and vanillin) were significantly enhanced. Based on the AEDA results, 36 key odorants were identified across LJF, LF and LJC SAFE extracts. Compounds with the highest FD-factors were 4-ethyl phenol, decanoic acid, 3-methyl-2-butenal, β-ionone, γ-octalactone, and vanillin, and the concentrations of key odorants among different honeysuckle species and parts were noted to be quite varied. PCA classified various honeysuckle extracted by HS-SPME and SAFE by using key odorants, and clustering was observed for flowers of the same species regardless of origins. The results concluded that the key odorants may potentially be used to discriminate honeysuckle by species. Therefore, this systematic analytical approach is important for determining the volatile compounds of various honeysuckles and would definitely provide the basis for extending the application of honeysuckle in food products.

Supporting information

S1 Table. Identification of volatile compounds in dry and brewed Lonicera japonica Flos, Lonicera japonica Caulis, Lonicera Flos, and Lonicera Caulis extracted using HS-SPME (80°C for 30 min).

(DOCX)

Click here for additional data file.^{(56.8KB, docx)}

S2 Table. Identification of volatile compounds and concentration (ng/mL) in Lonicera japonica Flos, Lonicera Flos, Lonicera japonica Caulis, and Lonicera Caulis extracted using SAFE.

(DOCX)

Click here for additional data file.^{(37.9KB, docx)}

Acknowledgments

The authors were grateful to Mane SEA Pte Ltd for providing technical assistance during the study. The authors are thankful to Martin Pelerentegui, Jenny Suwardi, Leong Kwong Chee and Carine Orecchioni for their effort and contribution to this project.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

Mane SEA Pte Ltd provided support in the form of salaries for BL, KHE, JS, and BY and research materials. The specific roles of each authors are articulated in the ‘author contributions’ section.

References

1.Shang X, Pan H, Li M, Miao X, Ding H. Lonicera japonica Thunb.: Ethnopharmacology, phytochemistry and pharmacology of an important traditional Chinese medicine. J Ethnopharmacol. 2011;138: 1–21. 10.1016/j.jep.2011.08.016 [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Brown L, Poudyal H, Panchal SK. Functional foods as potential therapeutic options for metabolic syndrome: Foods as the treatment of obesity. Obes Rev. 2015;16: 914–941. 10.1111/obr.12313 [DOI] [PubMed] [Google Scholar]
3.Yao X-H, Xu J-Y, Hao J-Y, Wan Y, Chen T, Zhang D-Y, et al. Microwave assisted extraction for the determination of chlorogenic acid in Flos Lonicerae by direct analysis in real time mass spectrometry (DART-MS). J Chromatogr B. 2018;1092: 82–87. 10.1016/j.jchromb.2018.05.045 [DOI] [PubMed] [Google Scholar]
4.Yang J, Farmer LM, Agyekum AAA, Elbaz-Younes I, Hirschi KD. Detection of an Abundant Plant-Based Small RNA in Healthy Consumers. Li M, editor. PLOS ONE. 2015;10: e0137516 10.1371/journal.pone.0137516 [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Hamilton AC. Medicinal plants, conservation and livelihoods. Biodivers Conserv. 2004;13: 1477–1517. 10.1023/B:BIOC.0000021333.23413.42 [DOI] [Google Scholar]
6.Ye J, Su J, Chen K, Liu H, Yang X, He Y, et al. Comparative investigation on chemical constituents of flower bud, stem and leaf of Lonicera japonica Thunb. by HPLC-DAD-ESI-MS/MS n and GC-MS. J Anal Chem. 2014;69: 777–784. 10.1134/S1061934814080036 [DOI] [Google Scholar]
7.Stephany M, Kapusi K, Bader-Mittermaier S, Schweiggert-Weisz U, Carle R. Odour-active volatiles in lupin kernel fibre preparations (Lupinus angustifolius L.): effects of thermal lipoxygenase inactivation. Eur Food Res Technol. 2016;242: 995–1004. 10.1007/s00217-015-2605-9 [DOI] [Google Scholar]
8.Lv S, Wu Y, Zhou J, Lian M, Li C, Xu Y, et al. The Study of Fingerprint Characteristics of Dayi Pu-Erh Tea Using a Fully Automatic HS-SPME/GC–MS and Combined Chemometrics Method. Amancio S, editor. PLOS ONE. 2014;9: e116428 10.1371/journal.pone.0116428 [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Xue J, Li H, Liu F, Jiang W, Hou F. Vortex-assisted matrix solid–liquid dispersive microextraction for the analysis of triazole fungicides in cotton seed and honeysuckle by gas chromatography. Food Chem. 2016;196: 867–876. 10.1016/j.foodchem.2015.09.093 [DOI] [PubMed] [Google Scholar]
10.Pawliszyn J. Handbook of solid phase microextraction. 2012. Available: http://www.sciencedirect.com/science/book/9780124160170
11.Belliardo F, Bicchi C, Cordero C, Liberto E, Rubiolo P, Sgorbini B. Headspace-solid-phase microextraction in the analysis of the volatile fraction of aromatic and medicinal plants. J Chromatogr Sci. 2006;44: 416–429. 10.1093/chromsci/44.7.416 [DOI] [PubMed] [Google Scholar]
12.Yang Z, Mao H, Long C, Sun C, Guo Z. Rapid determination of volatile composition from Polygala furcata Royle by MAE–HS-SPME followed by GC–MS. Eur Food Res Technol. 2010;230: 779–784. 10.1007/s00217-010-1214-x [DOI] [Google Scholar]
13.Engel W, Bahr W, Schieberle P. Solvent assisted flavour evaporation—a new and versatile technique for the careful and direct isolation of aroma compounds from complex food matrices. Eur Food Res Technol. 1999;209: 237–241. 10.1007/s002170050486 [DOI] [Google Scholar]
14.Mayuoni-kirshinbaum L, Tietel Z, Porat R, Ulrich D. Identification of aroma-active compounds in ‘wonderful’ pomegranate fruit using solvent-assisted flavour evaporation and headspace solid-phase micro-extraction methods. Eur Food Res Technol. 2012;235: 277–283. 10.1007/s00217-012-1757-0 [DOI] [Google Scholar]
15.Shimizu Y, Imayoshi Y, Kato M, Maeda K, Iwabuchi H, Shimomura K. Volatiles from leaves of field-grown plants and shoot cultures of Gynura bicolor DC. Flavour Fragr J. 2009;24: 251–258. 10.1002/ffj.1938 [DOI] [Google Scholar]
16.Feng Y, Cai Y, Sun-Waterhouse D, Cui C, Su G, Lin L, et al. Approaches of aroma extraction dilution analysis (AEDA) for headspace solid phase microextraction and gas chromatography–olfactometry (HS-SPME–GC–O): Altering sample amount, diluting the sample or adjusting split ratio? Food Chem. 2015;187: 44–52. 10.1016/j.foodchem.2015.03.138 [DOI] [PubMed] [Google Scholar]
17.Ravi R, Prakash M, Bhat KK. Aroma characterization of coriander (Coriandrum sativum L.) oil samples. Eur Food Res Technol. 2007;225: 367–374. 10.1007/s00217-006-0425-7 [DOI] [Google Scholar]
18.Li H, Lu C. Lonicera japonica Thunb (Jinyinhua, Honey Suckle) In: Liu Y, Wang Z, Zhang J, editors. Dietary Chinese Herbs. Vienna: Springer Vienna; 2015. pp. 693–702. 10.1007/978-3-211-99448-1_78 [DOI] [Google Scholar]
19.Wagner H, Bauer R, Melchart D, Xiao P-G, Staudinger A. Flos Lonicerae—Shanyinhua Flos Lonicerae Japonicae—Jinyinhua Caulis Lonicerae Japonicae—Rendongteng In: Wagner H, Bauer R, Melchart D, Xiao P-G, Staudinger A, editors. Chromatographic Fingerprint Analysis of Herbal Medicines. Vienna: Springer Vienna; 2011. pp. 587–600. doi: 10.1007/978-3-7091-0763-8_51 [Google Scholar]
20.Wong SW, Yu B, Curran P, Zhou W. Characterising the release of flavour compounds from chewing gum through HS-SPME analysis and mathematical modelling. Food Chem. 2009;114: 852–858. 10.1016/j.foodchem.2008.10.030 [DOI] [Google Scholar]
21.Lau H, Liu SQ, Xu YQ, Lassabliere B, Sun J, Yu B. Characterising volatiles in tea (Camellia sinensis). Part I: Comparison of headspace-solid phase microextraction and solvent assisted flavour evaporation. LWT. 2018;94: 178–189. 10.1016/j.lwt.2018.04.058 [DOI] [Google Scholar]
22.Pua A, Lau H, Liu SQ, Tan LP, Goh RMV, Lassabliere B, et al. Improved detection of key odourants in Arabica coffee using gas chromatography-olfactometry in combination with low energy electron ionisation gas chromatography-quadrupole time-of-flight mass spectrometry. Food Chem. 2020;302: 125370 10.1016/j.foodchem.2019.125370 [DOI] [PubMed] [Google Scholar]
23.Khio S-W, Cheong M-W, Zhou W, Curran P, Yu B. Characterization of the Volatility of Flavor Compounds in Alcoholic Beverages through Headspace Solid-Phase Microextraction (HS-SPME) and Mathematical Modeling. J Food Sci. 2012;77: C61–C70. 10.1111/j.1750-3841.2011.02474.x [DOI] [PubMed] [Google Scholar]
24.Akkad R, Kharraz E, Han J, House JD, Curtis JM. Characterisation of the volatile flavour compounds in low and high tannin faba beans (Vicia faba var. minor) grown in Alberta, Canada. Food Res Int. 2019;120: 285–294. 10.1016/j.foodres.2019.02.044 [DOI] [PubMed] [Google Scholar]
25.Ikeda N, Ishihara M, Tsuneya T, Kawakita M, Yoshihara M, Suzuki Y, et al. Volatile components of honeysuckle (lonicera japonica thunb.) Flowers. Flavour Fragr J. 1994;9: 325–331. 10.1002/ffj.2730090609 [DOI] [Google Scholar]
26.Wang J, Ji Z, Kang W. The volatile oil from different parts of lonicera japonica thumb (in Chinese). Fine Chem. 2008;25: 1075–1078. [Google Scholar]
27.Schlotzhauer WS, Pair SD, Horvat RJ. Volatile Constituents from the Flowers of Japanese Honeysuckle (Lonicera japonica). J Agric Food Chem. 1996;44: 206–209. 10.1021/jf950275b [DOI] [Google Scholar]
28.Xiao HY, Hou DY, Hui RH. SPME GCMS Analysis on Flowers of Lonicera Japonica. J Anshan Norm Univ. 2010. [Google Scholar]
29.Kim ES, Liang YR, Jin J, Sun QF, Lu JL, Du YY, et al. Impact of heating on chemical compositions of green tea liquor. Food Chem. 2007;103: 1263–1267. 10.1016/j.foodchem.2006.10.031 [DOI] [Google Scholar]
30.Wang Y, Xue X, Zhang F, Xu Q, Liang X. Separation and Analysis of Volatile Oil from Lonicera japonica thunb. World Sci Technol. 2008;10: 45–55. 10.1016/S1876-3553(10)60004-X [DOI] [Google Scholar]
31.Wu C, Wang F, Liu J, Zou Y, Chen X. A comparison of volatile fractions obtained from Lonicera macranthoides via different extraction processes: ultrasound, microwave, Soxhlet extraction, hydrodistillation, and cold maceration. Integr Med Res. 2015;4: 171–177. 10.1016/j.imr.2015.06.001 [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Ilie D-C, Radulescu V, Dutu L. Volatile constituents from the flowers of two species of honeysuckle (lonicera japonica and lonicera caprifolium). Farmacia. 2014;62: 8. [Google Scholar]
33.Kai L. Analysis of essential oil from lonicera japonica in Fujian. Agric J Jiangxi. 2009;21: 102–104. [Google Scholar]
34.Rahman A, Kang SC. In vitro control of food-borne and food spoilage bacteria by essential oil and ethanol extracts of Lonicera japonica Thunb. Food Chem. 2009;116: 670–675. 10.1016/j.foodchem.2009.03.014 [DOI] [Google Scholar]
35.Jolliffe I. Principal Component Analysis In: Lovric M, editor. International Encyclopedia of Statistical Science. Berlin, Heidelberg: Springer; 2011. pp. 1094–1096. 10.1007/978-3-642-04898-2_455 [DOI] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0237881.r001

Decision Letter 0

Tommaso Lomonaco

30 Jun 2020

PONE-D-20-11596

Identification of Key Odorounts in Honeysuckle by Headspace-Solid Phase Microextraction and Solvent-Assisted Flavour Evaporation with Gas Chromatography-Mass Spectrometry and Gas Chromatograph-Olfactometry in Combination with Chemometrics

PLOS ONE

Dear Dr. Bin Yu,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by 29 July. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Tommaso Lomonaco, Ph.D

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please can you provide the following information about the honeysuckle collection:

- the specific locations of the sites from which the honeysuckle samples were collected (including geographical coordinates)

- details of any permits/permissions obtained to collect the samples.

3. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

4. Thank you for stating the following in the Competing Interests section:

'The authors have declared that no competing interests exist'

We note that one or more of the authors are employed by a commercial company: Mane SEA Pte Ltd.

Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

2. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.

Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf.

Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests

5. Please include a copy of Table 3 which you refer to in your text on page 4.

Additional Editor Comments (if provided):

Dear Authors, the current version of the manuscript require major revisions.

Please address all the comments of the reviewers.

Best regards,

Tommaso Lomonaco

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The article is interesting, the experiment is well-planned and clearly described. The methods are adequately described; the choice of samples and components on chromatograms is justified. There are questions on statistical data processing: how many samples were investigated in parallel? Figures 1, 2 show very small ranges of variation for individual components, is this really so? In order for the intervals to be such, the number of parallel-studied samples should be large. If so, then the values in the statistical columns are significantly different and the corresponding p values should be calculated. Regarding PCA: it is not indicated whether the 2 main components are sufficient to separate the groups. Figures 3b are uninformative, it may be better to give a correlation circle or simply give the values of the correlation coefficients with the composition of the honeysuckle. The conclusions and annotations refer to classification, but no numerical characteristics of the classification are mentioned. It also seems to me that it would be appropriate to give an example of a chromatogram and give in the text of the article a table with the quantitative content of the determined components, since it is not very convenient to refer to additional materials. I believe that after making the appropriate changes, the article can be published.

Reviewer #2: Reviewer

In this paper, a full and systematic characterization of key odourants in honeysuckle is interestingly presented and discussed. The use of HS-SPME and SAFE in combination with GC-MS and AEDA with GC-O represents a valid approach for the chemical characterization of the volatile fraction of honeysuckle and the identification of the aroma, which is generally based on experienced tasters.

I would like to suggest some comments to improve the manuscript:

Main general comments:

i) Several sentences along the text are unclear or incorrect. Please improve your writing skills in English, thus restating the text more clearly.

ii) In the “Results and Discussion” section, the discussion is not extremely exhaustive. Please furnish a more comprehensive discussion of your data with respect to the literature.

Specific comments:

Abstract:

Line 29: Change “polarity” with “polar”

Line 34: “Finally, principal component analysis (PCA) analysed”. Unclear sentence, restate. PCA cannot be the subject of this sentence.

Line 35: In the sentence “Their results suggested that..”. What does “Their” refer to?

Line 38: Too vague.

Introduction:

Line 46: After reference [1] put a dot and start the following sentence as “It has been consumed…”

Line 47: Rephrase “In addition to treat pneumonia and respiratory infection and improve consumers’ health,..” as “In addition to the treatment of pneumonia and respiratory infection and the improvement of consumers' health,..”

Line 53: Rephrase “but the quality of honeysuckle is evaluated by professional tasters presently that..” as “. At present, the quality of honeysuckle is evaluated by professional tasters that..”

Line 59: Change “techniques” with “approaches”.

Line 66: Change “polarity” with “polar”

Line 75: Rephrase “Hence, the aromas of different honeysuckle species were difficult to distinguish..” as “ Hence, it was difficult to distinguish between the aromas of different honeysuckle species”

Line 82: Change “and analysed by GCMS” with “both coupled to GCMS”

Materials and methods:

Line 91: put a comma before “but”.

Line 98: “All samples were stored at 5 ℃ in airtight aluminium sachets until use.” How long? What about the influence of humidity? May be advisable the use of desiccants?

Line 102: “0.200 g of each honeysuckle species” do you refer to flowers, stem or both? Please specify in the text.

Line 104: If the numbers refer to nominal values, please express them as integer and change along the text.

Line 109: Specify the extraction conditions, i.e. time and temperature

Line 110: Change “followed by GC analysis” with “for the subsequent GC analysis”

Line 112: Rephrase “but the sample size was increased to 60.000 g and 600.0 g of Ultrapure water was used” as “but in this case 60 g of sample and 600 g of Ultrapure water were used”

Line 113: Internal standard solution (0.060 g). Does this weight correspond to 50 mL of the solution prepared in ethanol?

Line 114: “was added to 300 mL of filtrate” Is it the chilled filtrate? Where exactly was the filtrate contained? Which was the container? Please add this information.

Line 115: Why choosing butyl butyryl lactate as internal standard?

Line 135: What does “Mean values” refer to? Peak area? Peak intensity? Peak area corrected with respect to internal standard (if used)?

Line 141: Add a brief description of FD-factor.

Line 143: What do you mean exactly with odour quality?

Lines 146-148: Restate the sentence more clearly

Line 149: Why using the Benjamini-Hochberg Multiple Testing Correction?

Line 152: Rephrase “entity list were used” as “entity list and were used”.

Results and discussion

Line 157: Change “lead” with “could lead”

Line 158: Add a reference after “recovery.”

Line 160: Rephrase “volatile compounds of honeysuckle (dry and brewed)” as “volatile compounds from honeysuckle (both dry and brewed)”

Lines 165-168: Why did you make a list of five compounds as indicators and then you discussed the results with respect to some others, e.g. (e.g. 2-butenal, heptanal, trans-2-hexenal, benzaldehyde, geraniol, octanol, and 2-phenylethyl alcohol)? Please, restate sentence at line 161 and describe here the overall panel of indicators, with more or less 3-4 compounds for each chemical class.

Line 170: Hexanal or hexenal? Please correct.

Line 174: “higher peak areas” How much higher? Be specific and express in percentage with respect to the temperature.

Line 178: Rephrase “trends as peak areas increasing with extraction temperature increase after 60 °C” as “as the peak areas increase when the extraction temperature is above 60°C”.

Line 182: “various extraction times at 80 °C” Did you work one factor at a time? Why did you not use experimental design for the optimization of the extraction step?

Line 185: Largest??? What do you mean? Please change.

Line 187: “the peak area of hexanal first decreased and then increased” Be specific on the timeframe of decrease/increase.

Line 189: When referring to LJC and LJF, do you mean dry LJC and LJF? Please specify.

Line 190: “Because” is not required. Please change.

Line 193: Please add “both in dry and brewed samples” ate the end of the sentence.

Line 197: Tentatively identified? Did you check with pure standards? In some cases you did not, thus it is just a tentative identification. Please add this information

Line 198: Rephrase “were preferential” as “ were preferentially found”

Line 199: “identified”. This was true, only if the retention time and MS spectrum was matched with that of the corresponding pure standard. Some of the compounds reported in table were just tentatively identified.

Line 199: In S1 Table, does the mean ± dev std refer to the peak area? The peak area of the full scan or EIC chromatogram? If EIC, please specified the selected ion in the table?

Line 203: Restate the sentence more clearly

Line 207: “This might be due to different extraction methods..” Explain better. Which are the main differences between your extraction procedure and the common one?

Line 210: “they only focused on one species”. On which one? Be specific

Line 216: In S2 Table, does the mean value refer to a mean concentration? Explain more clearly in the materials and methods section how exactly it was obtained (preparation of calibration curves) and calculated

How did you obtained the concentration of compounds whose pure standards were not available in lab?

Did you normalize the data to the internal standard to control the analyte variations across the entire analytical procedure? If you did, please specify. If you did not, how could you assess the reliability of your experiments?

Line 217: Please insert in the text just few examples of the compounds listed in S2 Table and the agreement with chemicals reported the literature

Line 218: Change “polarity” with “polar”

Line 243: Not detected means a FD below XXX? Please specify.

Lines 273-274: “to pinpoint the main areas of variation among different species, parts and origins of honeysuckle in this study.” Please restate as a separate sentence.

Lines 275-276: Rephrase “In this present study, key odourants were identified by GC-O, which contributed to the aroma of honeysuckle” as “In the present study, the key odourants that were identified by GC-O and contributed to the aroma of honeysuckle”

Line277: Among the HS-SPME-GC/MS AND SAFE-GC/MS data, did you just select the compounds identified through GC-O? Compounds identified through the match with standard or also tentatively identified? Explain more clearly.

Line 287: Larger??? Please change.

Line 289: In which direction of the plot?

Line 293: In which direction? In correspondence of which variable?

Line 298: In which direction? Be specific

Line 299: Larger??? Please change.

Conclusion

Line 327: “approach is critical”. Please change “critical”. It has a negative meaning.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 Aug 20;15(8):e0237881. doi: 10.1371/journal.pone.0237881.r002

Author response to Decision Letter 0

22 Jul 2020

Reference Number: PONE-D-20-11596

Dear Editor and Reviewers,

We wish to thank you for the time and effort spent on reviewing our manuscript. Motivated by your extensive and insightful comments, we have modified the manuscript and hope that the revised version will be reconsidered. Our responses to your questions, comments and suggestions (repeated below for your convenience) are as follows:

Editor’s comments:

1. Please can you provide the following information about the honeysuckle collection:

- the specific locations of the sites from which the honeysuckle samples were collected (including geographical coordinates)

Authors’ answer:

Thank you for your suggestion. I have added the specific locations of the honeysuckle samples in the section of sample preparation.

- details of any permits/permissions obtained to collect the samples.

Authors’ answer:

Thank you for your suggestion. I have provided the permissions in the section of sample preparation.

2. Please include a copy of Table 3 which you refer to in your text on page 4.

Authors’ answer:

Thank you for your suggestion. I have corrected the error and changed the Table 3 with Table 1.

Reviewer #1’s comments:

General comments:

The article is interesting, the experiment is well-planned and clearly described. The methods are adequately described; the choice of samples and components on chromatograms is justified.

Authors’ answer:

Thank you for your affirmation of our work.

1. There are questions on statistical data processing: how many samples were investigated in parallel? Figures 1, 2 show very small ranges of variation for individual components, is this really so? In order for the intervals to be such, the number of parallel-studied samples should be large. If so, then the values in the statistical columns are significantly different and the corresponding p values should be calculated.

Authors’ answer:

Thanks for your question. Every sample was carried out in triplicate at each extraction condition. Among the six selected compounds in Figures 1 and 2, the compound with the greatest peak area was about 106 times larger than the compound with the smallest peak area. Hence, the log scale was chosen as the ordinate to show the trends of all six compounds clearly on the coordinate axis. Given your insight, we have changed the coordinates of Figures 1 and 2 for greater ease of understanding. As you suggested, the p-values have also been added in the figures.

2. Regarding PCA: it is not indicated whether the 2 main components are sufficient to separate the groups. Figures 3b are uninformative, it may be better to give a correlation circle or simply give the values of the correlation coefficients with the composition of the honeysuckle. The conclusions and annotations refer to classification, but no numerical characteristics of the classification are mentioned.

Authors’ answer:

PC 1 and PC 2 account for the vast majority of the variation. This indicates that a 2-D graph, using just PC 1 and PC 2, would be a sufficient representation of significant areas of variation and it would account for a considerable 72.6 % (Figure 4(a)), 59.9 % (Figure 4(b)), and 63.6 % (Figure 4(c)) of the total variation in the data. In addition, we noted that the samples in the 2-D graph can be well separated, and areas of variation are clear (as opposed to the 3-D graph). As suggested, we replotted the PCA by R programming language for added detail, and the correlation circle was added in the biplot.

3. It also seems to me that it would be appropriate to give an example of a chromatogram and give in the text of the article a table with the quantitative content of the determined components, since it is not very convenient to refer to additional materials.

Authors’ answer:

Thanks for your suggestion. The chromatogram has been added in the manuscript.

Reviewer #2’s comments:

General comments:

Authors’ answer:

Thank you for your affirmation of our work.

Main general comments:

i) Several sentences along the text are unclear or incorrect. Please improve your writing skills in English, thus restating the text more clearly.

Authors’ answer:

Thanks for your suggestion. We have corrected the unclear and incorrect sentences in the manuscript.

ii) In the “Results and Discussion” section, the discussion is not extremely exhaustive. Please furnish a more comprehensive discussion of your data with respect to the literature.

Authors’ answer:

Thanks for your suggestion. We have furnished the discussion of the results based on the corresponding references.

Specific comments:

Abstract:

1. Line 29: Change “polarity” with “polar”

Authors’ answer:

Thank you for your correction. We have corrected the word error on line 30.

2. Line 34: “Finally, principal component analysis (PCA) analysed”. Unclear sentence, restate. PCA cannot be the subject of this sentence.

Authors’ answer:

Thank you for pointing out our mistakes. We have corrected the unclear sentence on line 35.

3. Line 35: In the sentence “Their results suggested that..”. What does “Their” refer to?

Authors’ answer:

Thanks for your correction. We have changed “Their” with specific information on line 36.

4. Line 38: Too vague.

Authors’ answer:

Sorry to make you confused and thank you for your question. We have corrected the sentence on line 39.

Introduction:

1. Line 46: After reference [1] put a dot and start the following sentence as “It has been consumed…”

Authors’ answer:

Thanks for your suggestion. We have added a dot on line 48 after reference [1].

2. Line 47: Rephrase “In addition to treat pneumonia and respiratory infection and improve consumers’ health,..” as “In addition to the treatment of pneumonia and respiratory infection and the improvement of consumers' health,..”

Authors’ answer:

Thank you for pointing out our mistakes. We have corrected the sentence on line 49 and 50.

3. Line 53: Rephrase “but the quality of honeysuckle is evaluated by professional tasters presently that..” as “. At present, the quality of honeysuckle is evaluated by professional tasters that..”

Authors’ answer:

Thanks for your correction. We have corrected the sentence on line 55 and 56.

4. Line 59: Change “techniques” with “approaches”.

Authors’ answer:

Thank you for your suggestion. We have changed “techniques” with “approaches” on line 61.

5. Line 66: Change “polarity” with “polar”

Authors’ answer:

Thanks for your correction. We have changed “polarity” with “polar” on line 68.

6. Line 75: Rephrase “Hence, the aromas of different honeysuckle species were difficult to distinguish.” as “ Hence, it was difficult to distinguish between the aromas of different honeysuckle species”

Authors’ answer:

Thank you for pointing out our mistakes. We have corrected the sentence on line 77.

7. Line 82: Change “and analysed by GCMS” with “both coupled to GCMS”

Authors’ answer:

Thank you for your correction. We have corrected the sentence on line 84.

Materials and methods:

1. Line 91: put a comma before “but”.

Authors’ answer:

Thanks for your correction. We have added a comma before “but” on line 93.

2. Line 98: “All samples were stored at 5 ℃ in airtight aluminium sachets until use.” How long? What about the influence of humidity? May be advisable the use of desiccants?

Authors’ answer:

Thank you for your question. One-kilogram honeysuckle samples were sealed in a package. After opening, every 100 g sample was put into a small heat-sealed vacuum aluminium foil bag and stored in the 5 ℃ refrigerator. The aluminium foil bag can isolate the outside humidity, so no desiccant is needed. All samples are used within one week after repacking. Once each bag of sample was opened, it will not be used again. We have added the information on line 96-105.

3. Line 102: “0.200 g of each honeysuckle species” do you refer to flowers, stem or both? Please specify in the text.

Authors’ answer:

Thank you for your question. We have added the information on line 109. LJF (Shandong and Henan), LF (Huan and Hubei), LC, and LJC were all extracted by HS-SPME. Hence, 0.200 g of each sample was weighted and put in the headspace vial.

4. Line 104: If the numbers refer to nominal values, please express them as integer and change along the text.

Authors’ answer:

Thanks for your suggestion. The measurement scale of the balance we used is accurate to three decimal places or one decimal place. Hence, 0.200 g has three decimal places. The information of balance has been added on line 109.

5. Line 109: Specify the extraction conditions, i.e. time and temperature

Authors’ answer:

Thanks for your suggestion, the extraction time and temperature have been added in the manuscript on line 118 to 120.

6. Line 110: Change “followed by GC analysis” with “for the subsequent GC analysis”

Authors’ answer:

Thank you for pointing out our mistakes. We have corrected the sentence on line 120.

7. Line 112: Rephrase “but the sample size was increased to 60.000 g and 600.0 g of Ultrapure water was used” as “but in this case 60 g of sample and 600 g of Ultrapure water were used”

Authors’ answer:

Thanks for your correction. We have corrected the sentence on line 123.

8. Line 113: Internal standard solution (0.060 g). Does this weight correspond to 50 mL of the solution prepared in ethanol?

Authors’ answer:

Sorry to make you confused, and we have rephrased this sentence for a better understanding. 0.300 g butyl butyryl lactate was added in 250 mL volumetric flask, then dilute to the desired volume with ethanol. This solution was then referred to as the internal standard. Then, 0.060 g internal standard solution was added to 300 mL filtrate sample solution.

9. Line 114: “was added to 300 mL of filtrate” Is it the chilled filtrate? Where exactly was the filtrate contained? Which was the container? Please add this information.

Authors’ answer:

Sorry to make you confused, and we have rephrased this sentence and added the following information in the manuscript for a better understanding. 60.000 g of honeysuckle was added in 600.0 g of 80 ℃ Ultrapure water. After brewing, the mixture was filtered through a metal sieve and chilled in an ice bath for 10 min. 300 mL of the chilled filtrate was then transferred into a 500 mL beaker. Internal standard solution (0.060 g) was added to the 300 mL of filtrate and stirred for 5 min.

10. Line 115: Why choosing butyl butyryl lactate as internal standard?

Authors’ answer:

Thank you for your question. The peak of butyl butyryl lactate does not coelute with the other naturally-occurring compounds in honeysuckle (validated by overlaying the chromatograms for the internal standard, and for all samples of honeysuckle that were unspiked). In addition, the retention time of butyryl lactate is in the middle of all volatile compounds in honeysuckle based on our GC set-up and method applied. Hence, butyl butyryl lactate was chosen as internal standard.

11. Line 135: What does “Mean values” refer to? Peak area? Peak intensity? Peak area corrected with respect to internal standard (if used)?

Authors’ answer:

Thanks for your question. “Mean values” refer to the average peak area of each compound extracted by HS-SPME and average concentration of each compound extracted by SAFE. We have added the information under the S1 and S2 table.

12. Line 141: Add a brief description of FD-factor.

Authors’ answer:

Thanks for your suggestion. We have added the description of FD-factor in section 2.5 of the manuscript.

13. Line 143: What do you mean exactly with odour quality?

Authors’ answer:

Thanks for your question. The odour quality means odour description, such as floral and fruity. We have changed the “odour quality” with “odour description” for easier understanding in the manuscript.

14. Lines 146-148: Restate the sentence more clearly

Authors’ answer:

Thanks for your suggestion. We have rewritten part of section 2.6 in order to improve the clarity of the content.

15. Line 149: Why using the Benjamini-Hochberg Multiple Testing Correction?

Authors’ answer:

Thanks for your question. We have changed the method of the calculation of PCA using R instead. The data was processed and transformed with data.table and magrittr packages and visualized them with ggplot2, ggbiplot, and patchwork packages.

16. Line 152: Rephrase “entity list were used” as “entity list and were used”.

Authors’ answer:

Thanks for your correction. We have rewritten the part of section 2.6.

Results and discussion

1. Line 157: Change “lead” with “could lead”

Authors’ answer:

Thanks for your correction. We have added “could” before “lead”.

2. Line 158: Add a reference after “recovery.”

Authors’ answer:

Thanks for your suggestion. We have added the following references.

https://doi-org.libproxy1.nus.edu.sg/10.1111/j.1750-3841.2011.02474.x https://doi.org/10.1016/j.foodres.2019.02.044

3. Line 160: Rephrase “volatile compounds of honeysuckle (dry and brewed)” as “volatile compounds from honeysuckle (both dry and brewed)”

Authors’ answer:

Thank you for pointing out our mistakes. We have corrected the sentence on line 187.

4. Lines 165-168: Why did you make a list of five compounds as indicators and then you discussed the results with respect to some others, e.g. (e.g. 2-butenal, heptanal, trans-2-hexenal, benzaldehyde, geraniol, octanol, and 2-phenylethyl alcohol)? Please, restate sentence at line 161 and describe here the overall panel of indicators, with more or less 3-4 compounds for each chemical class.

Authors’ answer:

The comparison and grouping were conducted based on the chemical functional groups of the compounds. For example, hexanal was chosen as the representative of aldehyde compounds. 2-Butenal, heptanal, trans-2-hexenal, and benzaldehyde all contain an aldehylic functional group and were observed to undergo similar trends as hexanal. Geraniol, octanol, and 2-phenylethyl alcohol all contain the hydroxyl group and had the same trend with cis-3-hexenol and benzyl alcohol.

5. Line 170: Hexanal or hexenal? Please correct.

Authors’ answer:

Thanks for your correction. We have changed “hexenal” with “hexanal” in the manuscript.

6. Line 174: “higher peak areas” How much higher? Be specific and express in percentage with respect to the temperature.

Authors’ answer:

Thanks for your suggestion. We have calculated the corresponding p-values and added them on the figure 1 and 2. We hope that this makes it easier to see the differences between different extraction conditions.

7. Line 178: Rephrase “trends as peak areas increasing with extraction temperature increase after 60 °C” as “as the peak areas increase when the extraction temperature is above 60°C”.

Authors’ answer:

Thanks for your suggestion. We have rephrased the sentence on line 202.

8. Line 182: “various extraction times at 80 °C” Did you work one factor at a time? Why did you not use experimental design for the optimization of the extraction step?

Authors’ answer:

Thanks for your suggestion. It is more reasonable to use an experimental design for the optimization of the extraction step. However, considering that the focus of this manuscript is to determine the key aroma compounds and distinguish different types of honeysuckle, we referred to the optimization conditions of other natural plants instead. This led us to select and optimise some specific extraction times and temperatures. It was finally decided to determine the most suitable extraction temperature and time from the extraction conditions mentioned in the manuscript. The reference is shown below.

https://doi.org/10.1016/j.lwt.2018.04.058

9. Line 185: Largest??? What do you mean? Please change.

Authors’ answer:

Thanks for your correction. We have changed “largest” with “greatest”.

10. Line 187: “the peak area of hexanal first decreased and then increased” Be specific on the timeframe of decrease/increase.

Authors’ answer:

Thanks for your suggestion. We have specified the information in the manuscript.

11. Line 189: When referring to LJC and LJF, do you mean dry LJC and LJF? Please specify.

Authors’ answer:

Thanks for your question. We have specified the information on line 221 in the manuscript.

12. Line 190: “Because” is not required. Please change.

Authors’ answer:

Thanks for your suggestion. We have deleted “Because” on line 222.

13. Line 193: Please add “both in dry and brewed samples” ate the end of the sentence.

Authors’ answer:

Thanks for your suggestion. We have added the information on line 225.

14. Line 197: Tentatively identified? Did you check with pure standards? In some cases you did not, thus it is just a tentative identification. Please add this information

Authors’ answer:

Thanks for your suggestion. We have added the information on line 239 in the manuscript.

15. Line 198: Rephrase “were preferential” as “ were preferentially found”

Authors’ answer:

Thanks for your suggestion. We have rephrased the sentence on line 240.

16. Line 199: “identified”. This was true, only if the retention time and MS spectrum was matched with that of the corresponding pure standard. Some of the compounds reported in table were just tentatively identified.

Authors’ answer:

Thanks for your suggestion. We have replaced “identified compounds” by “results” on line 241.

17. Line 199: In S1 Table, does the mean ± dev std refer to the peak area? The peak area of the full scan or EIC chromatogram? If EIC, please specified the selected ion in the table?

Authors’ answer:

Thanks for your question. The mean ± dev std refers to the average peak area of each compound. Volatile compounds in honeysuckle were detected by GC-MS/FID. Hence, the peak area was obtained from FID detector.

18. Line 203: Restate the sentence more clearly

Authors’ answer:

Thanks for your suggestion. We have restated the sentence on line 246 to 248.

19. Line 207: “This might be due to different extraction methods..” Explain better. Which are the main differences between your extraction procedure and the common one?

Authors’ answer:

Thanks for your suggestion. We have specified the information on line 252.

20. Line 210: “they only focused on one species”. On which one? Be specific

Authors’ answer:

Thanks for your suggestion. We have specified the information on line 257.

21. Line 216: In S2 Table, does the mean value refer to a mean concentration? Explain more clearly in the materials and methods section how exactly it was obtained (preparation of calibration curves) and calculated

How did you obtained the concentration of compounds whose pure standards were not available in lab?

Authors’ answer

Thank you for your question. We have added the explanation in the materials and methods.

The calculation of the concentration of compounds based on the formula that the ratio of the peak area of internal standard to its concentration is equal to the ratio of the peak area of each compound to the concentration of the corresponding compound. In this formula only the concentration of the compound is unknown. Hence, we utilised the internal standard peak ratio to semi-quantify the other volatile compounds present.

As the concentration of each compound was further utilised when using PCA to compare different types of honeysuckle, these compounds were additionally verified by pure standards and GC-O simultaneously. Hence, the results are reliable.

22. Line 217: Please insert in the text just few examples of the compounds listed in S2 Table and the agreement with chemicals reported the literature

Authors’ answer

Thanks for your suggestion. We have added the compounds listed in S2 Table in the manuscript. To our best knowledge, this is the first time that volatile compounds were extracted by SAFE from honeysuckle. The results were well-corroborated as most of the volatile compounds extracted by SAFE are similar to those extracted by other extraction methods such as HS-SPME and SDE. The results were obtained by comparing S1 table and S2 table and referring to literature.

23. Line 218: Change “polarity” with “polar”

Authors’ answer

Thanks for your correction. We have changed “polarity” with “polar” on line 271.

24. Line 243: Not detected means a FD below XXX? Please specify.

Authors’ answer

Thanks for your suggestion. The FD factor of decanoic acid is 625 in LJF but this volatile compound could not be smelled by panellists even in the dilution of 50 and it cannot be detected by GC-MS. Hence, decanoic acid was not detected in LJC and LF.

25. Lines 273-274: “to pinpoint the main areas of variation among different species, parts and origins of honeysuckle in this study.” Please restate as a separate sentence.

Authors’ answer

Thanks for your suggestion. We have corrected the two sentences as suggested on line 325 and 328, respectively.

26. Line277: Among the HS-SPME-GC/MS AND SAFE-GC/MS data, did you just select the compounds identified through GC-O? Compounds identified through the match with standard or also tentatively identified? Explain more clearly.

Authors’ answer

Thanks for your question. Panellists described the odour of each compound and indicated the retention time of the odour by GC-O. The name of these compounds was identified by injection of pure standards. Hence, these key odourants we selected were identified through GC-O, MS, LRI and pure standards. We have added the information in the materials and method based on your question.

27. Line 287: Larger??? Please change.

Authors’ answer:

Thanks for your correction. We have change “larger” with “greater” on line 343.

28. Line 289: In which direction of the plot?

Line 293: In which direction? In correspondence of which variable?

Line 298: In which direction? Be specific

Authors’ answer:

Thanks for your question. We have added the information on line 345, 350, and 356.

29. Line 299: Larger??? Please change.

Authors’ answer:

Thanks for your correction. We have change “larger” with “greater” on line 357.

Conclusion

Line 327: “approach is critical”. Please change “critical”. It has a negative meaning.

Authors’ answer to conclusion:

Thanks for your suggestion. We have changed “critical” with “important” on line 395.

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(41.4KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0237881.r003

Decision Letter 1

Tommaso Lomonaco

5 Aug 2020

PONE-D-20-11596R1

Dear Dr. Bin Yu,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Tommaso Lomonaco, Ph.D

Academic Editor

PLOS ONE

Dear Author, all the questions have been addressed and then I suggest to accept the article in the present form.

Regards,

Tommaso Lomonaco

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: The authors made all the changes in accordance with the recommendations of the reviewers, which significantly improved the article. In its present form, the article can be accepted for publication.

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

PLoS One. doi: 10.1371/journal.pone.0237881.r004

Acceptance letter

Tommaso Lomonaco

10 Aug 2020

PONE-D-20-11596R1

Identification of Key Odorants in Honeysuckle by Headspace-Solid Phase Microextraction and Solvent-Assisted Flavour Evaporation with Gas Chromatography-Mass Spectrometry and Gas Chromatograph-Olfactometry in Combination with Chemometrics

Dear Dr. Yu:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Tommaso Lomonaco

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

(DOCX)

Click here for additional data file.^{(56.8KB, docx)}

S2 Table. Identification of volatile compounds and concentration (ng/mL) in Lonicera japonica Flos, Lonicera Flos, Lonicera japonica Caulis, and Lonicera Caulis extracted using SAFE.

(DOCX)

Click here for additional data file.^{(37.9KB, docx)}

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(41.4KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

[pone.0237881.ref001] 1.Shang X, Pan H, Li M, Miao X, Ding H. Lonicera japonica Thunb.: Ethnopharmacology, phytochemistry and pharmacology of an important traditional Chinese medicine. J Ethnopharmacol. 2011;138: 1–21. 10.1016/j.jep.2011.08.016 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237881.ref002] 2.Brown L, Poudyal H, Panchal SK. Functional foods as potential therapeutic options for metabolic syndrome: Foods as the treatment of obesity. Obes Rev. 2015;16: 914–941. 10.1111/obr.12313 [DOI] [PubMed] [Google Scholar]

[pone.0237881.ref003] 3.Yao X-H, Xu J-Y, Hao J-Y, Wan Y, Chen T, Zhang D-Y, et al. Microwave assisted extraction for the determination of chlorogenic acid in Flos Lonicerae by direct analysis in real time mass spectrometry (DART-MS). J Chromatogr B. 2018;1092: 82–87. 10.1016/j.jchromb.2018.05.045 [DOI] [PubMed] [Google Scholar]

[pone.0237881.ref004] 4.Yang J, Farmer LM, Agyekum AAA, Elbaz-Younes I, Hirschi KD. Detection of an Abundant Plant-Based Small RNA in Healthy Consumers. Li M, editor. PLOS ONE. 2015;10: e0137516 10.1371/journal.pone.0137516 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237881.ref005] 5.Hamilton AC. Medicinal plants, conservation and livelihoods. Biodivers Conserv. 2004;13: 1477–1517. 10.1023/B:BIOC.0000021333.23413.42 [DOI] [Google Scholar]

[pone.0237881.ref006] 6.Ye J, Su J, Chen K, Liu H, Yang X, He Y, et al. Comparative investigation on chemical constituents of flower bud, stem and leaf of Lonicera japonica Thunb. by HPLC-DAD-ESI-MS/MS n and GC-MS. J Anal Chem. 2014;69: 777–784. 10.1134/S1061934814080036 [DOI] [Google Scholar]

[pone.0237881.ref007] 7.Stephany M, Kapusi K, Bader-Mittermaier S, Schweiggert-Weisz U, Carle R. Odour-active volatiles in lupin kernel fibre preparations (Lupinus angustifolius L.): effects of thermal lipoxygenase inactivation. Eur Food Res Technol. 2016;242: 995–1004. 10.1007/s00217-015-2605-9 [DOI] [Google Scholar]

[pone.0237881.ref008] 8.Lv S, Wu Y, Zhou J, Lian M, Li C, Xu Y, et al. The Study of Fingerprint Characteristics of Dayi Pu-Erh Tea Using a Fully Automatic HS-SPME/GC–MS and Combined Chemometrics Method. Amancio S, editor. PLOS ONE. 2014;9: e116428 10.1371/journal.pone.0116428 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237881.ref009] 9.Xue J, Li H, Liu F, Jiang W, Hou F. Vortex-assisted matrix solid–liquid dispersive microextraction for the analysis of triazole fungicides in cotton seed and honeysuckle by gas chromatography. Food Chem. 2016;196: 867–876. 10.1016/j.foodchem.2015.09.093 [DOI] [PubMed] [Google Scholar]

[pone.0237881.ref010] 10.Pawliszyn J. Handbook of solid phase microextraction. 2012. Available: http://www.sciencedirect.com/science/book/9780124160170

[pone.0237881.ref011] 11.Belliardo F, Bicchi C, Cordero C, Liberto E, Rubiolo P, Sgorbini B. Headspace-solid-phase microextraction in the analysis of the volatile fraction of aromatic and medicinal plants. J Chromatogr Sci. 2006;44: 416–429. 10.1093/chromsci/44.7.416 [DOI] [PubMed] [Google Scholar]

[pone.0237881.ref012] 12.Yang Z, Mao H, Long C, Sun C, Guo Z. Rapid determination of volatile composition from Polygala furcata Royle by MAE–HS-SPME followed by GC–MS. Eur Food Res Technol. 2010;230: 779–784. 10.1007/s00217-010-1214-x [DOI] [Google Scholar]

[pone.0237881.ref013] 13.Engel W, Bahr W, Schieberle P. Solvent assisted flavour evaporation—a new and versatile technique for the careful and direct isolation of aroma compounds from complex food matrices. Eur Food Res Technol. 1999;209: 237–241. 10.1007/s002170050486 [DOI] [Google Scholar]

[pone.0237881.ref014] 14.Mayuoni-kirshinbaum L, Tietel Z, Porat R, Ulrich D. Identification of aroma-active compounds in ‘wonderful’ pomegranate fruit using solvent-assisted flavour evaporation and headspace solid-phase micro-extraction methods. Eur Food Res Technol. 2012;235: 277–283. 10.1007/s00217-012-1757-0 [DOI] [Google Scholar]

[pone.0237881.ref015] 15.Shimizu Y, Imayoshi Y, Kato M, Maeda K, Iwabuchi H, Shimomura K. Volatiles from leaves of field-grown plants and shoot cultures of Gynura bicolor DC. Flavour Fragr J. 2009;24: 251–258. 10.1002/ffj.1938 [DOI] [Google Scholar]

[pone.0237881.ref016] 16.Feng Y, Cai Y, Sun-Waterhouse D, Cui C, Su G, Lin L, et al. Approaches of aroma extraction dilution analysis (AEDA) for headspace solid phase microextraction and gas chromatography–olfactometry (HS-SPME–GC–O): Altering sample amount, diluting the sample or adjusting split ratio? Food Chem. 2015;187: 44–52. 10.1016/j.foodchem.2015.03.138 [DOI] [PubMed] [Google Scholar]

[pone.0237881.ref017] 17.Ravi R, Prakash M, Bhat KK. Aroma characterization of coriander (Coriandrum sativum L.) oil samples. Eur Food Res Technol. 2007;225: 367–374. 10.1007/s00217-006-0425-7 [DOI] [Google Scholar]

[pone.0237881.ref018] 18.Li H, Lu C. Lonicera japonica Thunb (Jinyinhua, Honey Suckle) In: Liu Y, Wang Z, Zhang J, editors. Dietary Chinese Herbs. Vienna: Springer Vienna; 2015. pp. 693–702. 10.1007/978-3-211-99448-1_78 [DOI] [Google Scholar]

[pone.0237881.ref019] 19.Wagner H, Bauer R, Melchart D, Xiao P-G, Staudinger A. Flos Lonicerae—Shanyinhua Flos Lonicerae Japonicae—Jinyinhua Caulis Lonicerae Japonicae—Rendongteng In: Wagner H, Bauer R, Melchart D, Xiao P-G, Staudinger A, editors. Chromatographic Fingerprint Analysis of Herbal Medicines. Vienna: Springer Vienna; 2011. pp. 587–600. doi: 10.1007/978-3-7091-0763-8_51 [Google Scholar]

[pone.0237881.ref020] 20.Wong SW, Yu B, Curran P, Zhou W. Characterising the release of flavour compounds from chewing gum through HS-SPME analysis and mathematical modelling. Food Chem. 2009;114: 852–858. 10.1016/j.foodchem.2008.10.030 [DOI] [Google Scholar]

[pone.0237881.ref021] 21.Lau H, Liu SQ, Xu YQ, Lassabliere B, Sun J, Yu B. Characterising volatiles in tea (Camellia sinensis). Part I: Comparison of headspace-solid phase microextraction and solvent assisted flavour evaporation. LWT. 2018;94: 178–189. 10.1016/j.lwt.2018.04.058 [DOI] [Google Scholar]

[pone.0237881.ref022] 22.Pua A, Lau H, Liu SQ, Tan LP, Goh RMV, Lassabliere B, et al. Improved detection of key odourants in Arabica coffee using gas chromatography-olfactometry in combination with low energy electron ionisation gas chromatography-quadrupole time-of-flight mass spectrometry. Food Chem. 2020;302: 125370 10.1016/j.foodchem.2019.125370 [DOI] [PubMed] [Google Scholar]

[pone.0237881.ref023] 23.Khio S-W, Cheong M-W, Zhou W, Curran P, Yu B. Characterization of the Volatility of Flavor Compounds in Alcoholic Beverages through Headspace Solid-Phase Microextraction (HS-SPME) and Mathematical Modeling. J Food Sci. 2012;77: C61–C70. 10.1111/j.1750-3841.2011.02474.x [DOI] [PubMed] [Google Scholar]

[pone.0237881.ref024] 24.Akkad R, Kharraz E, Han J, House JD, Curtis JM. Characterisation of the volatile flavour compounds in low and high tannin faba beans (Vicia faba var. minor) grown in Alberta, Canada. Food Res Int. 2019;120: 285–294. 10.1016/j.foodres.2019.02.044 [DOI] [PubMed] [Google Scholar]

[pone.0237881.ref025] 25.Ikeda N, Ishihara M, Tsuneya T, Kawakita M, Yoshihara M, Suzuki Y, et al. Volatile components of honeysuckle (lonicera japonica thunb.) Flowers. Flavour Fragr J. 1994;9: 325–331. 10.1002/ffj.2730090609 [DOI] [Google Scholar]

[pone.0237881.ref026] 26.Wang J, Ji Z, Kang W. The volatile oil from different parts of lonicera japonica thumb (in Chinese). Fine Chem. 2008;25: 1075–1078. [Google Scholar]

[pone.0237881.ref027] 27.Schlotzhauer WS, Pair SD, Horvat RJ. Volatile Constituents from the Flowers of Japanese Honeysuckle (Lonicera japonica). J Agric Food Chem. 1996;44: 206–209. 10.1021/jf950275b [DOI] [Google Scholar]

[pone.0237881.ref028] 28.Xiao HY, Hou DY, Hui RH. SPME GCMS Analysis on Flowers of Lonicera Japonica. J Anshan Norm Univ. 2010. [Google Scholar]

[pone.0237881.ref029] 29.Kim ES, Liang YR, Jin J, Sun QF, Lu JL, Du YY, et al. Impact of heating on chemical compositions of green tea liquor. Food Chem. 2007;103: 1263–1267. 10.1016/j.foodchem.2006.10.031 [DOI] [Google Scholar]

[pone.0237881.ref030] 30.Wang Y, Xue X, Zhang F, Xu Q, Liang X. Separation and Analysis of Volatile Oil from Lonicera japonica thunb. World Sci Technol. 2008;10: 45–55. 10.1016/S1876-3553(10)60004-X [DOI] [Google Scholar]

[pone.0237881.ref031] 31.Wu C, Wang F, Liu J, Zou Y, Chen X. A comparison of volatile fractions obtained from Lonicera macranthoides via different extraction processes: ultrasound, microwave, Soxhlet extraction, hydrodistillation, and cold maceration. Integr Med Res. 2015;4: 171–177. 10.1016/j.imr.2015.06.001 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0237881.ref032] 32.Ilie D-C, Radulescu V, Dutu L. Volatile constituents from the flowers of two species of honeysuckle (lonicera japonica and lonicera caprifolium). Farmacia. 2014;62: 8. [Google Scholar]

[pone.0237881.ref033] 33.Kai L. Analysis of essential oil from lonicera japonica in Fujian. Agric J Jiangxi. 2009;21: 102–104. [Google Scholar]

[pone.0237881.ref034] 34.Rahman A, Kang SC. In vitro control of food-borne and food spoilage bacteria by essential oil and ethanol extracts of Lonicera japonica Thunb. Food Chem. 2009;116: 670–675. 10.1016/j.foodchem.2009.03.014 [DOI] [Google Scholar]

[pone.0237881.ref035] 35.Jolliffe I. Principal Component Analysis In: Lovric M, editor. International Encyclopedia of Statistical Science. Berlin, Heidelberg: Springer; 2011. pp. 1094–1096. 10.1007/978-3-642-04898-2_455 [DOI] [Google Scholar]

PERMALINK

Identification of key odorants in honeysuckle by headspace-solid phase microextraction and solvent-assisted flavour evaporation with gas chromatography-mass spectrometry and gas chromatograph-olfactometry in combination with chemometrics

Keran Su

Xin Zhang

Shao Quan Liu

LiHui Jia

Benjamin Lassabliere

Kim Huey Ee

Aileen Pua

Rui Min Vivian Goh

Jingcan Sun

Bin Yu

XiaoXue Hu

Roles

Abstract

Introduction

Materials and methods

Sample preparation

HS-SPME procedure

SAFE procedure

GC-MS/FID analysis

AEDA

Data processing and statistical analysis

Results and discussion

The selection of HS-SPME extraction conditions

Fig 1.

Fig 2.

Volatile profiles of different species and parts of honeysuckle by HS-SPME

Fig 3. GC-MS/FID chromatogram of Lonicera japonica Flos (Shandong) extracted by HS-SPME at the extraction conditions of 80°C and 30 min.

Volatile profiles of different species and parts of honeysuckle extracted by SAFE

Key odorants of honeysuckle

Table 1. Key odorants (flavour dilution factor ≥ 1) with their respective flavour dilution factors in SAFE extracts of Lonicera japonica Flos, Lonicera Flos and Lonicera japonica Caulis.

Principal component analysis

Fig 4.

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Tommaso Lomonaco

Roles

Author response to Decision Letter 0

Decision Letter 1

Tommaso Lomonaco

Roles

Acceptance letter

Tommaso Lomonaco

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases