The Difference of Volatile Compounds in Female and Male Buds of Trichosanthes anguina L. Based on HS-SPME-GC-MS and Multivariate Statistical Analysis

Trichosanthes anguina L. (family Cucurbitaceae) is a monoecious and diclinous plant that can be consumed as a vegetable and has anti-inflammatory and antioxidant effects. The chemical composition and content of volatile compounds in female and male buds of T. anguina were explored by headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) technology combined with multivariate statistical analysis. The results showed that the content of the volatile compounds was different between female and male buds. 2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-ol and 2,2,6-trimethyl-6-vinyldihydro-2H-pyran-3(4H)-one were the main volatile compounds in both female and male buds. Based on the multivariate statistical analysis of orthogonal projections to latent structures discriminant analysis (OPLS-DA) and t-test, the content of seven compounds was significantly different between female and male buds. The content of three compounds in male buds was higher than that in female, i.e., (E)-4,8-dimethyl-1,3,7-nonatriene, 1,5,9,9-tetramethyl-1,4,7-cycloundecatriene, and (E)-caryophyllene. Conversely, the content of (Z)-4-hexen-1-ol, (Z)-3-hexenyl benzoate, (Z)-3-hexenyl salicylate, and 2-hexen-1-ol in female buds was higher than that in male buds. This is the first report on the difference in the volatile compounds between female and male buds of T. anguina, which enriches the basic research on the monoecious and diclinous plant and provides a reference for the study of plant sex differentiation.


Introduction
Trichosanthes anguina L. (family Cucurbitaceae), an annual climbing herb, is known as snake gourd or snake bean for its slender (up to 200 cm in length and 3 cm in diameter), twisted, and snake-like shape fruit. It is originated in India and Malaysia and commonly cultivated in tropical and subtropical areas and northern China [1]. Its tender fruit is a popular vegetable, which contains proteins, carbohydrates, cellulose, fat, and a variety of minerals [2]. The fruit, seed, and root of T. anguina can be used in traditional Chinese medicine for some effects in clearing heat and generating fluid, moisturizing the lung, eliminating dampness, and destroying parasites [3]. It has anti-inflammatory and antioxidant activities and can be used for the treatment of malaria and bronchitis [4]. In addition, two proteins of TR3 from the root and of TS3 from the seed have cytotoxic activity on cancer cell lines [5].
The buds, just before blooming, were picked up as experimental samples for the detection of volatile compounds in order to avoid any contamination of external substances. The HS-SPME-GC-MS method was used to detect and identify the volatile compounds from the female and male buds of T. anguina. The multivariate statistical analysis of OPLS-DA and t-test were carried out to screen out the differential compounds between female and male buds for revealing the difference in the chemical composition of genders of monoecious and diclinous plants.

Volatile Compounds of GC-MS Analysis
Both female and male fresh buds of T. anguina just before blooming were individually picked up at night. The samples were weighed as f1 0.1063 g, f2 0.1057 g, f3 0.0975 g, m1 0.0842 g, m2 0.0826 g, and m3 0.0849 g. The volatile compounds were analyzed by HS-SPME-GC-MS successfully. The total ion chromatography is shown in Figure 2. A total of 53 compounds were identified on the base of mass spectrum and retention index (RI). The relative content of these volatile compounds was calculated with peak area normalization and shown in Table 1. The total ion chromatograms of volatile compounds for three female and three male bud samples are shown in Figure 3. Solid-phase microextraction (SPME) is a sample treatment method invented by Pawliszyn and Arthur in the 1990s [17], which needs no solvent to extract the sample components. The SPME fiber is used to extract samples directly whose weight is usually less than 5 mg [18]. Headspace (HS) technology combined with SPME is a convenient extraction method. The sample is directly placed in a sealed headspace bottle, and an SPME fiber is put into the headspace bottle and located above the sample. During the heating and extraction process, volatile substances diffuse from the sample to the SPME fiber and are enriched in the fiber. This extraction method is so quick, less than one hour, and does not destroy the sample or require a liquid solvent. HS-SPME is often combined with gas chromatography-mass spectrometry (GC-MS) for widespread use in some volatile substances analysis [19,20].
The material of SPME fiber coating is crucial to the extraction, which determines the fiber's affinity to compounds in the sample. Since the introduction of SPME, many coating materials have been employed in the analysis. Polydimethylsiloxane (PDMS) fiber could be used to determine the content of acrylamide from coffee beans [21]. Polyacrylate (PA) fiber was suitable for the quantification of sesquiterpenes in Zingiber zerumbet L. (family Zingiberaceae) volatiles [22]. Polydimethylsiloxane/divinylbenzene (PDMS/DVB) could be applied for the determination of the esters of carboxylic acids in insect lipids [23]. Carboxen/polydimethylsiloxane (CAR/PDMS) fiber was used for the analysis of volatile compounds from European ciders [24]. Divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber was able to extract more chemical diverse volatile compounds, including ketones, aldehydes, alcohols, terpenoids, and others from Medicago sativa L. (family Leguminosae) compared with PDMS and CAR/PDMS fibers [25] and to analyze a variety of plants and their oils such as sweet potato, tomato, and olive oils [26][27][28]. In this study, DVB/CAR/PDMS fiber was selected due to its better ability to provide a more comprehensive chemical profile of plants and its great extraction effect in the pre-experiment.
The buds, just before blooming, were picked up as experimental samples for the detection of volatile compounds in order to avoid any contamination of external substances. The HS-SPME-GC-MS method was used to detect and identify the volatile compounds from the female and male buds of T. anguina. The multivariate statistical analysis of OPLS-DA and t-test were carried out to screen out the differential compounds between female and male buds for revealing the difference in the chemical composition of genders of monoecious and diclinous plants.

Volatile Compounds of GC-MS Analysis
Both female and male fresh buds of T. anguina just before blooming were individually picked up at night. The samples were weighed as f1 0.1063 g, f2 0.1057 g, f3 0.0975 g, m1 0.0842 g, m2 0.0826 g, and m3 0.0849 g. The volatile compounds were analyzed by HS-SPME-GC-MS successfully. The total ion chromatography is shown in Figure 2. A total of 53 compounds were identified on the base of mass spectrum and retention index (RI). The relative content of these volatile compounds was calculated with peak area normalization and shown in Table 1. The total ion chromatograms of volatile compounds for three female and three male bud samples are shown in Figure 3. The buds, just before blooming, were picked up as experimental samples for the detection of volatile compounds in order to avoid any contamination of external substances. The HS-SPME-GC-MS method was used to detect and identify the volatile compounds from the female and male buds of T. anguina. The multivariate statistical analysis of OPLS-DA and t-test were carried out to screen out the differential compounds between female and male buds for revealing the difference in the chemical composition of genders of monoecious and diclinous plants.

Volatile Compounds of GC-MS Analysis
Both female and male fresh buds of T. anguina just before blooming were individually picked up at night. The samples were weighed as f1 0.1063 g, f2 0.1057 g, f3 0.0975 g, m1 0.0842 g, m2 0.0826 g, and m3 0.0849 g. The volatile compounds were analyzed by HS-SPME-GC-MS successfully. The total ion chromatography is shown in Figure 2. A total of 53 compounds were identified on the base of mass spectrum and retention index (RI). The relative content of these volatile compounds was calculated with peak area normalization and shown in Table 1. The total ion chromatograms of volatile compounds for three female and three male bud samples are shown in Figure 3.     The volatile components of female and male buds of T. anguina included alcohols, ketones, aromatic esters, non-aromatic esters, monoterpenes, sesquiterpenes, diterpenes, alkenes, oximes, heterocycles, alkanes, and acids. The detailed results are shown in Table  2. Among them, the type of heterocycles was the highest content component, with a total relative content of 69.75-75.49% for female and of 69.58-73.39% for male buds. The total content of alcohols was the second highest, with 9.48-12.36% for females and 7.59-8.89% for male buds. Interestingly, there were seven sesquiterpenes whose content in male buds (average 3.56%) was significantly higher than that in female buds (average 2.66%) with p < 0.05 in the t-test. In addition, the content of four alkenes in male buds (average 5.34%) was significantly higher than that in female buds (average 2.25%), with p < 0.05 in the ttest. These results showed that there was a significant difference between female and male buds in terms of the content of sesquiterpenes and alkenes.  The volatile components of female and male buds of T. anguina included alcohols, ketones, aromatic esters, non-aromatic esters, monoterpenes, sesquiterpenes, diterpenes, alkenes, oximes, heterocycles, alkanes, and acids. The detailed results are shown in Table 2. Among them, the type of heterocycles was the highest content component, with a total relative content of 69.75-75.49% for female and of 69.58-73.39% for male buds. The total content of alcohols was the second highest, with 9.48-12.36% for females and 7.59-8.89% for male buds. Interestingly, there were seven sesquiterpenes whose content in male buds (average 3.56%) was significantly higher than that in female buds (average 2.66%) with p < 0.05 in the t-test. In addition, the content of four alkenes in male buds (average 5.34%) was significantly higher than that in female buds (average 2.25%), with p < 0.05 in the t-test. These results showed that there was a significant difference between female and male buds in terms of the content of sesquiterpenes and alkenes.

Multivariate Statistical Analysis
An OPLS-DA model of multivariate statistical analysis was carried out in order to further explore the difference in volatile compounds between female and male buds. This model was verified by 200 times permutation tests. Figure 4a showed that the R 2 and Q 2 values generated by any random arrangement on the left end were smaller than those on the right end, the slope of the regression line was large, and the lower regression line intersected the negative half-axis of the Y-axis, indicating that the model was not overfitting and could be used to find differential compounds. The OPLS-DA score diagram (Figure 4b) showed that the points of f1−f3 of female samples and m1−m3 of male samples were separated along the t1 axis. The value of R 2 was 0.944, and that of Q 2 was 0.838, both of which were greater than 0.5, indicating that the model had suitable interpretation and prediction ability. further explore the difference in volatile compounds between female and male buds. This model was verified by 200 times permutation tests. Figure 4a showed that the R 2 and Q 2 values generated by any random arrangement on the left end were smaller than those on the right end, the slope of the regression line was large, and the lower regression line intersected the negative half-axis of the Y-axis, indicating that the model was not overfitting and could be used to find differential compounds. The OPLS-DA score diagram (Figure 4b) showed that the points of f1−f3 of female samples and m1−m3 of male samples were separated along the t1 axis. The value of R 2 was 0.944, and that of Q 2 was 0.838, both of which were greater than 0.5, indicating that the model had suitable interpretation and prediction ability.

Heat Map
The content distribution of seven differential compounds in the female and male buds of T. anguina was visualized in the form of a heat map. Horizontal columns represented different samples (f1-3 and m1-3), and vertical columns represented different compounds (no. 4, 19, 21, 39, 46, 9, and 10). If the color of the block was red, the deeper the red was, the higher the content of the compound in the sample was; if the color was blue, the deeper the blue was, the lower the content of the compound was. As shown in Figure 5, six samples were clearly separated into two groups, i.e., all three female samples were classified into a group on the left, and three male samples were classified into a group on the right. Therefore, the different content levels of seven differential compounds in the female and male buds could be intuitively and clearly observed in Figure 5.   Table 1.

Heat Map
The content distribution of seven differential compounds in the female and male buds of T. anguina was visualized in the form of a heat map. Horizontal columns represented different samples (f1-3 and m1-3), and vertical columns represented different compounds (no. 4, 19, 21, 39, 46, 9, and 10). If the color of the block was red, the deeper the red was, the higher the content of the compound in the sample was; if the color was blue, the deeper the blue was, the lower the content of the compound was. As shown in Figure 5, six samples were clearly separated into two groups, i.e., all three female samples were classified into a group on the left, and three male samples were classified into a group on the right. Therefore, the different content levels of seven differential compounds in the female and male buds could be intuitively and clearly observed in Figure 5.  Table 1.

Discussion
As a monoecious and diclinous plant, the structure and function of female and male buds and flowers of T. anguina are quite different. The buds just before opening were collected for our experiment, which confirmed some differences in the volatile compounds between female and male buds. The results must be more accurate and reliable than those of opening flowers, which can avoid the loss of bud volatile compounds and the pollution of external substances [13]. This is the first report to study the volatile compounds from the buds and to find the differential compounds between female and male buds of T. anguina.
Diisobutyl phthalate (no. 51) is usually used as a plasticizer, which is often detected in soil, water, and air, besides in plastics. It is a detection index component of environmental pollution [54]. In this experiment, the fresh buds were treated by HS-SPME Figure 5. The heatmap of the differential compounds in female and male buds of T. anguina. The numbers of the heat map were consistent with the no. in Table 1.

Discussion
As a monoecious and diclinous plant, the structure and function of female and male buds and flowers of T. anguina are quite different. The buds just before opening were collected for our experiment, which confirmed some differences in the volatile compounds between female and male buds. The results must be more accurate and reliable than those of opening flowers, which can avoid the loss of bud volatile compounds and the pollution of external substances [13]. This is the first report to study the volatile compounds from the buds and to find the differential compounds between female and male buds of T. anguina.
Diisobutyl phthalate (no. 51) is usually used as a plasticizer, which is often detected in soil, water, and air, besides in plastics. It is a detection index component of environmental pollution [54]. In this experiment, the fresh buds were treated by HS-SPME without any solvent extraction or plastics exposure. Therefore, it was suspected to be derived from the soil during growth or from the air.
(E)-4,8-Dimethyl-1,3,7-nonatriene (DMNT) is one of the floral compounds of some night-flowering plants [55]. DMNT can attract insects to pollinate [56] and has the function of attracting natural enemies of pests to avoid pest invasion [57]. The average content of DMNT in males (3.48%) was 6.14 times that in female buds (0.57%), whose content gap between genders was the largest. The results pointed out that high DMNT content in male buds was more conducive to attracting insects to pollinate and improving the reproductive capacity of T. anguina.
(E)-Caryophyllene, a bicyclic sesquiterpene, has many pharmacological effects such as anti-inflammation, antidepression, and anti-convulsion [58]. Its average content in male (2.37%) was higher than that in female buds (1.81%), which is a significant difference with p < 0.05 in the t-test between female and male buds. The result indicated that the male buds should have stronger pharmacological activities than the female buds of T. anguina.
(Z)-3-Hexenyl benzoate and (Z)-3-hexenyl salicylate are two (Z)-3-hexenol esters of aromatic acids. The average content of the former was 1.26% in females and 0.64% in males, and of the latter was 0.92% in female and 0.47% in male buds. Both of their content in females was about two times that in male buds. (Z)-3-Hexenyl benzoate showed specific binding to odorant-binding proteins of Halyomorpha halys and Plautia stali, which was similar to alarm pheromones of both two pests with the function of repelling [60]. The higher content of (Z)-3-hexenylbenzoate in female buds indicated that the compound might help female flowers avoid pests and protect female flowers' pollination and development. (Z)-3-Hexenyl salicylate is a useful compound for people, often used as fragrance ingredients for fine fragrances, shampoos, toilet soaps, and household cleaners [61].
The chemical components of some dioecious plants were reported to be different between females and males. The essential oils of the female and male aerial parts of Baccharis tridentata Vahl. (family Asteraceae) were explored with GC-MS analysis. α-Pinene was the main compound in the essential oil of both genders, which was presented at higher content in males (1173 ± 60 µg/L) than that in females (794 ± 40 µg/L). Conversely, the concentrations of α-phellandrene, α-terpinene, and trans-verbenol in female essential oil were higher than those in males [42]. The chemical components from female and male flowers of Schisandra chinensis (family Magnoliaceae) were differentiated with GC-MS analysis. The results showed that 16 compounds were found only in female flowers (including α-farnesene, α-pinene, and 3-carene), and 19 compounds (including p-xylene, 3-pyridinecarboxaldehyde, and 1,2-epoxydodecane) were detected only in male flowers. A number of compounds detected both in female and male flowers were quantitatively different; for example, the content of β-pinene in females was 6.36 times that in male flowers (0.70 ± 0.06% and 0.11 ± 0.01%, respectively) [51].
The plant T. kirilowii, a different species of the same genus of T. anguina, was a dioecious plant. The highest content component of T. kirilowii flowers was alcohol, the same as T. anguina buds. Some same compounds of linalool, benzyl alcohol, and (E)-linalool oxide were found both in T. anguina and T. kirilowii. However, the highest content compound in females was linalool, but in male flowers of T. kirilowii was benzyl alcohol. Some compounds, such as β-myrcene and α-ocimene, were only detected in females, and ben-zaldehyde was only detected in male flowers of T. kirilowii [14]. The difference between female and male flowers of T. kirilowii was larger than that between female and male buds of T. anguina. In other words, the difference in volatile compounds between the two genders from the dioecious plant was larger than that from the monoecious and diclinous plants. Dioecious plants have more advanced evolution than monoecious and diclinous plants in plant sexology [63]. From the perspective of the flowers and buds of sexual organs, the higher inequality was found based on their volatile compounds.

Sample Collection
The

Sample Preparation
The fresh bud samples were collected and placed in 15 mL glass headspace bottles. Each headspace bottle was placed in one of the bud samples, tightly covered, weighed, and subtracted from the bottle's body weight to obtain the sample weight. Moreover, the material of the headspace bottle gasket was polytetrafluoroethylene (PTFE). The 50/30 µm CAR/PDMS/DVB SPME fiber was aged at 260 • C for 30 min, extended through the needle, exposed into the headspace bottle to adsorb volatile compounds at 50 • C for 30 min, and immediately injected into the gas chromatography injection port at 250 • C for 3 min to desorb volatile compounds.

Chromatographic Conditions
The volatile compounds of the samples were analyzed by the GC-MS method. A GC-MS-QP 2010 plus gas chromatography-mass spectrometer was used coupled to polyethylene glycol capillary chromatographic column Agilent HP-INNOWAX (30 m × 0.25 mm, 0.25 µm). The splitless injection mode was used. The carrier gas was high-purity helium, which was used at a constant flow rate of 1.01 mL·min −1 . The temperature of the injection port was set at 250 • C. The heating program was as follows: the initial temperature was 40 • C maintained for 8 min, raised to 160 • C at a rate of 3 • C·min −1 , and subsequently raised to 240 • C at a rate of 10 • C·min −1 and held for 5 min.

MS Conditions
The electron ionization (EI) source was used and operated at 70 eV. The ion source temperature was 200 • C. The interface temperature was 220 • C. Moreover, the scanning range was m/z 29-350.

Data Processing
The volatile compounds from samples were identified with the mass spectrum and RI. The mass spectra obtained from the GC-MS experiments were compared with the National Institute of Standards and Technology (NIST) 14 spectrum library. The RIs were calculated according to the peak retention time of these volatile compounds and of the series of n-alkanes (C 11 -C 32 ) under the same temperament condition and were compared with the values in the previous reports. The peak area normalization was carried out for semi-quantitative analysis and for comparison of the difference of the volatile compounds between female and male samples.

Statistical Analysis
OPLS-DA is a supervised discriminant analysis that can effectively distinguish the difference between groups. The experimental data were imported into SIMCA-P software (version 14.1, Umetrics, Malmö, Sweden) to establish an OPLS-DA model to distinguish the female and male buds. The experimental data were also imported into SPSS (version 19.0, IBM, America), analyzed with a t-test, and then combined with OPLS-DA results to identify the significantly different compounds of the volatile components from female and male buds of T. anguina.

Heat Map
After data were imported into the Metabo Analyst 5.0 website, the heat map was generated to visualize the distribution of different compounds in different samples so that readers could intuitively observe the content gap of these compounds in female and male samples.

Conclusions
In this study, HS-SPME-GC-MS combined with multivariate statistical analysis was used to explore some differences in volatile compounds of T. anguina buds, and it was found that there was a significant difference between female and male buds. A total of 53 volatile compounds were identified by GC-MS. There were the same volatile compounds from female and male buds, but their content was different. Based on multivariate statistical analysis, seven different compounds were screened out. Among them, the content of (E)-4,8-dimethyl-1,3,7-nonatriene, (E)-caryophyllene, and 1,5,9,9-tetramethyl-1,4,7-cycloundecatriene from male was higher than that from female buds, and the content of (Z)-4-hexen-1-ol, (Z)-3-hexenyl benzoate, (Z)-3-hexenyl salicylate, and 2-hexen-1-ol from male was lower than that from female buds. Further comparison between the monoecious and diclinous plant of T. anguina and the dioecious plant of T. kirilowii, an opinion suggested that the difference of volatile compounds between female and male buds of the monoecious and diclinous plant was smaller than that of the dioecious plant. This opinion was consistent with the evolutionary view of plant sex [63]. This is the first report that the difference in volatile compounds between female and male buds of T. anguina was analyzed. Furthermore, a comparison between monoecious and diclinous plants and dioecious plants was first carried out.