Study on the Variation Patterns of Main Components and Chromaticity During the Developmental Process of Magnoliae Flos (Magnolia biondii)

Abstract

Analyze the quality differences of Magnoliae Flos (MF) at different developmental stages and determine its optimal harvest period. In this study, a detection method for the main chemical components of MF was established based on GC-MS and UPLC, and the volatile oil and lignan components were determined. The quality differences between MF at different developmental stages were compared based on chemical composition. Chromaticity values of MF samples were measured using electronic eye technology, followed by correlation analysis to reveal the relationship between internal compositional changes and external color differences. The results indicated that the harvesting period significantly affected the chemical composition of MF. Specifically, the contents of volatile oils and lignans initially increased and then decreased as the flower buds developed. There are obvious correlations between six different volatile components and some lignans of MF and their chromaticity values (p < 0.05). This study clarified the dynamic changes in relevant indicators during the development of MF, which can provide a reference for the rational utilization and scientific harvesting of MF resources.

1. Introduction

Magnoliae Flos (MF), the dry flower buds of Magnolia biondii Pamp. with the Chinese name “Xinyi”, is a traditional Chinese medicine for the treatment of nasal diseases [1]. MF has the effects of dispersing wind and cold and clearing the nasal passages. It is often used to treat headaches caused by wind and cold, nasal congestion and runny nose, allergic rhinitis, and sinusitis [2]. Modern research shows that MF contains chemical components such as volatile oils, lignans, alkaloids, and flavonoids [3]. Among them, volatile oil components such as 1,8-cineole, farnesol, and β-pinene have anti-inflammatory, antibacterial, and antioxidant effects and can inhibit the inflammatory response of the airways of asthmatic mice and achieve the effect of relieving asthma [4]. Lignan components such as magnolin, pinoresinol dimethyl ether, and fargesin have anti-inflammatory, anti-allergic, and insecticidal effects and can antagonize the activity of platelet activation factor and reduce nasal lesions in mice [5,6].

MF is mainly distributed in Henan, Hubei, Shanxi, Sichuan, and Anhui provinces in China. Nanzhao County in Henan Province is recognized as the authentic Dao-di production region for MF. The harvesting period typically begins in September and extends until February of the following year, representing an exceptionally prolonged collection window. Current research indicates that the bioactive constituents of traditional Chinese herbs exhibit significant correlations with their harvesting periods [7,8]. Kim et al. [9] confirmed that the volatile components of samples at different developmental stages are the same, but their content ratios are significantly different, and the corresponding pharmacological effects are also different. Marzocchi et al. [10] verified that the maturity stage of Bergamot Essential Oil has a significant impact on the composition of volatile components. Li et al. [11] analyzed the relationship between the individual development of oil cells and the yield and composition of essential oils. Therefore, investigating the optimal harvesting period for MF is of paramount importance. At present, some scholars have studied the volatile oil components of MF at different harvesting periods and reported the differences in volatile oil yield and composition at different developmental stages, as well as the effects of cell density and oil accumulation on yield. However, traditional Chinese medicine emphasizes the holistic concept, and the study of only volatile oil components is not comprehensive enough.

In recent years, some scholars have applied electronic sensory equipment to the quality evaluation of sample appearance characteristics. Among them, the electronic eye is an electronic visual analysis system that can simulate the human eye and can identify and detect the appearance, color, and shape of the sample [12]. The color of Chinese medicinal materials is the external manifestation of the internal chemical composition, which is closely related to the variety, origin, harvest time, processing method, and storage conditions of the medicinal materials. The determination of chromaticity values by electronic eyes can objectively and quantitatively evaluate the quality of medicinal materials, which is of great significance to the quality control of Chinese medicine. Therefore, this study used electronic eye technology to measure the color of MF from the perspective of appearance and objectively characterized the quality differences of MF at different development stages through the indicators of lightness (L*), red-green value (a*), yellow-blue value (b*), and total color value (E*ab).

Therefore, the samples of MF in different developmental stages were collected from Nanzhao County, Henan Province. This study used GC-MS and UPLC to establish a detection method for the main chemical components of MF, determine the content of volatile oils and lignans, and combine the color data of medicinal materials to explore the correlation between the accumulation of chemical components and color changes in MF during development. In order to reveal the dynamic changes in relevant indicators in the development of MF and provide a reference for the harvesting, evaluation, and application of MF.

2. Materials and Methods

2.1. Materials and Reagents

The reference standards of pinoresinol dimethyl ether, magnolin, epimagnolin A, and fargesin were obtained from Weikeqi (Sichuan, China). The compounds had purities higher than 98%. Mass spectrometry grade formic acid and acetonitrile were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Deionized water was prepared by a Milli-Q system (Millipore, Burlington, MA, USA).

The samples of MF used in this study were collected from Nanzhao County, Nanyang City, Henan Province (longitude 112.754286°, latitude 33.47847099°). They were picked from 30 September 2023, every 15 days, and ended on 15 February 2024. They were numbered S1~S10, and 3 batches were picked in parallel on each date, marked as S1–1, S1–2, S1–3, etc. The detailed information of the MF samples is shown in Table 1. All the samples were authenticated by Professor Suiqing Chen (Henan University of Chinese Medicine). After picking, the samples were naturally dried, crushed, sieved through a No. 2 sieve, and sealed to avoid light.

2.2. Extraction of Volatile Oil

According to the volatile oil determination method in the Pharmacopoeia of the People’s Republic of China 2020, weigh 25.00 g of powder (Through No. 2 sieve), add 500 mL of ultrapure water, soak for 1 h, place in an electric heating mantle and slowly heat to boiling, keep it at a slight boil for 5 h, and record the content of volatile oil after cooling [13,14]. Collect the extracted volatile oil into an EP tube, dehydrate and dry it with anhydrous sodium sulfate, accurately draw 10 µL of volatile oil, dissolve it with n-hexane and make it up to 1 mL, shake it well, and wait for GC-MS detection.

2.3. GC-MS Analysis Conditions

GC analysis was performed on a TSQ 8000 Evo gas spectrometer (Thermo Finnigan, San Jose, CA, USA). The chromatographic column is a TG-5MS (30 m × 0.25 mm, 0.25 μm) quartz capillary column; the carrier gas is helium, and the carrier gas flow rate is 1 mL/min; Heating program: initial temperature 50 °C, keep for 3 min, 5 °C/min heating to 80 °C, keep for 2 min, then at 4 °C/min heating to 125 °C, keep for 10 min, then 5 °C/min heating up to 150 °C, keep for 2 min, and finally at 30 °C/min heating up to 300 °C, hold for 5 min; The injection port and detector temperature were both set to 250 °C, the split ratio was 50:1, and the injection volume was 1 μL. The mass spectrometry conditions were EI ion source, collision energy 70 eV, interface temperature: 280 °C, data acquisition in full scan mode, and the scan range was m/z 35–550 [15,16].

2.4. UPLC Chromatographic Conditions

UPLC analysis was performed on a Waters ACQUITY UPLC I-Class ultra-high performance liquid chromatograph (Waters Corporation, Milford, MA, USA). The chromatographic column was Waters ACQUITY UPLC® BEH C18 (2.1 × 100 mm, 1.7 μm); the mobile phase was acetonitrile−0.1% formic acid aqueous solution (40: 60), and the isocratic elution mode was adopted; the detection wavelength was 278 nm; the column temperature was 30 °C; the flow rate was 0.3 mL/min; and the injection volume was 2 μL.

2.5. Mass Spectrometry Conditions

MS identification was integrated on an UltiMate3000-LTQ Orbitrap liquid-mass spectrometer (Thermo Fisher Scientific, USA). An electrospray ion source (ESI) was used in positive ion mode detection: ion spray voltage 4.2 KV; capillary voltage 20 V; tube lens voltage 90 V; capillary temperature 350 °C; sheath gas (N₂) flow rate 40 arb; auxiliary gas (He) flow rate 10 arb; mass scan range: m/z 50–1500.

2.6. Preparation of Reference Solution

Accurately weigh appropriate amounts of pinoresinol dimethyl ether, magnolin, epimagnolin A, and fargesin reference substances, and add methanol to prepare mixed reference substance solutions with concentrations of 1.74, 3.26, 0.96, and 1.08 mg/mL, respectively.

2.7. Preparation of Test Solution

Accurately weigh 0.5 g of MF sample powder, place in a stoppered conical flask, add 20 mL of chloroform, stopper, weigh, soak for 0.5 h, ultrasonically treat (power 250 W, frequency 40 kHz) for 0.5 h, take out, cool, weigh again, make up the lost weight with chloroform, shake well, filter, accurately measure 10 mL of the filtrate, evaporate to dryness, dissolve the residue with 10 mL of methanol, and filter through a 0.22 μm microporous filter membrane to obtain.

3. Results

3.1. Characterization of Volatile Compounds

3.1.1. Comparison of Volatile Oil Content in MF at Different Developmental Stages

A photo of the MF (Figure 1A). To compare volatile oils extracted from MF at different developmental stages and understand the variations during development. It can be found that the total amount of volatile oil in different developmental stages of MF is significantly different. The volatile oil content has been increasing since September, reaching its highest point at the end of November. After December, the content no longer increases and begins to show a slight downward trend, as shown in Figure 1B. However, all 30 batches of samples meet the requirements of the Pharmacopoeia that the volatile oil content should not be less than 1.0% (mL/g) [2].

3.1.2. Analysis of Chemical Components in Volatile Oils from MF at Different Developmental Stages

On the basis of comparing the total volatile oil content of MF at different developmental stages, the GC-MS method was further used to qualitatively analyze its volatile components [17]. The total ion chromatograms (TICs) of volatile oils from MF at different developmental stages are shown in Figure 2. The volatile oil of MF was qualitatively identified by combining the 14-mass spectral library of the National Institute of Standards and Technology (NIST) with the retention index, and the compounds with the highest mass spectrum similarity and retention index proximity were selected as the final identification results [18,19]. Finally, 85 volatile components were identified from MF at different developmental stages. The relative content of each volatile component was calculated by the peak area normalization method, as shown in Table 2. From the GC-MS analysis results, it can be seen that there is basically no difference in the types of volatile components of the MF at different developmental stages, and all contain the 85 components identified. Among the identified components, 1,8-cineole has the highest relative content (14.29~24.19%), followed by (2E,6E)-farnesol (3.23~19.29%) and α-terpineol (5.16~9.87%). In addition, the relative contents of α-cadinol, (+)-δ-cadinene, β-pinene, (−)-camphor, linalool, etc. are also relatively high, all above 1%.

3.1.3. Analysis of Differences in Volatile Components

By calculating the predicted variable importance for the projection (VIP), we can measure the influence and explanatory power of each chemical component on the classification and discrimination of each group of samples. The larger the VIP value of a compound, the greater its contribution to the main component as a key differential metabolite [20]. In order to further screen the differential components of the volatile oil of MF at different developmental stages, the relative mass fractions of 85 common peaks in 30 batches of samples were imported into SIMCA14.1 software for supervised partial least squares discriminant analysis (PLS-DA), and 20 compounds with VIP values greater than 1 were obtained, as shown in Figure 3.

Screening with VIP value >2 as the condition, 6 differential components were obtained, namely farnesol, sabinene, β-pinene, eucalyptol, limonene, and (−)-camphor. The relative content change trends are shown in Figure 4. Further comparison of the dynamic change trends of these 6 components showed that: with the delay of the harvest period, the relative percentage content of 1,8-cineole, β-pinene, sabinene, and limonene generally showed an upward trend, while the relative percentage content of (2E,6E)-farnesol and (−)-camphor showed a gradual downward trend, as shown in Figure 4.

3.2. Research on Lignan Compounds

3.2.1. Establishment of the Characteristic Map of MF and Similarity Evaluation

Thirty batches of MF test solution were sampled and tested, and the chromatograms were recorded and imported into the “Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System”. The S5–3 sample was used as the reference spectrum (R), and the time window width was set to 0.1 min. The median method and multi-point correction method were used to automatically match and establish the characteristic spectrum, as shown in Figure 5. According to the matching results, a total of 9 common peaks were determined. By comparing with the reference substances, 4 of them were identified, namely peak 3 for pinoresinol dimethyl ether, peak 4 for magnolinone, peak 6 for epimagnolinone A, and peak 9 for magnolinone, as shown in Figure 6. The calculation results of similarity showed that the similarity of the 30 batches of MF spectrum was high, ranging from 0.968 to 0.999, indicating that the overall change trend of the main components of MF during its development process was basically the same.

3.2.2. Mass Spectrometry Qualitative Analysis

The supernatant of the test solution was injected for analysis, and the total ion chromatogram of the positive ion mode of MF was obtained, as shown in Figure 7. Based on the information of multi-stage mass spectrometry ion fragments, combined with the comparison of reference materials and related literature, 8 components were identified [21,22]. They are peak 1: eudesmin; peak 3: pinoresinol dimethyl ether; peak 4: magnolin; peak 5: lirioresinol B dimethyl ether; peak 6: epimagnolin A; peak 7: demethoxyaschantin; peak 8: aschantin; and peak 9: fargesin, all of which are lignan components. Details are shown in Table 3.

Relevant information was found to have a diquaternary tetrahydrofuran ring structure in all eight lignans, and it was speculated that these components had similar cracking rules [23]. The rules can be found by observing the mass spectra of these eight components: in the primary mass spectra of each component, the abundance of the ion peak [M]⁺ or [M + H]⁺ is relatively small, while the abundance of [M + H-H₂O]⁺ is the largest. It is speculated that this is because the furan lignans contain two furan rings, which are easy to combine with “H” atoms, thereby removing a “H₂O” molecule.

3.2.3. Multi-Component Content Determination

The standard curves of pinoresinol dimethyl ether, magnolin, epimagnolin A, and fargesin were drawn by diluting the mixed reference solution to different concentrations, with R² > 0.999, indicating a good linear correlation (Table 4). In addition, the relative standard deviations of precision (0.84–1.09%), repeatability (2.82–2.96%), stability (2.31–2.86%), and recovery rate (1.53–2.15%) of this method are all below 3.00%, and the recovery rate is 99.88–101.27%. The above results show that this method is effective and accurate for quantitative analysis of lignans.

Samples of 30 batches of test solutions of MF at different developmental stages were injected for detection, and the contents (%) of four lignans in the samples were calculated (Figure 8). The results showed that the contents of pinoresinol dimethyl ether, magnolin, epimagnolin A, and fargesin in 30 batches of samples were 1.92~4.78%, 4.69~9.15%, 0.91~2.47%, and 0.24~0.79%, respectively. The Chinese Pharmacopoeia stipulates that MF must contain no less than 0.40% magnolin (as the reference standard) [2]. All 30 tested batches complied with this requirement. The content change trend of the above four components on different harvest dates is basically the same, showing a trend of first rising and then falling, and the content accumulation reaches the highest between 15 November and 15 December.

3.3. Determination of Colorimetric Values in MF at Different Developmental Stage

Figure 9 shows the powder samples of MF at different developmental stages. Visual observation of the powder samples of 30 batches of MF found that the color of the powder samples showed a trend of dark brown, brownish yellow, and yellowish brown as the harvest period was delayed. In order to analyze the color of medicinal materials more quickly and intuitively, this study used the IRIS visual analyzer to measure the samples [12]. The instrument was turned on for 20 s. min, put the 24-color color calibration plate into the instrument for calibration, using Basler-12 mm lens, light source D65, and top and bottom lighting, and repeat the shooting mode. Take an appropriate amount of MF powder and place it evenly in the sample plate to collect images. Repeat the measurement 3 times and take the average value. Calculate the lightness (L*), red-green color (a*), yellow-blue color (b*), and total chromaticity (E*ab) of each sample. Details are shown in Table 5. The analysis results show that with the development of MF, L* first rises and then decreases slightly, indicating that the color of MF first becomes brighter and then slightly darker; a* has no obvious change; b* first rises and then decreases slightly and is positive, indicating that as the MF grows, its color tends to be more yellow; and E*ab first rises significantly and then decreases slightly, indicating that the color of MF powder is gradually becoming lighter.

3.4. Correlation Analysis Between Main Components and Chromaticity Values in MF

During the development of MF, the chemical components and chromaticity values contained in MF are changing. Is there any correlation between the two? In order to further explore this issue, the relative contents of 6 differential compounds, the peak areas of 9 common peaks, and 4 chromaticity values (L*, a*, b*, E*ab) in MF were imported into SPSS 26.0 software for Pearson correlation analysis, and a heat map was made, as shown in Figure 10. Red indicates that the two are positively correlated, and blue indicates that the two are negatively correlated. The Pearson correlation coefficients with significant differences are marked in the figure. The results showed that peak 5, peak 8, sabinene, β-pinene, limonene, and 1,8-cineole were significantly positively correlated with L*, indicating that the increase in the content of these chemical components will cause the sample color to become brighter; peak 1, (2E,6E)-farnesol, and (−)-camphor were significantly negatively correlated with L*, indicating that the increase in the content of these chemical components will cause the sample color to become darker. Peak 1, (2E,6E)-farnesol, and (−)-camphor are significantly positively correlated with a* and significantly negatively correlated with b*, indicating that the increase in these chemical components will lead to an increase in the redness value of the sample and a decrease in the yellowness value; sabinene, β-pinene, limonene, and 1,8-cineole are significantly negatively correlated with a* and significantly positively correlated with b*, indicating that the increase in these chemical components will lead to a decrease in the redness value of the sample and an increase in the yellowness value. Peak 8, sabinene, β-pinene, limonene, and 1,8-cineole are significantly positively correlated with E*ab, indicating that the increase in these chemical components will cause the sample color to become lighter; Peak 1, (2E,6E)-farnesol, and (−)-camphor are significantly negatively correlated with E*ab, indicating that the increase in these chemical components will cause the sample color to become darker.

4. Discussion

The harvesting time of Chinese herbal medicine is an important factor in determining the content of its active ingredients [24]. The volatile oil in MF is its main medicinal ingredient [1]. In this study, steam distillation was employed to extract volatile oils from 30 batches of MF samples collected across 10 different developmental stages. The results showed that the volatile oil content initially increased and then decreased as the harvesting period progressed. It is speculated that this trend is mainly the result of the combined effects of plant physiological metabolism and environmental conditions. In September, when the buds of MF are in the early stage of growth, the photosynthetic products and secondary metabolites of the plants begin to accumulate, but the total amount is low. As time goes by, the plant metabolism gradually reaches its peak, and the volatile oil content is the highest at this time. When the buds are close to opening, the volatile oil components may be converted into other substances, so they gradually decrease. Similarly, the volatile component content of Magnolia kobus flowers is different in the early flowering period and the full flowering period [9]. This result is also confirmed. GC-MS analysis was conducted to compare the volatile components, leading to the identification of 85 chemical compounds, predominantly terpenes and terpene alcohols. Among the identified constituents, the major components in MF volatile oil included 1,8-cineole, (2E,6E)-farnesol, α-terpineol, α-cadinol, (+)-δ-cadinene, β-pinene, and (−)-camphor. Studies report that 1,8-cineole and (2E,6E)-farnesol are the main bioactive compounds, exhibiting antibacterial, anti-inflammatory, antioxidant, and anti-allergic properties, making them effective in treating allergic asthma [25,26]. Further analysis revealed that while the types of volatile compounds remained consistent across different developmental stages, their relative concentrations varied significantly [27]. A comparative study identified six differential components among the 85 volatile compounds: (2E,6E)-farnesol, sabinene, β-pinene, 1,8-cineole, limonene, and (−)-camphor. Investigation into their concentration trends showed distinct fluctuation patterns, the underlying causes of which warrant further exploration.

Lignans are another major active ingredient of MF [6]. This study investigated the extraction solvent, extraction method, mobile phase, and detection wavelength and established a detection method when the extraction efficiency was high, the baseline was stable, and the separation was good. The UPLC characteristic spectrum of MF was obtained, and a total of 9 common peaks were calibrated. The similarity evaluation results showed that the similarity of 30 batches of MF samples was above 0.968, indicating that the types of chemical components did not change during the development of MF, and the overall change trend of the content of the components contained was basically the same [28]. Quantitative analysis was performed on four lignan constituents in MF: pinoresinol dimethyl ether, magnolin, epimagnolin A, and fargesin, with their temporal variation patterns examined. Finally, it was found that with the development of the flower buds of MF, the lignan and volatile oil components increased first and then decreased. Likewise, it is speculated that this result is similar to the changes in volatile oils, which are mainly related to the laws of plant development and the regulation of secondary metabolism.

With the development of MF, in addition to the changes in chemical composition, the color of its powder is also changing accordingly. The contents of total volatile oil, pinoresinol dimethyl ether, magnolin, epimagnolin A, and magnolin in 30 batches of MF reached the highest level between 15 November and 30 December. At this time, the color of the sample was brighter and more yellowish. The L* value, b* value, and E*ab value were all larger. In order to further explore the relationship between the powder color of MF at different developmental stages and its internal chemical composition, the six differential components of volatile oil of MF, the nine common peaks of the characteristic spectrum, and the four chromaticity values of L*, a*, b*, and E*ab were analyzed for correlation. The results showed that the color of MF was significantly correlated with the relative content of the six volatile components and the peak area of some chromatographic peaks in the characteristic spectrum, indicating that color, as an important indicator for evaluating the quality of medicinal materials, can reflect the changes in endogenous substances of medicinal materials to a certain extent.

The results showed that the content of active ingredients in MF from 15 November to 30 December was higher and the quality of the medicinal materials was the best. This conclusion was consistent with the actual harvesting period of the origin. This study took samples of MF at different developmental stages as the research objects, systematically analyzed the dynamic changes in volatile oil components and lignan components, and further explained the correlation between the changes in the intrinsic chemical composition of MF and the chromaticity value. The optimal bud-picking period of MF as a medicinal “Xinyi” was clarified, which provided an important reference value for the quality control and scientific picking of MF medicinal materials and had certain practical significance.

5. Conclusions

The flower buds of MF contain a diverse array of bioactive phytochemicals. The chemical constituents of MF in 10 developmental stages are basically the same, but the contents of volatile oil and lignans in each developmental stage are obviously different. The harvest time will have a certain impact on the samples of MF. With the development of the buds of MF, the internal chemical composition and appearance color are changing, and there is an obvious correlation between them. The medicinal component content of MF is the highest in November and December, which can be used as the optimal harvesting period. This study established a more comprehensive quality evaluation method, which facilitates a better understanding of the chemical composition differences in MF at different developmental stages. It provides guidance for the rational clinical selection and use of medicinal materials and offers data support for determining the optimal harvesting period of MF.

Author Contributions

Conceptualization, C.B. and Q.Z.; data curation, C.B., Q.Z. and X.S.; formal analysis, C.B. and Q.Z.; writing—first draft preparation, C.B. and. S.C.; writing—review and editing, C.B., Q.Z., X.S. and S.C.; funding acquisition, S.C. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

All data supporting the findings of this study are include within the article.

Conflicts of Interest

The authors declare no competing financial interest.

Abbreviations

The following abbreviations are used in this manuscript:

MF	Magnoliae Flos
GC-MS	gas chromatography-mass spectrometry
UPLC	ultra-high performance liquid chromatography
TICs	total ion chromatograms
NIST	National Institute of Standards and Technology
VIP	variable importance for the projection
PLS-DA	partial least squares discriminant analysis

Footnotes

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Figures and Tables

Figure 1 The Photo of MF (A) and Volatile oil content in MF at different developmental stages (B).

Figure 2 Total ion chromatograms of volatile oil from MF.

Figure 3 VIP diagram of MF PLS-DA at different developmental stages.

Figure 4 Relative content trend of 6 volatile components.

Figure 5 UPLC characteristic spectra of 30 batches of MF samples.

Figure 6 UPLC chromatograms of (a) mixed reference standards and (b) MF sample. 3: pinoresinol dimethyl ether; 4: magnolin; 6: epimagnolin A; 9: fargesin.

Figure 7 Total ion chromatogram of MF in positive ion mode.

Figure 8 Temporal variation in lignan constituents in MF.

Figure 9 10 groups of MF sample powders at different developmental stage.

Figure 10 Correlation heat map between main components and chromaticity values of MF. Note: * indicates significant correlation at the 0.05 level (bilateral); ** indicates significant correlation at the 0.01 level (bilateral).

Table 1

Information on MF with different developmental stages.

No.	Harvest Date
S1–1~S1–3	2023.9.30
S2–1~S2–3	2023.10.15
S3–1~S3–3	2023.10.30
S4–1~S4–3	2023.11.15
S5–1~S5–3	2023.11.30
S6–1~S6–3	2023.12.15
S7–1~S7–3	2023.12.30
S8–1~S8–3	2024.1.15
S9–1~S9–3	2024.1.30
S10–1~S10–3	2024.2.15

Table 2

Summary of volatile compounds in MF identified by GC-MS (mean ± SD, n = 3).

NO.	Compound	RI	Relative Content (%)
			S1	S2	S3	S4	S5	S6	S7	S8	S9	S10
Alkene
1	Cycloheptatriene	778	0.02 ± 0.00	0.02 ± 0.00	0.02 ± 0.00	0.02 ± 0.00	0.02 ± 0.00	0.02 ± 0.00	0.01 ± 0.00	0.01 ± 0.00	0.02 ± 0.00	0.01±0.00
2	α-Thujene	924	0.04 ± 0.00	0.03 ± 0.02	0.06 ± 0.02	0.04 ± 0.02	0.05 ± 0.01	0.25 ± 0.17	0.20 ± 0.05	0.19 ± 0.03	0.21 ± 0.04	0.20 ± 0.01
3	α-Pinene	930	0.52 ± 0.09	0.48 ± 0.22	0.80 ± 0.20	0.68 ± 0.26	0.93 ± 0.19	2.69 ± 1.80	2.70 ± 0.46	2.28 ± 0.30	2.76 ± 0.40	2.90 ± 0.27
4	Camphene	942	0.26 ± 0.06	0.15 ± 0.06	0.28 ± 0.04	0.21 ± 0.08	0.28 ± 0.04	0.29 ± 0.09	0.64 ± 0.35	0.33 ± 0.06	0.51 ± 0.36	1.14 ± 1.01
5	Sabinene	964	0.74 ± 0.09	0.74 ± 0.20	1.42 ± 0.11	1.41 ± 0.31	1.85 ± 0.60	4.37 ± 2.64	5.27 ± 0.15	5.72 ± 0.91	5.16 ± 0.87	5.37 ± 1.27
6	β-Pinene	966	2.09 ± 0.21	2.09 ± 0.73	3.25 ± 0.67	2.84 ± 0.85	3.59 ± 0.71	6.70 ± 3.45	6.69 ± 0.87	6.22 ± 0.48	6.97 ± 0.76	7.06 ± 0.58
7	Myrcene	980	0.25 ± 0.01	0.19 ± 0.05	0.46 ± 0.21	0.28 ± 0.07	0.31 ± 0.06	1.33 ± 0.97	1.26 ± 0.14	1.33 ± 0.17	1.26 ± 0.18	1.34 ± 0.24
8	α-Phellandrene	992	0.03 ± 0.00	0.02 ± 0.01	0.04 ± 0.00	0.03 ± 0.01	0.03 ± 0.01	0.17 ± 0.13	0.13 ± 0.04	0.12 ± 0.03	0.13 ± 0.01	0.09 ± 0.04
9	Carene	997	0.01 ± 0.00	0.01 ± 0.00	0.01 ± 0.00	0.01 ± 0.00	0.01 ± 0.00	0.06 ± 0.04	0.05 ± 0.02	0.04 ± 0.01	0.05 ± 0.00	0.03 ± 0.02
10	α-Terpinene	1004	0.39 ± 0.07	0.32 ± 0.11	0.57 ± 0.16	0.36 ± 0.11	0.40 ± 0.04	1.16 ± 0.69	0.90 ± 0.24	0.93 ± 0.12	0.94 ± 0.11	0.79 ± 0.21
11	o-Cymene	1012	0.35 ± 0.02	0.25 ± 0.05	0.39 ± 0.05	0.41 ± 0.16	0.43 ± 0.15	1.49 ± 1.11	1.79 ± 0.56	1.31 ± 0.41	1.55 ± 0.04	1.19 ± 0.50
12	Limonene	1017	0.71 ± 0.21	0.71 ± 0.10	1.31 ± 0.13	0.95 ± 0.43	1.15 ± 0.19	4.73 ± 3.63	4.80 ± 1.19	3.79 ± 0.76	4.79 ± 0.14	4.39 ± 1.16
14	(Z)-β-Ocimene	1040	0.02 ± 0.00	0.02 ± 0.01	0.04 ± 0.00	0.03 ± 0.01	0.03 ± 0.01	0.12 ± 0.08	0.12 ± 0.02	0.11 ± 0.02	0.10 ± 0.00	0.10 ± 0.03
15	γ-Terpinene	1052	1.00 ± 0.13	0.83 ± 0.23	1.46 ± 0.33	1.05 ± 0.32	1.15 ± 0.14	2.97 ± 1.91	2.36 ± 0.58	2.48 ± 0.41	2.59 ± 0.18	1.98 ± 0.71
17	Terpinolene	1087	0.32 ± 0.03	0.21 ± 0.04	0.35 ± 0.05	0.24 ± 0.06	0.25 ± 0.02	0.38 ± 0.11	0.39 ± 0.02	0.38 ± 0.02	0.39 ± 0.10	0.48 ± 0.18
19	4-Thujanol	1098	0.08 ± 0.07	0.15 ± 0.09	0.14 ± 0.08	0.27 ± 0.10	0.24 ± 0.07	0.22 ± 0.17	0.18 ± 0.05	0.32 ± 0.09	0.14 ± 0.02	0.21 ± 0.10
39	δ-Elemene	1336	0.02 ± 0.01	0.04 ± 0.02	0.03 ± 0.00	0.04 ± 0.02	0.05 ± 0.01	0.02 ± 0.01	0.04 ± 0.00	0.03 ± 0.02	0.03 ± 0.01	0.02 ± 0.02
40	(-)-α-Cubebene	1348	0.21 ± 0.03	0.27 ± 0.02	0.31 ± 0.09	0.30 ± 0.08	0.32 ± 0.03	0.22 ± 0.01	0.20 ± 0.02	0.24 ± 0.04	0.23 ± 0.04	0.17 ± 0.04
41	α-Copaene	1375	0.21 ± 0.12	0.25 ± 0.11	0.34 ± 0.05	0.31 ± 0.16	0.36 ± 0.01	0.22 ± 0.04	0.30 ± 0.03	0.20 ± 0.04	0.29 ± 0.04	0.20 ± 0.13
42	β-Cubebene	1389	0.04 ± 0.02	0.06 ± 0.03	0.06 ± 0.01	0.07 ± 0.03	0.10 ± 0.01	0.06 ± 0.02	0.09 ± 0.01	0.09 ± 0.06	0.08 ± 0.01	0.06 ± 0.04
43	β-Elemene	1391	0.12 ± 0.06	0.17 ± 0.07	0.19 ± 0.02	0.21 ± 0.10	0.24 ± 0.04	0.12 ± 0.02	0.19 ± 0.03	0.12 ± 0.05	0.16 ± 0.06	0.11 ± 0.07
44	α-cis-Bergamotene	1414	0.04 ± 0.02	0.07 ± 0.03	0.05 ± 0.00	0.06 ± 0.04	0.11 ± 0.02	0.06 ± 0.02	0.09 ± 0.01	0.05 ± 0.02	0.07 ± 0.02	0.05 ± 0.04
45	β-Caryophyllene	1417	2.30 ± 1.01	3.61 ± 1.33	2.74 ± 0.39	3.37 ± 1.46	4.75 ± 0.88	3.14 ± 1.26	3.86 ± 0.91	2.29 ± 0.83	3.46 ± 0.87	2.77 ± 2.01
46	β-Copaene	1425	0.08 ± 0.04	0.11 ± 0.05	0.11 ± 0.02	0.10 ± 0.06	0.12 ± 0.00	0.05 ± 0.01	0.06 ± 0.01	0.04 ± 0.01	0.07 ± 0.01	0.04 ± 0.04
47	α-Bergamotene	1432	0.03 ± 0.02	0.06 ± 0.03	0.06 ± 0.00	0.06 ± 0.02	0.08 ± 0.01	0.05 ± 0.01	0.07 ± 0.01	0.04 ± 0.01	0.06 ± 0.01	0.04 ± 0.03
48	cis-Muurola-3,5-diene	1441	0.04 ± 0.03	0.04 ± 0.02	0.07 ± 0.02	0.06 ± 0.05	0.05 ± 0.02	0.03 ± 0.00	0.03 ± 0.00	0.03 ± 0.00	0.04 ± 0.00	0.02 ± 0.01
49	trans-Muurola-3,5-diene	1445	0.05 ± 0.03	0.04 ± 0.02	0.08 ± 0.03	0.06 ± 0.05	0.06 ± 0.02	0.04 ± 0.00	0.05 ± 0.01	0.04 ± 0.02	0.05 ± 0.00	0.03 ± 0.02
50	α-Caryophyllene	1448	0.52 ± 0.24	0.67 ± 0.21	0.52 ± 0.07	0.57 ± 0.23	0.79 ± 0.09	0.49 ± 0.17	0.62 ± 0.08	0.37 ± 0.07	0.53 ± 0.09	0.43 ± 0.27
51	(E)-β-Farnesene	1453	0.62 ± 0.39	0.97 ± 0.51	0.65 ± 0.15	0.75 ± 0.42	1.28 ± 0.24	0.57 ± 0.26	1.06 ± 0.15	0.52 ± 0.22	0.75 ± 0.20	0.54 ± 0.48
52	Alloaromadendrene	1455	0.28 ± 0.15	0.30 ± 0.13	0.50 ± 0.03	0.42 ± 0.26	0.40 ± 0.08	0.24 ± 0.03	0.34 ± 0.05	0.29 ± 0.15	0.36 ± 0.02	0.24 ± 0.16
53	cis-Muurola-4(15),5-diene	1456	0.11 ± 0.06	0.10 ± 0.04	0.15 ± 0.04	0.13 ± 0.12	0.09 ± 0.07	0.04 ± 0.02	0.04 ± 0.00	0.04 ± 0.03	0.07 ± 0.04	0.03 ± 0.02
54	γ-Muurolene	1473	0.39 ± 0.23	0.49 ± 0.21	0.73 ± 0.13	0.60 ± 0.33	0.70 ± 0.02	0.43 ± 0.06	0.66 ± 0.08	0.45 ± 0.13	0.59 ± 0.07	0.44 ± 0.29
55	Germacrene D	1476	1.66 ± 0.83	2.94 ± 1.31	3.00 ± 0.56	3.85 ± 1.25	5.31 ± 0.70	3.54 ± 1.18	4.76 ± 0.52	3.39 ± 0.60	3.94 ± 0.78	3.39 ± 1.77
56	α-Curcumene	1480	0.05 ± 0.02	0.05 ± 0.02	0.07 ± 0.00	0.06 ± 0.03	0.08 ± 0.01	0.04 ± 0.00	0.05 ± 0.00	0.05 ± 0.01	0.05 ± 0.00	0.04 ± 0.02
57	Bicyclosesquiphellandrene	1487	0.10 ± 0.06	0.09 ± 0.04	0.16 ± 0.04	0.13 ± 0.09	0.13 ± 0.03	0.10 ± 0.01	0.13 ± 0.02	0.08 ± 0.04	0.11 ± 0.01	0.07 ± 0.05
59	Bicyclogermacrene	1493	0.63 ± 0.27	1.05 ± 0.42	0.91 ± 0.08	1.09 ± 0.40	1.37 ± 0.16	0.75 ± 0.32	0.95 ± 0.12	0.69 ± 0.16	0.96 ± 0.17	0.72 ± 0.48
60	α-Muurolene	1499	0.98 ± 0.52	0.97 ± 0.34	1.35 ± 0.22	1.20 ± 0.64	1.29 ± 0.16	0.86 ± 0.04	1.18 ± 0.17	0.86 ± 0.27	1.10 ± 0.10	0.83 ± 0.46
61	α-Bulnesene	1504	0.04 ± 0.02	0.04 ± 0.02	0.06 ± 0.01	0.05 ± 0.02	0.07 ± 0.01	0.06 ± 0.02	0.08 ± 0.01	0.06 ± 0.02	0.06 ± 0.00	0.05 ± 0.03
62	Farnesene	1510	0.26 ± 0.18	0.19 ± 0.10	0.61 ± 0.17	0.41 ± 0.24	0.40 ± 0.10	0.23 ± 0.11	0.21 ± 0.04	0.29 ± 0.13	0.23 ± 0.01	0.20 ± 0.14
63	(+)-γ-Cadinene	1513	1.28 ± 0.69	1.32 ± 0.50	1.96 ± 0.31	1.79 ± 1.23	1.68 ± 0.30	1.01 ± 0.24	1.18 ± 0.19	0.97 ± 0.32	1.17 ± 0.06	0.85 ± 0.46
64	(+)-δ-Cadinene	1526	4.42 ± 2.50	4.26 ± 1.65	6.93 ± 1.02	5.77 ± 3.30	5.76 ± 0.89	4.28 ± 0.66	5.28 ± 0.77	4.06 ± 1.70	5.09 ± 0.30	3.86 ± 2.17
65	Cadinadiene	1534	0.10 ± 0.06	0.09 ± 0.04	0.15 ± 0.03	0.12 ± 0.08	0.11 ± 0.02	0.08 ± 0.00	0.10 ± 0.01	0.08 ± 0.02	0.11 ± 0.00	0.07 ± 0.04
66	α-Cadinene	1537	0.21 ± 0.13	0.20 ± 0.09	0.31 ± 0.05	0.28 ± 0.21	0.24 ± 0.06	0.16 ± 0.01	0.20 ± 0.03	0.15 ± 0.06	0.21 ± 0.02	0.14 ± 0.08
67	α-Calacorene	1545	0.03 ± 0.00	0.04 ± 0.01	0.04 ± 0.01	0.04 ± 0.02	0.04 ± 0.00	0.03 ± 0.01	0.02 ± 0.00	0.02 ± 0.01	0.02 ± 0.00	0.02 ± 0.00
68	Germacrene B	1558	0.03 ± 0.00	0.04 ± 0.01	0.03 ± 0.01	0.05 ± 0.02	0.05 ± 0.01	0.02 ± 0.00	0.03 ± 0.00	0.02 ± 0.00	0.02 ± 0.01	0.02 ± 0.00
74	Humulene epoxide ii	1604	0.03 ± 0.01	0.02 ± 0.00	0.02 ± 0.00	0.02 ± 0.00	0.03 ± 0.00	0.01 ± 0.00	0.01 ± 0.00	0.01 ± 0.00	0.02 ± 0.00	0.02 ± 0.00
Alcohol
13	1,8-Cineole	1020	14.47 ± 3.76	14.29 ± 2.70	18.54 ± 3.77	18.64 ± 5.86	17.51 ± 1.74	21.61 ± 3.22	18.13 ± 1.30	24.19 ± 1.90	19.44 ± 3.07	20.86 ± 2.38
16	(Z)-Sabinene hydrate	1062	0.11 ± 0.10	0.21 ± 0.12	0.18 ± 0.09	0.38 ± 0.15	0.35 ± 0.14	0.32 ± 0.26	0.28 ± 0.09	0.47 ± 0.14	0.19 ± 0.02	0.28 ± 0.12
20	Linalool	1100	5.22 ± 1.69	5.76 ± 1.85	3.20 ± 1.15	4.32 ± 1.93	4.15 ± 0.15	3.60 ± 0.60	3.10 ± 0.60	2.90 ± 1.84	2.86 ± 0.60	2.78 ± 1.30
21	Fenchol	1113	0.09 ± 0.06	0.13 ± 0.02	0.02 ± 0.01	0.06 ± 0.04	0.07 ± 0.00	0.03 ± 0.02	0.05 ± 0.02	0.04 ± 0.02	0.04 ± 0.00	0.03 ± 0.03
22	(Z)-ρ-Menth-2-en-1-ol	1122	0.14 ± 0.02	0.11 ± 0.03	0.13 ± 0.00	0.11 ± 0.04	0.10 ± 0.02	0.12 ± 0.05	0.08 ± 0.01	0.11 ± 0.03	0.09 ± 0.02	0.13 ± 0.07
23	(E)-ρ-Menth-2-en-1-ol	1140	0.05 ± 0.01	0.06 ± 0.02	0.05 ± 0.02	0.05 ± 0.02	0.04 ± 0.00	0.05 ± 0.01	0.03 ± 0.01	0.05 ± 0.02	0.04 ± 0.01	0.04 ± 0.01
25	Camphene hydrate	1148	1.29 ± 0.54	0.14 ± 0.05	0.47 ± 0.38	0.23 ± 0.19	0.31 ± 0.36	0.20 ± 0.31	0.25 ± 0.21	0.12 ± 0.06	0.18 ± 0.24	0.54 ± 0.68
27	δ-Terpineol	1167	0.45 ± 0.09	0.50 ± 0.18	0.65 ± 0.14	0.54 ± 0.16	0.45 ± 0.09	0.61 ± 0.57	0.30 ± 0.05	0.46 ± 0.08	0.33 ± 0.08	0.36 ± 0.11
28	Terpinine-4-ol	1177	1.85 ± 0.31	1.78 ± 0.70	1.93 ± 0.59	1.72 ± 0.61	1.45 ± 0.29	1.96 ± 0.47	1.25 ± 0.16	1.82 ± 0.44	1.55 ± 0.39	1.77 ± 0.59
29	2-(4-Methylphenyl)propan-2-ol	1185	0.06 ± 0.04	0.01 ± 0.00	0.02 ± 0.01	0.01 ± 0.00	0.01 ± 0.00	0.01 ± 0.00	0.01 ± 0.00	0.01 ± 0.00	0.01 ± 0.00	0.02 ± 0.01
30	α-Terpineol	1191	7.41 ± 1.74	8.45 ± 2.95	9.87 ± 2.85	9.25 ± 2.09	7.75 ± 0.97	7.69 ± 4.85	5.16 ± 0.44	7.62 ± 0.95	5.64 ± 1.08	6.44 ± 1.48
31	(Z)-Piperitol	1195	0.03 ± 0.01	0.03 ± 0.02	0.03 ± 0.01	0.03 ± 0.01	0.02 ± 0.00	0.03 ± 0.01	0.02 ± 0.00	0.03 ± 0.00	0.03 ± 0.00	0.03 ± 0.00
32	(E)-Piperitol	1208	0.03 ± 0.01	0.03 ± 0.01	0.03 ± 0.01	0.03 ± 0.01	0.02 ± 0.00	0.03 ± 0.02	0.02 ± 0.00	0.03 ± 0.00	0.03 ± 0.00	0.03 ± 0.01
34	Citronellol	1229	2.86 ± 1.30	4.12 ± 1.18	0.63 ± 0.38	1.39 ± 0.80	1.43 ± 0.12	0.57 ± 0.36	0.75 ± 0.16	0.58 ± 0.50	0.62 ± 0.16	0.42 ± 0.52
36	Geraniol	1256	2.19 ± 1.44	3.13 ± 1.01	0.65 ± 0.09	1.24 ± 0.64	1.04 ± 0.10	0.55 ± 0.11	0.44 ± 0.10	0.52 ± 0.29	0.45 ± 0.07	0.40 ± 0.30
58	epi-Cubebol	1492	0.16 ± 0.07	0.25 ± 0.09	0.30 ± 0.05	0.30 ± 0.23	0.25 ± 0.06	0.17 ± 0.07	0.04 ± 0.02	0.14 ± 0.10	0.19 ± 0.03	0.14 ± 0.04
69	Nerolidol	1566	0.07 ± 0.02	0.08 ± 0.02	1.23 ± 1.62	0.05 ± 0.01	0.08 ± 0.04	0.44 ± 0.63	0.03 ± 0.00	0.37 ± 0.45	0.34 ± 0.37	0.39 ± 0.53
70	Spathulenol	1575	1.13 ± 0.31	2.05 ± 0.81	2.77 ± 1.16	4.37 ± 2.57	3.63 ± 0.65	2.89 ± 2.22	2.94 ± 1.16	3.85 ± 0.66	2.12 ± 0.35	2.51 ± 1.36
72	Gleenol	1586	0.06 ± 0.00	0.08 ± 0.02	0.08 ± 0.02	0.10 ± 0.04	0.07 ± 0.01	0.05 ± 0.04	0.04 ± 0.01	0.05 ± 0.01	0.05 ± 0.01	0.05 ± 0.00
75	1,10-di-epi-Cubenol	1612	0.16 ± 0.06	0.15 ± 0.02	0.18 ± 0.04	0.15 ± 0.10	0.12 ± 0.04	0.07 ± 0.03	0.07 ± 0.01	0.07 ± 0.02	0.08 ± 0.00	0.07 ± 0.00
76	1-epi-Cubenol	1627	0.32 ± 0.10	0.30 ± 0.03	0.37 ± 0.10	0.31 ± 0.17	0.22 ± 0.08	0.16 ± 0.08	0.16 ± 0.02	0.16 ± 0.06	0.18 ± 0.00	0.16 ± 0.00
77	T-Muurolol	1642	3.66 ± 0.98	3.45 ± 0.27	4.75 ± 0.92	4.41 ± 2.61	3.41 ± 0.55	2.60 ± 1.40	2.55 ± 0.58	2.75 ± 1.13	2.82 ± 0.25	2.69 ± 0.35
78	α-Muurolol	1644	0.55 ± 0.13	0.53 ± 0.06	0.71 ± 0.16	0.71 ± 0.32	0.54 ± 0.09	0.39 ± 0.22	0.38 ± 0.09	0.42 ± 0.16	0.43 ± 0.05	0.41 ± 0.06
79	α-Cadinol	1654	5.30 ± 1.38	5.40 ± 0.83	7.37 ± 1.39	7.07 ± 3.28	5.43 ± 0.56	4.64 ± 2.93	4.36 ± 1.12	4.85 ± 1.99	4.78 ± 0.71	4.90 ± 0.93
80	Eudesma-4(15),7-dien-1β-ol	1689	0.04 ± 0.00	0.05 ± 0.00	0.02 ± 0.01	0.03 ± 0.00	0.04 ± 0.01	0.01 ± 0.00	0.02 ± 0.00	0.01 ± 0.00	0.03 ± 0.01	0.02 ± 0.00
81	Shyobunol	1691	0.10 ± 0.01	0.15 ± 0.02	0.10 ± 0.03	0.15 ± 0.03	0.13 ± 0.02	0.08 ± 0.05	0.08 ± 0.01	0.08 ± 0.01	0.07 ± 0.03	0.07 ± 0.00
82	4(15),5,10(14)-Germacratrien-1-ol	1697	0.07 ± 0.01	0.10 ± 0.01	0.04 ± 0.02	0.04 ± 0.00	0.04 ± 0.01	0.02 ± 0.01	0.03 ± 0.00	0.02 ± 0.01	0.03 ± 0.01	0.02 ± 0.00
83	Farnesol	1714	0.03 ± 0.01	0.03 ± 0.00	0.01 ± 0.00	0.02 ± 0.00	0.02 ± 0.01	0.03 ± 0.02	0.01 ± 0.00	0.02 ± 0.02	0.03 ± 0.03	0.02 ± 0.01
84	(2E,6E)-Farnesol	1724	15.79 ± 2.96	19.29 ± 0.58	5.84 ± 5.52	8.62 ± 3.00	9.03 ± 2.12	3.23 ± 1.71	5.18 ± 0.52	3.70 ± 2.65	5.48 ± 3.68	3.70 ± 2.40
Ketone
18	2-Nonanone	1093	0.03 ± 0.01	0.04 ± 0.02	0.03 ± 0.00	0.03 ± 0.02	0.03 ± 0.01	0.04 ± 0.02	0.06 ± 0.01	0.08 ± 0.04	0.04 ± 0.01	0.05 ± 0.01
24	(−)-Camphor	1143	11.60 ± 2.96	1.84 ± 0.35	4.88 ± 2.74	2.73 ± 1.82	3.25 ± 3.06	2.00 ± 2.68	2.80 ± 2.26	1.49 ± 0.62	2.18 ± 2.66	5.61 ± 6.62
73	Salvial-4(14)-en-1-one	1591	0.02 ± 0.00	0.03 ± 0.00	0.01 ± 0.00	0.01 ± 0.01	0.02 ± 0.00	0.01 ± 0.00	0.01 ± 0.00	0.01 ± 0.00	0.01 ± 0.00	0.01 ± 0.00
Aldehyde
26	(+)-Citronellal	1154	0.07 ± 0.03	0.10 ± 0.01	0.02 ± 0.02	0.03 ± 0.02	0.04 ± 0.01	0.07 ± 0.07	0.09 ± 0.02	0.05 ± 0.04	0.07 ± 0.04	0.05 ± 0.05
85	(2-trans,6-trans)-Farnesal	1746	0.17 ± 0.16	0.10 ± 0.02	0.02 ± 0.02	0.03 ± 0.02	0.02 ± 0.01	0.06 ± 0.04	0.04 ± 0.01	0.04 ± 0.04	0.06 ± 0.06	0.04 ± 0.02
Phenol
38	Carvacrol	1304	0.04 ± 0.00	0.06 ± 0.02	0.03 ± 0.00	0.03 ± 0.00	0.03 ± 0.00	0.02 ± 0.01	0.03 ± 0.00	0.04 ± 0.01	0.03 ± 0.00	0.03 ± 0.01
Ester
33	Fenchyl acetate	1221	0.03 ± 0.00	0.03 ± 0.01	0.01 ± 0.00	0.02 ± 0.00	0.02 ± 0.00	0.02 ± 0.01	0.02 ± 0.00	0.02 ± 0.00	0.02 ± 0.00	0.02 ± 0.01
37	(+)-Bornyl acetate	1286	0.64 ± 0.22	0.18 ± 0.02	0.38 ± 0.18	0.24 ± 0.07	0.31 ± 0.18	0.25 ± 0.25	0.18 ± 0.10	0.19 ± 0.07	0.26 ± 0.27	0.55 ± 0.71
Ether
35	2-Isopropyl-5-methylanisole	1236	0.04 ± 0.00	0.04 ± 0.00	0.05 ± 0.02	0.04 ± 0.01	0.04 ± 0.00	0.03 ± 0.01	0.03 ± 0.01	0.04 ± 0.01	0.04 ± 0.01	0.03 ± 0.00
71	Caryophyllene oxide	1581	0.22 ± 0.05	0.26 ± 0.03	0.10 ± 0.05	0.17 ± 0.04	0.19 ± 0.02	0.11 ± 0.02	0.12 ± 0.01	0.08 ± 0.01	0.07 ± 0.02	0.07 ± 0.03

Table 3

Identification of chemical components in MF (positive ion mode).

No.	tR/min	Compound Formula	MS (ESI+)	Error/ppm	MS2 (ESI+)	Name
1	1.31	C22H26O6	387.1800 [M + H]+/369.1695 [M + H-H2O]+	−0.659	351.1591, 339.1590, 231.1009, 151.0754	eudesmin
3	4.14	C22H26O6	386.1721 [M]+/369.1693 [M + H-H2O]+	−0.621	351.1592, 339.1592, 231.1015, 151.0752	pinoresinol dimethyl ether
4	4.6	C23H28O7	417.1903 [M + H]+/399.1801 [M + H-H2O]+	−1.054	381.1698, 350.1514, 261.1121, 231.1015, 181.0858, 151.0752	magnolin
5	5.1	C24H30O8	447.2002 [M + H]+/429.1906 [M + H-H2O]+	−0.895	411.1801, 380.1620, 261.1119, 231.1014, 181.0856	lirioresinol B dimethyl ether
6	5.75	C23H28O7	416.1825 [M]+/399.1800 [M + H-H2O]+	−1.02	381.1696, 350.1512, 261.1120, 231.1015, 181.0857, 151.0751	epimagnolin A
7	6.83	C21H22O6	370.1419 [M]+/353.1383 [M + H-H2O]+	2.135	335.1277, 323.1269, 135.0435	demethoxyaschantin
8	7.63	C22H24O7	401.1517 [M + H]+/383.1489 [M + H-H2O]+	0.114	365.139	aschantin
9	8.77	C21H22O6	370.1414 [M + H]+/353.1384 [M + H-H2O]+	0.811	335.1276, 283.0961	fargesin

Table 4

Linear relationship and range of four lignans.

Compound	Regression	R2	Linear Range/(mg·mL−1)
Pinoresinol diMethyl ether	y = 5,719,722 x + 82,768	0.9992	0.0272–1.74
magnolin	y = 3,198,608 x + 81,412	0.9996	0.0510–3.26
epimagnolin A	y = 3,278,152 x + 3689	1.0000	0.0150–0.96
fargesin	y = 6,435,286 x − 7258	1.0000	0.0169–1.08

Table 5

Expected chromaticity values of MF samples at different development stages.

No.	L*	a*	b*	E*ab
S1–1	47.01	2.15	25.88	53.71
S1–2	43.08	2.81	25.40	50.09
S1–3	48.85	2.39	28.62	56.67
S2–1	51.52	1.74	26.16	57.81
S2–2	48.56	1.82	27.82	55.99
S2–3	48.58	1.90	26.53	55.39
S3–1	46.09	2.66	29.77	54.93
S3–2	50.24	2.01	30.26	58.68
S3–3	56.21	1.34	29.89	63.68
S4–1	56.12	1.57	35.26	66.30
S4–2	59.58	1.37	32.77	68.01
S4–3	53.32	1.95	35.48	64.08
S5–1	60.41	1.58	39.42	72.15
S5–2	59.43	2.05	39.73	71.52
S5–3	57.20	1.88	40.25	69.97
S6–1	65.33	1.16	42.40	77.89
S6–2	55.92	1.74	40.27	68.93
S6–3	61.85	1.62	39.49	73.40
S7–1	60.84	1.60	42.14	74.03
S7–2	58.26	1.90	42.26	72.00
S7–3	58.03	1.59	41.40	71.30
S8–1	55.78	1.14	38.62	67.85
S8–2	56.45	1.28	37.37	67.71
S8–3	57.32	1.44	40.09	69.96
S9–1	55.26	0.99	39.65	68.02
S9–2	57.98	1.08	37.88	69.27
S9–3	55.35	1.55	36.79	66.48
S10–1	57.57	1.01	37.38	68.65
S10–2	57.25	1.26	37.61	68.51
S10–3	57.23	1.27	38.50	68.99

References

1. Hu, M.L.; Bai, M.; Ye, W.; Wang, Y.L.; Wu, H. Variations in Volatile Oil Yield and Composition of “Xin-yi” (Pamp. Flower Buds) at Different Growth Stages. J. Oleo Sci.; 2018; 67, pp. 779-787. [DOI: https://dx.doi.org/10.5650/jos.ess17229]

2. Pharmacopoeiacommittee of the Ministry of Health of the People’s Republic of China. Pharmacopoeia of the People’s Republic of China; China Pharmaceutical Science and Technology: Beijing, China, 2020; Volume Part I pp. 189-190.

3. Shen, Y.; Li, C.G.; Zhou, S.F.; Pang, E.C.K.; Story, D.F.; Xue, C.C.L. Chemistry and bioactivity of Flos Magnoliae, a Chinese herb for rhinitis and sinusitis. Curr. Med. Chem.; 2008; 15, pp. 1616-1627. [DOI: https://dx.doi.org/10.2174/092986708784911515]

4. Sun, Y.Y.; Cai, X.J.; Cao, J.X.; Wu, Z.; Pan, D.D. Effects of 1,8-cineole on Carbohydrate Metabolism Related Cell Structure Changes of Salmonella. Front Microbiol.; 2018; 9, 1078. [DOI: https://dx.doi.org/10.3389/fmicb.2018.01078]

5. Bhuia, M.S.; Wilairatana, P.; Chowdhury, R.; Rakib, A.I.; Kamli, H.; Shaikh, A.; Coutinho, H.D.M.; Islam, M.T. Anticancer Potentials of the Lignan Magnolin: A Systematic Review. Molecules; 2023; 28, 3671. [DOI: https://dx.doi.org/10.3390/molecules28093671]

6. Park, R.; Park, E.J.; Cho, Y.Y.; Lee, J.Y.; Kang, H.C.; Song, I.S.; Lee, H.S. Tetrahydrofurofuranoid Lignans, Eudesmin, Fargesin, Epimagnolin A, Magnolin, and Yangambin Inhibit UDP-Glucuronosyltransferase 1A1 and 1A3 Activities in Human Liver Microsomes. Pharmaceutics; 2021; 13, 187. [DOI: https://dx.doi.org/10.3390/pharmaceutics13020187]

7. Hu, J.M.; Cai, J.H.; Cheng, Q.Q.; Wang, L.J.; Hu, X.R.; Wang, W.Z.; Liao, Z.F.; Tao, X.H. Multi-Indicator Comprehensive Quality Evaluation of Turpinia arguta (Lindl.) Seem Herbs at Different Harvesting Periods. Agronomy; 2024; 14, 2658. [DOI: https://dx.doi.org/10.3390/agronomy14112658]

8. Lee, J.; Lee, D.; Jang, D.S.; Nam, J.W.; Kim, J.P.; Park, K.H.; Yang, M.S.; Seo, E.K. Two new stereoisomers of tetrahydrofuranoid lignans from the flower buds of. Chem. Pharm. Bull.; 2007; 55, pp. 137-139. [DOI: https://dx.doi.org/10.1248/cpb.55.137]

9. Kim, H.K.; Seo, J.W.; Kim, G.H. Various effects of volatile constituents from Magnolia kobus flowers against Aedes albopictus (Diptera: Culicidae). Ind. Crop Prod.; 2020; 145, 112109. [DOI: https://dx.doi.org/10.1016/j.indcrop.2020.112109]

10. Marzocchi, S.; Baldi, E.; Crucitti, M.C.; Toselli, M.; Caboni, M.F. Effect of Harvesting Time on Volatile Compounds Composition of Bergamot (Citrus × Bergamia) Essential Oil. Flavour. Frag. J.; 2019; 34, pp. 426-435. [DOI: https://dx.doi.org/10.1002/ffj.3520]

11. Li, Y.Q.; Kong, D.X.; Huang, R.S.; Liang, H.L.; Xu, C.G.; Wu, H. Variations in essential oil yields and compositions of innamomum cassia leaves at different developmental stages. Ind. Crop Prod.; 2013; 47, pp. 92-101. [DOI: https://dx.doi.org/10.1016/j.indcrop.2013.02.031]

12. Liu, Z.D.; Lan, J.X.; Chen, S.Q. Quality analysis of Citri Trifoliatae Fructus in different harvest time by HPLC combined with electronic eye technology. Zhongguo Zhong Yao Za Zhi; 2021; 46, pp. 5253-5259. [DOI: https://dx.doi.org/10.19540/j.cnki.cjcmm.20210720.102]

13. Miyazawa, M.; Nakashima, Y.; Nakahashi, H.; Hara, N.; Nakagawa, H.; Usami, A.; Chavasiri, W. Volatile Compounds with Characteristic Odor of Essential Oil from Leaves by Hydrodistillation and Solvent-assisted Flavor Evaporation. J. Oleo Sci.; 2015; 64, pp. 999-1007. [DOI: https://dx.doi.org/10.5650/jos.ess15114]

14. Calín-Sánchez, A.; Lech, K.; Szumny, A.; Figiel, A.; Carbonell-Barrachina, A.A. Volatile composition of sweet basil essential oil (Ocimum basilicum L.) as affected by drying method. Food Res. Int.; 2012; 48, pp. 217-225. [DOI: https://dx.doi.org/10.1016/j.foodres.2012.03.015]

15. Georgieva, P.; Rusanov, K.; Rusanova, M.; Kitanova, M.; Atanassov, I. Construction of Simple Sequence Repeat-Based Genetic Linkage Map and Identification of QTLs for Accumulation of Floral Volatiles in Lavender (Lavandula angustifolia Mill.). Int. J. Mol. Sci.; 2025; 26, 3705. [DOI: https://dx.doi.org/10.3390/ijms26083705]

16. Kunc, N.; Hudina, M.; Osterc, G.; Grohar, M.C. Determination of Volatile Compounds in Blossoms Rosa spinosissima, Rosa pendulina, Rosa gallica, and Their Cultivars. Agriculture; 2024; 14, 253. [DOI: https://dx.doi.org/10.3390/agriculture14020253]

17. Ning, J.R.; Wang, K.H.; Yang, W.L.; Liu, M.S.; Tian, J.Y.; Wei, M.Y.; Zheng, G.D. Qualitative and quantitative analyses of chemical components of Citri Sarcodactylis Fructus from different origins based on UPLC-Q-Exactive Orbitrap-MS and GC-MS. Food Sci. Nutr.; 2022; 10, pp. 2057-2070. [DOI: https://dx.doi.org/10.1002/fsn3.2822]

18. Zeng, Z.; Xie, R.Q.; Zhang, T.; Zhang, H.; Chen, J.Y. Analysis of Volatile Compositions of Pamp by Steam Distillation and Headspace Solid Phase Micro-extraction. J. Oleo Sci.; 2011; 60, pp. 591-596. [DOI: https://dx.doi.org/10.5650/jos.60.591]

19. Cirlini, M.; Mena, P.; Tassotti, M.; Herrlinger, K.A.; Nieman, K.M.; Dall’Asta, C.; Del Rio, D. Phenolic and Volatile Composition of a Dry Spearmint (Mentha spicata L.) Extract. Molecules; 2016; 21, 1007. [DOI: https://dx.doi.org/10.3390/molecules21081007]

20. Feng, X.; Shi, H.; Yang, G.Y.; Chang, Y.Q.; Zhang, D.; Zheng, Y.G.; Guo, L. Dynamic changes of volatile components in Forsythia suspensa at different harvest periods based on GC-MS and chemometrics analysis. Zhongguo Zhong Yao Za Zhi; 2022; 47, pp. 54-61. [DOI: https://dx.doi.org/10.19540/j.cnki.cjcmm.20211025.104]

21. Schühly, W.; Skarbina, J.; Kunert, O.; Nandi, O.I.; Bauer, R. Chemical Characterization of Magnolia biondii (Flos Magnoliae, Xin Yi). Nat. Prod. Commun.; 2009; 4, pp. 231-234. [DOI: https://dx.doi.org/10.1177/1934578X0900400213]

22. Lu, Y.; He, Z.X.; Wang, Q.; Lei, T.; Ning, N.; Chen, X.Y.; Wu, X.; Wang, S.P.; Wan, L.; Cao, J.L. An advanced strategy for quality evaluation of Xinyi Biyan Pill by UPLC-DAD fingerprinting combined with multi-components UPLC-MS/MS analysis. J. Pharm. Biomed.; 2023; 239, 115858. [DOI: https://dx.doi.org/10.1016/j.jpba.2023.115858] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/38042092]

23. Kim, J.S.; Kim, J.Y.; Lee, H.J.; Lim, H.J.; Lee, D.Y.; Kim, D.H.; Ryu, J.H. Suppression of Inducible Nitric Oxide Synthase Expression by Furfuran Lignans from Flower Buds of in BV-2 Microglial Cells. Phytother. Res.; 2010; 24, pp. 748-753. [DOI: https://dx.doi.org/10.1002/ptr.3028] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/19943243]

24. Senatore, F. Influence of harvesting time on yield and composition of the essential oil of a thyme (Thymus pulegioides L.) growing wild in Campania (Southern Italy). J. Agr. Food Chem.; 1996; 44, pp. 1327-1332. [DOI: https://dx.doi.org/10.1021/jf950508z]

25. Juergens, L.J.; Worth, H.; Juergens, U.R. New Perspectives for Mucolytic, Anti-inflammatory and Adjunctive Therapy with 1,8-Cineole in COPD and Asthma: Review on the New Therapeutic Approach. Adv. Ther.; 2020; 37, pp. 1737-1753. [DOI: https://dx.doi.org/10.1007/s12325-020-01279-0]

26. Gibbs, J.E.M. Essential oils, asthma, thunderstorms, and plant gases: A prospective study of respiratory response to ambient biogenic volatile organic compounds (BVOCs). J. Asthma Allergy; 2019; 12, pp. 169-181. [DOI: https://dx.doi.org/10.2147/JAA.S193211]

27. Phan, H.; Nam, Y.; Kim, H.; Woo, J.; NamKung, W.; Nam, J.; Kim, W. In-vitro and in-vivo anti-allergic effects of magnolol on allergic rhinitis via inhibition of ORAI1 and ANO1 channels. J. Ethnopharmacol.; 2022; 289, 115061. [DOI: https://dx.doi.org/10.1016/j.jep.2022.115061]

28. Tang, Q.; Zhang, R.; Zhou, J.; Zhao, K.; Lu, Y.; Zheng, Y.; Wu, C.; Chen, F.; Mu, D.; Ding, Z. . The levels of bioactive ingredients in Citrus aurantium L. at different harvest periods and antioxidant effects on H₂O₂-induced RIN-m5F cells. J. Sci. Food Agric.; 2021; 101, pp. 1479-1490. [DOI: https://dx.doi.org/10.1002/jsfa.10761]