DOI: 10.31038/IMROJ.2021624

Abstract

In performing molecular profiling of secondary metabolites, a lot of research has focused on biogenic volatile organic compounds with medium to low polarity. In this study, chemical composition similarity relationships among the various organs of the Ilex cornuta Lindl. & Paxton were assessed based on the analysis of hydrophilic volatile compounds. GC-MS analysis was conducted to characterize and classify the chemical compounds. A total of 36, 46, 42, 25, 64, 26 compounds have been respectively extracted from the root, stem, stem skin, leaf, flower and fruit. The six organs have 3 common compounds and large percentages of exclusive compounds ranging from 36.0% to 62.5% with a mean of 49.8%, indicating substantial component differences among the different organs. The percentage of overlapping compounds between each of the two organs ranges from 10.9% to 44.0%, which is relatively small, further demonstrating the strong organ specificity of the chemical composition. The overlapping index is used to reveal the similarity among the organs. The stem shares the maximum similarity while the fruit the minimum similarity with the other organs. Aside from fruit, the average overlapping indices between each of the other two organs correlate well to their physical proximity. In conclusion, hydrophilic volatile metabolites are a class of natural products that are rarely investigated but constitute a significant part of the plant chemical composition. Chemical profiling of these metabolites could provide a valuable tool for the plant taxonomy and help understand the chemically mediated biological phenomena.

Keywords

Chemical composition similarity, GC-MS, Hydrophilic volatile compounds, Ilex cornuta Lindl. & Paxton, Plant taxonomy

Introduction

Plant taxonomy is traditionally conducted based on macroscopic and microscopic morphological characteristics. Growing evidence suggests that many biologically relevant entities could be missed in the studies that rely solely on morphological traits, particularly since speciation is not always accompanied by morphological change [1,2]. In recent years, plant chemical taxonomy has been developed to perform classification based on a wide array of biologically active secondary metabolites [3]. The expression of secondary metabolites could vary due to convergent evolution or differential gene expression [4], suggesting that the metabolite content of plants may reveal more information on the bioactive pattern of plants in comparison to morphology characterization [5].

In performing molecular profiling of secondary metabolites, a lot of research has focused on biogenic volatile organic compounds with medium to low polarity [6-9]. Volatile compounds are secreted and part of them are volatilized immediately after secretion [10,11]. The remaining part is stored in the special structure of the plant as in the case of essential oils [12-14]. Additionally, Berlinck and collaborators found that the vast majority of new compounds from natural sources reported in recent literature are compounds of medium to low polarity. Water-soluble, volatile, minor and photosensitive natural products are yet poorly known. One of the possible reasons for this trend could be that organic solvents of medium to low polarity used in isolation procedures require less time and less sophisticated instrumentation to be evaporated [15]. The author speculates that there is a class of hydrophilic volatile compounds in plants that are dispersed or dissolved in the water phase, evaporated with water vapor, and whose polarity and volatility are somewhere between essential oils and water-soluble compounds. To protect this type of ingredients from loss during extraction, water vapor distillation is used to collect volatile compounds that are dispersed or dissolved in the plant’s water phase. The root, stem, stem skin, leaf, flower and fruit of the Ilex cornuta Lindl. & Paxton were analyzed as study samples. Volatile essential oils were removed by using Soxhlet extraction method. Hydrophilic volatile compounds obtained by water reflux extraction are characterized and classified by quantitative GC-MS. The study revealed the potential use of hydrophilic volatile metabolites in the plant taxonomy and understanding the chemically mediated biological phenomena.

Materials and Methods

Material

Ilex cornuta Lindl. & Paxton was collected in Nanjing, China. Its roots, stems, stem skins, leaves, flowers and fruits were washed, cut into pieces, dried at 30°C and stored at 2-8°C prior to use.

Chemicals and Reagents

Ethyl acetate was purchased from Xilong Chemical Co., Ltd (Shantou, China). Hexane was purchased from Shanghai Titan Scientific Co., Ltd (Shanghai, China). Activated carbon was purchased from Shanghai Chemical Reagent Procurement Center (Shanghai, China). C7-C40 saturated alkanes standard was purchased from Anpel Laboratory Technologies Inc. (Shanghai, China).

Sample Preparation

Each sample was sliced and dried at 30°C. After ground into powder, the samples were sieved through 80 mesh followed by 180 mesh. Approximately 6 g of the sample were subjected to Soxhlet extractor method with hexane for 24 hrs to remove essential oils and other lipophilic compounds. The remainder was then removed and dried at 30°C in the ventilation cabinet. Approximately 4 g of the dried powder was then added into a 6 x 7 cm nonwoven bag together with three glass balls of 4 cm diameter. At least 3 segments of thread were used to separate and tighten the bag into 3 parts, each containing a glass ball and even amount of the dried powder. The bag was then placed in a flask and 2100 mL of water was subsequently added to soak the powder for about 2 hrs. After reflux extraction for 6 hrs, 2 L of distilled water was collected. The same reflux extraction was repeated to collect another 1 L of distilled water for a total of 3 L. After cooling, activated carbon (4 g) was added to absorb the active ingredients from the 3 L of distilled water for about 8 hrs. The activated carbon containing the active ingredients was then filtered and dried at 30°C for 12 hrs. Ethyl acetate was subsequently added to isolate the active ingredients from the activated carbon using Soxhlet extractor method for 8 hrs. The resulting ethyl acetate extract was left in the ventilation cabinet to dry at 30°C. The dried active ingredients were finally re-dissolved using ethyl acetate, filtrated through 0.22 µm filter and analyzed using GC-MS.

GC-MS Analysis

Analysis of hydrophilic volatile compounds was performed using Shimadzu GCMS-QP2010 Single Quadrupole GC-MS (Kyoto, Japan). A Rxi-1 ms GC capillary column (30 cm length, 0.25 mm inner diameter and 0.25 µm thick film) from Shimadzu (Kyoto, Japan) was used for analysis.

One microliter of sample was injected in split mode with split ratio of 5 to 1. GC inlet temperature is set at 280°C. High purity nitrogen (≥99.999%) was used as carrier gas in constant flow mode at 1 mL/min. The initial temperature of the GC oven is set at 60°C and held for 1 min, then ramped at 4°C/min to 160°C and held for 3 mins, followed by 2°C/min to 280°C and held for 6 mins. Finally, the temperature is raised to 300°C at 4°C/min and held for 6 mins. The mass spectrometer was operated in positive electron ionization mode at 70 eV and all spectra were recorded in full scan with a mass range of 40-700 Da. The interface temperature is set at 280°C and ion source temperature is set at 250°C.

Data Processing and Compound Identification

The GC-MS data processing was done with Shimazdzu GCMS Solution software. Compound identification was performed by applying several assignments, e.g., reference standard analysis, retention index calculation, and by NIST08 Spectrum Library comparison. Only peaks with area greater than 3 million are analyzed. The overlapping percentage is calculated by the number of overlapping compounds divided by the total number of hydrophilic volatile compounds from each of the two organ and times 100. Overlapping index is calculated by the number of overlapping compounds squared and divided by the total number of hydrophilic volatile compounds from each of the two organs. In addition, hierarchical clustering analysis was performed with Python to assess the similarities between each of the two organs by analyzing the number of overlapping hydrophilic volatile compounds.

Results and Discussion

The root, stem, stem skin, leaf, flower and fruit of the Ilex cornuta Lindl. & Paxton contain compounds that are water soluble and can volatilize with water vapor. These hydrophilic compounds do not separate from the water phase and possess greater polarity than essential oils. The largest number (64) of hydrophilic volatile compounds are isolated from the flower and the smallest (25) from the leaf, indicating that the number of hydrophilic volatile compounds varies greatly from organ to organ. The hydrophilic volatile compounds include aromatics, fatty acids, furans, heterocycle, esters, alkanes, ketones, halogens and other types of small molecular compounds. This is a diverse group of molecules that could contribute to the expression of biological information about the plant. Tables 1-6 present the lists of hydrophilic volatile compounds identified from the root, stem, stem skin, leaf, flower and fruit, respectively. The bold and italic fonts in the table are used to refer to exclusive compounds that are only found in the specific organ and not contained in any other organ.

Table 1: List of the hydrophilic volatile compounds identified from the root of the Ilex cornuta Lindl. & Paxton.

No	RT	RI	Compound	Formula
1	8.434	1041	2(3H)-Furanone, dihydro-4-hydroxy-	C4H6O3
2	8.555	1044	2-Oxo-n-valeric acid	C5H8O3
3	8.623	1047	2,3-Anhydro-d-galactosan	C6H8O4
4	9.159	1064	Acetic acid, hexyl ester	C8H16O2
5	9.767	1084	2-Cyclopenten-1-one, 3-ethyl-2-hydroxy-	C7H10O2
6	12.753	1173	Octanoic Acid	C8H16O2
7	13.662	1199	2-Furancarboxaldehyde,5-(hydroxymethyl)-	C6H6O3
8	16.053	1269	Nonanoic acid	C9H18O2
9	19.301	1364	Benzaldehyde, 4-(methylthio)-	C8H8OS
10	23.459	1492	1H-2-Benzopyran-1-one, 3,4-dihydro-8-hydroxy-3-methyl-	C10H10O3
11	23.660	1498	3-Acetoxydodecane	C14H28O2
12	25.161	1546	7-Hydroxy-3-(1,1-dimethylprop-2-enyl) coumarin	C14H14O3
13	25.496	1557	Dodecanoic acid	C12H24O2
14	25.696	1564	Estra-1,3,5(10)-trien-17. beta. – ol	C18H24O
15	26.109	1577	Butyric acid, 3-tridecyl ester	C17H34O2
16	26.829	1600	Hexadecane	C16H34
17	27.305	1613	Ethanone, 1-[2-(5-hydroxy-1,1-dimethylhexyl)-3-methyl-2-cyclopropen-1-yl]-	C14H24O2
18	28.020	1631	Thieno[3,2-c]pyridin-4(5H)-one	C7H5NOS
19	28.671	1649	Dodecanoic acid, 3-hydroxy-	C12H24O3
20	30.636	1700	2-Bromotetradecane	C14H29Br
21	32.780	1750	7-Methyl-Z-tetradecen-1-ol acetate	C17H32O2
22	35.860	1821	1,2-Benzenedicarboxylic acid, bis(2-methylpropyl) ester	C16H22O4
23	37.894	1867	2a-isopropyl-9,10a-dimethyl-6-methylenedodecahydro-1H-cyclopenta[4′,5′]cycloocta[1′,2′:1,5]cyclopenta[1,2-b]oxiren-4-ol	C20H32O2
24	39.903	1912	1,2-Benzenedicarboxylic acid, butyl octyl ester	C20H30O4
25	41.687	1951	n-Hexadecanoic acid	C16H32O2
26	49.181	2119	7-Hexadecenal, (Z)-	C16H30O
27	50.098	2139	9-Octadecenamide, (Z)-	C18H35NO
28	50.483	2148	Octadecanoic acid	C18H36O2
29	59.601	2362	2-Methyloctadecan-7,8-diol	C19H40O2
30	65.148	2499	1,2-Benzenedicarboxylic acid, diisooctyl ester	C24H38O4
31	73.761	2726	13-Docosenamide, (Z)-	C22H43NO
32	77.825	2840	3-Phenyl-2-ethoxypropylphthalimide	C19H19NO3
33	83.874	3017	9,10-Secocholesta-5,7,10(19)-triene-3,24,25-triol, (3.beta.,5Z,7E)-	C27H44O3
34	89.708	3197	Heptanoic acid, docosyl ester	C29H58O2
35	92.123	3265	Isophthalic acid, allyl pentadecyl ester	C26H40O4
36	102.267	3561	Benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy-, octadecyl ester	C35H62O3

Note: The bold and italic fonts are used to refer to exclusive compounds. RT: Retention time. RI: Reflex index.

Table 2: List of the hydrophilic volatile compounds identified from the stem of the Ilex cornuta Lindl. & Paxton.

No	RT	RI	Compound	Molecular
1	13.608	1198	2-Furancarboxaldehyde, 5-(hydroxymethyl)-	C6H6O3
2	19.258	1363	4-Hydroxy-2-methoxybenaldehyde	C8H8O3
3	21.565	1433	Cyclopentanemethanol,.alpha.-(1-methylethyl)-2-nitro-, [1.alpha.(S*),2.alpha.]-	C9H17NO3
4	23.85	1504	4,8-Decadienal, 5,9-dimethyl-	C12H20O
5	24.743	1533	Megastigmatrienone	C13H18O
6	25.469	1556	Dodecanoic acid	C12H24O2
7	25.681	1563	1-Cyclohexene-1-methanol, .alpha.,2,6,6-tetramethyl-	C11H20O
8	26.105	1577	Pentanoic acid, 2,2,4-trimethyl-3-carboxyisopropyl, isobutyl ester	C16H30O4
9	26.245	1581	Phenol, 3,4,5-trimethoxy-	C9H12O4
10	26.495	1589	2-Methyl-4-(2,6,6-trimethylcyclohex-1-enyl)-but-2-en-1-ol	C14H24O
11	27.127	1608	Benzaldehyde, 4-hydroxy-3,5-dimethoxy-	C9H10O4
12	27.37	1614	Ethanone, 1-[2-(5-hydroxy-1,1-dimethylhexyl)-3-methyl-2-cyclopropen-1-yl]-	C14H24O2
13	27.88	1628	Thieno[3,2-c]-pyridin-4(5H)-one	C7H5NOS
14	28.27	1638	Spiro-[4.5]-decan-7-one, 1,8-dimethyl-8,9-epoxy-4-isopropyl-	C15H24O2
15	28.685	1649	2-Bromo dodecane	C12H25Br
16	29.172	1662	Ethanol, 2-(octadecyloxy)-	C20H42O2
17	29.971	1683	1-(2-Hydroxy-4,5-dimethoxy-phenyl)-ethanone	C10H12O4
18	30.271	1691	2-Propenal, 3-(4-hydroxy-3-methoxyphenyl)-	C10H10O3
19	30.399	1694	Butanol, 1-[2,2,3,3-tetramethyl-1-(3-methyl-1-penynyl)-cyclopropyl]-	C17H30O
20	30.641	1700	Heptadecane	C17H36
21	31.037	1710	Hexadecane, 2,6,10,14-tetramethyl-	C20H42
22	31.345	1717	4a-Dichloromethyl-4,4a,5,6,7,8-hexahydro-3H-naphthalen-2-one	C11H14Cl2O
23	31.75	1726	Adamantane, 1-thiocyanatomethyl-	C12H17NS
24	32.052	1733	9-(3,3-Dimethyloxiran-2-yl)-2,7-dimethylnona-2,6-dien-1-ol	C15H26O2
25	32.512	1744	1-Decanol, 2-hexyl-	C16H34O
26	32.788	1750	Cyclopropane, 1-(1-hydroxy-1-heptyl)-2-methylene-3-pentyl-	C16H30O
27	33.683	1771	3-Isobutyryl-6-isopropyl-2,3-dihydropyran-2,4-dione	C12H16O4
28	34.92	1800	Heneicosane	C21H44
29	35.489	1813	Heptadecane, 2,6,10,15-tetramethyl-	C21H44
30	35.86	1821	1,2-Benzenedicarboxylic acid, bis(2-methylpropyl) ester	C16H22O4
31	37.316	1854	1-Hexadecanol	C16H34O
32	37.898	1867	2a-isopropyl-9,10a-dimethyl-6-methylenedodecahydro-1H-cyclopenta[4′,5′]-cycloocta[1′,2′:1,5]-cyclopenta-[1,2-b]oxiren-4-ol	C20H32O2
33	39.907	1912	1,2-Benzenedicarboxylic acid, butyl 8-methylnonyl ester	C22H34O4
34	41.693	1951	n-Hexadecanoic acid	C16H32O2
35	43.899	2000	Eicosane	C20H42
36	49.179	2119	12-Methyl-E,E-2,13-octadecadien-1-ol	C19H36O
37	50.464	2148	Octadecanoic acid	C18H36O2
38	59.603	2362	2-Methyloctadecan-7,8-diol	C19H40O2
39	65.152	2499	1,2-Benzenedicarboxylic acid, diisooctyl ester	C24H38O4
40	73.757	2726	13-Docosenamide, (Z)-	C22H43NO
41	83.861	3016	Ethyl iso-allocholate	C26H44O5
42	89.711	3197	Heptanoic acid, docosyl ester	C29H58O2
43	92.16	3266	Isophthalic acid, allyl pentadecyl ester	C26H40O4
44	92.66	3280	17-(1,5-Dimethylhexyl)-10,13-dimethyl-3-styrylhexadecahydrocyclopenta[a]phenanthren-2-one	C35H52O
45	94.571	3331	4-Norlanosta-17(20),24-diene-11,16-diol-21-oic acid, 3-oxo-16,21-lactone	C29H42O4
46	102.263	3561	Benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy-, octadecyl ester	C35H62O3

Note: The bold and italic fonts are used to refer to exclusive compounds. RT: Retention time. RI: Reflex index.

Table 3: List of the hydrophilic volatile component identified from the stem skin of the Ilex cornuta Lindl. & Paxton.

No	RT	RI	Compound	Molecular
1	12.765	1173	Octanoic Acid	C8H16O2
2	13.818	1204	2-Furancarboxaldehyde, 5-(hydroxymethyl)-	C6H6O3
3	19.283	1364	Benzaldehyde, 3-hydroxy-4-methoxy-	C8H8O3
4	21.526	1432	2H-Pyran-2-one, 5,6-dihydro-6-pentyl-	C10H16O2
5	23.372	1489	4,6-di-tert-Butyl-m-cresol	C15H24O
6	23.599	1496	12-Oxa-[tetracyclo[5.2.1.1(2,6).1(8,11)]]dodecan-10-ol, 3-acetoxy-	C13H18O4
7	23.856	1504	2,6-Dimethoxybenzoquinone	C8H8O4
8	25.171	1547	1H-Benzocyclohepten-7-ol, 2,3,4,4a,5,6,7,8-octahydro-1,1,4a,7-tetramethyl-, cis-	C15H26O
9	25.317	1551	2(5H)-Furanone, 4-methyl-5,5-bis(2-methyl-2-propenyl)-	C13H18O2
10	25.462	1556	Dodecanoic acid	C12H24O2
11	25.694	1564	2-Oxabicyclo[3.3.0]oct-7-en-3-one, 7-(1-hydroxypentyl)-	C12H18O3
12	25.922	1571	Dodecane, 2,6,10-trimethyl-	C15H32
13	26.114	1577	Pentanoic acid, 2,2,4-trimethyl-3-carboxyisopropyl, isobutyl ester	C16H30O4
14	26.335	1584	3-Butyl-4-nitro-pent-4-enoic acid, methyl ester	C10H17NO4
15	26.514	1590	2-Dodecen-1-yl(-)succinic anhydride	C16H26O3
16	26.838	1600	Heptadecane	C17H36
17	27.227	1611	Benzaldehyde, 4-hydroxy-3,5-dimethoxy-	C9H10O4
18	27.929	1629	2,6,10,10-Tetramethyl-1-oxaspiro-[4.5]decan-6-ol	C13H24O2
19	28.288	1638	4-Isobenzofuranol, octahydro-3a,7a-dimethyl-, (3a.alpha.,4.beta.,7a.alpha.)-(.+-.)-	C10H18O2
20	29.187	1662	Ethanol, 2-(hexadecyloxy)-	C18H38O2
21	29.827	1679	2-Cyclohexen-1-one, 3-(3-hydroxybutyl)-2,4,4-trimethyl-	C13H22O2
22	29.956	1682	Cyclopentanone, 2-(1-adamantyl)-	C15H22O
23	30.308	1692	alpha. Isomethyl ionone	C14H22O
24	30.649	1700	2-Bromotetradecane	C14H29Br
25	31.047	1710	Hexadecane, 2,6,10,14-tetramethyl-	C20H42
26	31.774	1727	Adamantane, 1-thiocyanatomethyl-	C12H17NS
27	32.083	1734	E,E-6,8-Tridecadien-2-ol, acetate	C15H26O2
28	32.522	1744	1-Decanol, 2-hexyl-	C16H34O
29	32.801	1751	7-Methyl-Z-tetradecen-1-ol acetate	C17H32O2
30	33.682	1771	7-Bromo-3a,6,6-trimethyl-hexahydro-benzofuran-2(3H)-one	C11H17BrO2
31	35.475	1813	Heptadecane, 2,6,10,15-tetramethyl-	C21H44
32	35.876	1822	1,2-Benzenedicarboxylic acid, bis(2-methylpropyl) ester	C16H22O4
33	37.903	1867	Dodecane, 1,2-dibromo-	C12H24Br2
34	39.916	1912	Dibutyl phthalate	C16H22O4
35	41.653	1951	n-Hexadecanoic acid	C16H32O2
36	65.178	2500	1,2-Benzenedicarboxylic acid, diisooctyl ester	C24H38O4
37	73.760	2726	13-Docosenamide, (Z)-	C22H43NO
38	93.411	3301	1,2-Benzenedicarboxylic acid, diundecyl ester	C30H50O4
39	93.650	3307	Isophthalic acid, allyl pentadecyl ester	C26H40O4
40	100.522	3505	9-Octadecenoic acid (Z)-, phenylmethyl ester	C25H40O2
41	101.150	3525	2,6-Lutidine 3,5-dichloro-4-dodecylthio-	C19H31Cl2NS
42	102.301	3562	Benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy-, octadecyl ester	C35H62O3

Note: The bold and italic fonts are used to refer to exclusive compounds. RT: Retention time. RI: Reflex index.

Table 4: List of the hydrophilic volatile component of the leaf of the Ilex cornuta Lindl. & Paxton.

No	RT	RI	Compound	Molecular
1	13.543	1196	2-Furancarboxaldehyde, 5-(hydroxymethyl)-	C6H6O3
2	14.110	1212	2-Furancarboxaldehyde, 6-(hydroxymethyl)-	C6H6O4
3	14.318	1218	2-Furancarboxaldehyde, 7-(hydroxymethyl)-	C6H6O5
4	25.089	1544	Bicyclo[3.2.0]heptan-6-one, 2-acetyl-3,3-dimethyl-7-(1-methylethyl)-	C14H22O2
5	25.453	1556	Dodecanoic acid	C12H24O2
6	25.692	1564	trans-Z-.alpha.-Bisabolene epoxide	C15H24O
7	26.117	1577	4,6,10,10-Tetramethyl-5-oxatricyclo[4.4.0.0(1,4)]dec-2-en-7-ol	C13H20O2
8	26.493	1589	7-Heptadecene, 1-chloro-	C17H33Cl
9	26.831	1600	Hexadecane	C16H34
10	28.088	1633	3-Pyridinecarboxylic acid, 1,6-dihydro-4-hydroxy-2-methyl-6-oxo-, ethyl ester	C9H11NO4
11	30.644	1700	Heptadecane	C17H36
12	31.038	1710	Hexadecane, 2,6,11,15-tetramethyl-	C20H42
13	32.440	1742	2-Cyclohexen-1-one, 4-hydroxy-3,5,6-trimethyl-4-(3-oxo-1-butenyl)-	C13H18O3
14	32.801	1751	7-Methyl-Z-tetradecen-1-ol acetate	C17H32O2
15	34.479	1790	Pentadecyl trifluoroacetate	C17H31F3O2
16	34.925	1800	Heptadecane, 2,6,10,15-tetramethyl-	C21H44
17	35.476	1813	Nonadecane	C19H40
18	35.872	1822	1,2-Benzenedicarboxylic acid, bis(2-methylpropyl) ester	C16H22O4
19	41.601	1949	n-Hexadecanoic acid	C16H32O2
20	43.458	1991	1-Nonadecene	C19H38
21	59.608	2362	2-Methyloctadecan-7,8-diol	C19H40O2
22	73.760	2726	13-Docosenamide, (Z)-	C22H43NO
23	92.239	3268	Isophthalic acid, allyl pentadecyl ester	C26H40O4
24	93.235	3296	1,2-Benzenedicarboxylic acid, 2-butoxyethyl butyl ester	C18H26O5
25	94.006	3317	Phthalic acid, propyl octadecyl ester	C29H48O4

Note: The bold and italic fonts are used to refer to exclusive compounds. RT: Retention time. RI: Reflex index.

Table 5: List of the hydrophilic volatile component identified from the flower of the Ilex cornuta Lindl. & Paxton.

No	RT	RI	Compound	Molecular
1	9.359	1070	2,2-Dimethyl-3-vinyl-bicyclo[2.2.1]heptane	C11H18
2	9.987	1091	Cyclohex-3-enecarboxaldehyde, 2,4,6-trimethyl-, oxime	C10H17NO
3	12.197	1156	Phenol, 3-ethyl-	C8H10O
4	12.649	1170	Benzoic acid	C7H6O2
5	12.797	1174	Glucosamine, N-acetyl-N-benzoyl-	C15H19NO7
6	13.333	1190	Benzothiazole	C7H5NS
7	15.613	1256	Phenol, 2,3,5-trimethyl-	C9H12O
8	16.214	1273	5H-Inden-5-one, 1,2,3,6,7,7a-hexahydro-	C9H12O
9	16.640	1286	Hydroquinone	C6H6O2
10	17.145	1300	Cyclohexanol, 1-methyl-4-(1-methylethylidene)-	C10H18O
11	17.280	1304	Cyclohexanol, 2-methyl-5-(1-methylethenyl)-, (1.alpha.,2.beta.,5.alpha.)-	C10H18O
12	17.772	1319	2,7-Octadiene-1,6-diol, 2,6-dimethyl-	C10H18O2
13	18.160	1330	trans-Z-.alpha.-Bisabolene epoxide	C15H24O
14	18.430	1338	(3S,4R,5R,6R)-4,5-Bis(hydroxymethyl)-3,6-dimethylcyclohexene	C10H18O2
15	19.298	1364	4-Hydroxy-2-methoxybenaldehyde	C8H8O3
16	19.508	1370	2-Cyclopenten-1-one, 4-hydroxy-3-methyl-2-(2-propenyl)-	C9H12O2
17	21.040	1417	Phenol, 2-pentyl-	C11H16O
18	21.311	1425	2-Propen-1-ol, 2-methyl-3-(2,6,6-trimethyl-2-cyclohexen-1-yl)-, (E)-	C13H22O
19	21.602	1434	3-(2-Hydroxy-cyclopentylidene)-2-methyl-propionic acid	C9H14O3
20	21.838	1442	5-​Benzofuranacetic acid, 6-​ethenyl-​2,​4,​5,​6,​7,​7a-​hexahydro-​3,​6-​dimethyl-​α-​methylene-​2-​oxo-​, methyl ester	C16H20O4
21	23.259	1486	8-Methylenecyclooctene-3,4-diol	C9H14O2
22	23.514	1494	1-(3,6,6-Trimethyl-1,6,7,7a-tetrahydrocyclopenta[c]pyran-1-yl)ethanone	C13H18O2
23	24.011	1509	1-Acetamido-1,2-dihydro-2-oxopyridine	C7H8N2O2
24	24.675	1531	cis-Z-.alpha.-Bisabolene epoxide	C15H24O
25	24.767	1534	Cyclopentan-1-al, 4-isopropylidene-2-methyl-	C10H16O
26	25.085	1544	Ethanone, 1-(1a,2,3,5,6a,6b-hexahydro-3,3,6a-trimethyloxireno[g]benzofuran-5-yl)-	C13H18O3
27	25.514	1558	Dodecanoic acid	C12H24O2
28	25.685	1563	Bicyclo[3.3.1]nonan-9-one, 1,2,4-trimethyl-3-nitro-, (2-endo,3-exo,4-exo)-(.+-.)-	C12H19NO3
29	25.899	1570	2-Cyclohexen-1-one, 3-(3-hydroxybutyl)-2,4,4-trimethyl-	C13H22O2
30	26.127	1578	Ledol	C15H26O
31	26.498	1590	1-Hexadecanol	C16H34O
32	26.840	1600	Hexadecane	C16H34
33	27.155	1609	Spiro[androst-5-ene-17,1′-cyclobutan]-2′-one, 3-hydroxy-, (3.beta.,17.beta.)-	C22H32O2
34	27.486	1617	Bicyclo[3.1.0]hexane-6-methanol, 2-hydroxy-1,4,4-trimethyl-	C10H18O2
35	28.099	1634	3-Pyridinecarboxylic acid, 1,6-dihydro-4-hydroxy-2-methyl-6-oxo-, ethyl ester	C9H11NO4
36	28.615	1647	Bromoacetic acid, dodecyl ester	C14H27BrO2
37	28.684	1649	Chloroacetic acid, 4-tetradecyl ester	C16H31ClO2
38	29.178	1662	2-Dodecen-1-yl(-)succinic anhydride	C16H26O3
39	29.777	1678	2-Hydroxy-1,1,10-trimethyl-6,9-epidioxydecalin	C13H22O3
40	29.951	1682	1-Cyclopropene-1-pentanol, .alpha.,.epsilon.,.epsilon.,2-tetramethyl-3-(1-methylethenyl)-	C15H26O
41	30.651	1701	2-Bromotetradecane	C14H29Br
42	31.045	1710	Tetradecane, 1-chloro-	C14H29Cl
43	31.355	1717	5.beta.,7.beta.H,10.alpha.-Eudesm-11-en-1.alpha.-ol	C15H26O
44	31.582	1722	7-Hexadecenal, (Z)-	C16H30O
45	31.771	1727	Pentane-2,4-dione, 3-(1-adamantyl)-	C15H22O2
46	32.092	1734	Butanol, 1-[2,2,3,3-tetramethyl-1-(3-methyl-1-penynyl)-cyclopropyl]-	C17H30O
47	32.468	1743	Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)-	C11H18N2O2
48	32.769	1750	Tetradecanoic acid	C14H28O2
49	33.645	1771	1-Decanol, 2-hexyl-	C16H34O
50	34.485	1790	Pentadecyl trifluoroacetate	C17H31F3O2
51	34.932	1801	Heptadecane, 2,6,10,15-tetramethyl-	C21H44
52	35.479	1813	1-Octanol, 2-butyl-	C12H26O
53	35.873	1822	1,2-Benzenedicarboxylic acid, bis(2-methylpropyl) ester	C16H22O4
54	36.928	1845	5,10-Diethoxy-2,3,7,8-tetrahydro-1H,6H-dipyrrolo[1,2-a;1′,2′-d]pyrazine	C14H22N2O2
55	37.910	1867	2-Hexadecene, 3,7,11,15-tetramethyl-, [R-[R*,R*-(E)]]-	C20H40
56	39.389	1900	Nonadecane	C19H40
57	41.675	1951	n-Hexadecanoic acid	C16H32O2
58	43.480	1991	1-Nonadecene	C19H38
59	46.996	2069	3-Chloropropionic acid, heptadecyl ester	C20H39ClO2
60	48.984	2114	9,12-Octadecadienoic acid (Z,Z)-	C18H32O2
61	49.228	2120	9-Octadecenal, (Z)-	C18H34O
62	50.660	2152	Ethyl iso-allocholate	C26H44O5
63	52.394	2192	9-Tricosene, (Z)-	C23H46
64	73.770	2727	13-Docosenamide, (Z)-	C22H43NO

Note: The bold and italic fonts are used to refer to exclusive compounds. RT: Retention time. RI: Reflex index.

Table 6: List of the hydrophilic volatile component identified from the fruit of the Ilex cornuta Lindl. & Paxton.

No	RT	RI	Compound	Molecular
1	9.133	1063	Mequinol	C7H8O2
2	9.303	1069	Phenol, 4-methyl-	C7H8O
3	9.430	1073	Hexane, 3-bromo-	C6H13Br
4	9.923	1089	Phenylethyl Alcohol	C8H10O
5	10.510	1107	4-Acetylbutyric acid	C6H10O3
6	12.643	1169	Benzoic acid	C7H6O2
7	13.559	1196	2-Furancarboxaldehyde, 5-(hydroxymethyl)-	C6H6O3
8	15.378	1249	1,5-Cyclooctadien-4-one	C8H10O
9	17.652	1315	Phenol, 2,6-dimethoxy-	C8H10O3
10	19.180	1361	Benzaldehyde, 3-hydroxy-4-methoxy-	C8H8O3
11	21.852	1442	2-Ethoxyphenylacetonitrile	C10H11NO
12	22.103	1450	Benzeneacetonitrile, 4-hydroxy-	C8H7NO
13	22.466	1461	Coumarin, 8-methyl-	C10H8O2
14	25.187	1547	1,4-Benzenediol, 2-(1,1-dimethylethyl)-	C10H14O2
15	25.508	1558	Dodecanoic acid	C12H24O2
16	25.876	1569	3,5-Octadienoic acid, 7-hydroxy-2-methyl-, [R*,R*-(E,E)]-	C9H14O3
17	25.938	1571	2-Cyclopenten-1-one, 4-hydroxy-3-methyl-2-(2-propenyl)-	C9H12O2
18	26.125	1577	1b,5,5,6a-Tetramethyl-octahydro-1-oxa-cyclopropa[a]inden-6-one	C13H20O2
19	26.492	1589	4-Chloro-3-n-hexyltetrahydropyran	C11H21ClO
20	27.323	1613	Ethanone, 1-[2-(5-hydroxy-1,1-dimethylhexyl)-3-methyl-2-cyclopropen-1-yl]-	C14H24O2
21	30.643	1700	Heptadecane	C17H36
22	32.760	1750	Tetradecanoic acid	C14H28O2
23	35.878	1822	1,2-Benzenedicarboxylic acid, bis(2-methylpropyl) ester	C16H22O4
24	41.694	1952	n-Hexadecanoic acid	C16H32O2
25	48.863	2111	9,12-Octadecadienoic acid, methyl ester	C19H34O2
26	49.244	2120	9-Octadecenal, (Z)-	C18H34O

Note: The bold and italic fonts are used to refer to exclusive compounds. RT: Retention time. RI: Reflex index.

As shown in Table 7, the total number of hydrophilic volatile compounds isolated from the six organs ranges from 25 to 64. There are 3 common compounds in the six organs, i.e. Dodecanoic acid, 1,2-Benzenedicarboxylic acid, bis(2-methylpropyl) ester and n-Hexadecanoic acid. This accounts for 12.0% of total hydrophilic volatile compounds for the leaf and 4.7% for the flower with an average of 8.4% for all the six organs, indicating the little commonality of the six organs. Each organ also has its exclusive compounds which are not found in any other organ. The percentage of exclusive compounds follows the order of flower > fruit > stem skin > root > stem > leaf. The flower has the largest number and percentage of the exclusive compounds, 40 and 62.5%, respectively. The leaf has the smallest number and percentage of the exclusive compounds, 9 and 36.0%, respectively. The stem and stem skin display medium numbers of exclusive compounds. The average percentage of the exclusive compounds in the six organs was 49.8%, nearly half, indicating strong organ specificity. These results provide evidence to support the practice of the traditional herbal medicine to treat the diseases using either the whole plant or part of the plants depending on which part contains the substances that can be used for therapeutic purposes.

Table 7: The number and percentage of the common and exclusive hydrophilic volatile compounds identified from the six organs.

Organ	Root	Stem	Stem Skin	Leaf	Flower	Fruit
Total Compounds	36	46	42	25	64	26
Common Compounds	3
Percentage of Common Compounds	8.3%	6.5%	7.1%	12.0%	4.7%	11.5%
Exclusive Compounds	17	21	21	9	40	15
Percentage of Exclusive Compounds	47.2%	45.7%	50.0%	36.0%	62.5%	57.7%

Table 8 presents the number of overlapping compounds, overlapping percentage and overlapping index. The stem and stem skin share the largest number (15) of overlapping compounds. The overlapping percentage is calculated to be 32.6% for the stem and 35.7% for the stem skin. The smallest number (5) of overlapping compounds are found between root and fruit, leaf and fruit. The percentage of overlapping compounds between each of the two organs ranges from 10.9% to 44.0%, which is relatively small, further demonstrating substantial component differences among the different organs. The overlapping index is used to reveal the similarity among the organs. Two organs share the same number of overlapping compounds, but the overlapping index could be different if the total number of the hydrophilic volatile compounds differs. The more total number of the hydrophilic volatile compounds, the less the percentage of the overlapping compounds and smaller the overlapping index. That is why the average overlapping indices between the two organs is introduced to normalize the difference. In addition, total average overlapping indices is derived to calculate the mean of the average overlapping indices between each organ and the other five organs. Based on Table 8, the total average overlapping indices for each organ follows the order of stem > stem skin > root > leaf > flower > fruit. The total average overlapping indices for the stem is the greatest at 3.056, indicating the stem share the maximum similarity with the plant. The total average overlapping indices for the fruit was the smallest at 1.090, indicating that the fruit share the minimum similarity with the plant. And there is not much difference in the average overlapping indices between fruit and the other five organs. Except fruit, the average overlapping indices between each of the two organs correlate well to their physical proximity. The root, stem and stem skin are the organs that the plant survive and grow, and their total average overlapping indices are greater than 2.5. The overlapping index differences among these three organs are small, and they share the most in common. As an evergreen plant, the leaf is symbiotically related to the plant although the relationship between each leaf and the plant is cyclical, so the leaf is secondarily related to the plant. The flower and fruit are also cyclically related to the plant and have the most distant relationship. The leaf, flower and fruit are necessary but not survival organs for the growth of the plant. The relationship between the organs and the plant generated from the analysis of the hydrophilic volatile compounds is consistent with their biological function.

Table 8: The number of overlapping compounds, overlapping percentage and overlapping index.

Organ1	Organ 2	Number of overlapping compounds	Overlapping percentage	Overlapping index for Organ 1	Overlapping index for Organ 2	Average overlapping indices between organ 1 and 2	Total average overlapping Indices
Root	Stem	14	38.9%	5.444	4.261	4.853	2.522
	Stem skin	11	30.6%	3.361	2.881	3.121
	Leaf	9	25.0%	2.250	3.240	2.745
	Flower	7	19.4%	1.361	0.766	1.064
	Fruit	5	13.9%	0.694	0.962	0.828
Stem	Root	14	30.4%	4.261	5.444	4.853	3.056
	Stem skin	15	32.6%	4.891	5.357	5.124
	Leaf	9	19.6%	1.761	3.240	2.501
	Flower	10	21.7%	2.174	1.266	1.720
	Fruit	6	13.0%	0.783	1.385	1.084
Stem skin	Root	11	26.2%	2.881	3.361	3.121	2.710
	Stem	15	35.7%	5.357	4.891	5.124
	Leaf	9	21.4%	1.929	3.240	2.585
	Flower	10	21.4%	1.929	1.266	1.598
	Fruit	6	14.3%	0.857	1.385	1.121
Leaf	Root	9	36.0%	3.240	2.250	2.745	2.435
	Stem	9	36.0%	3.240	1.761	2.501
	Stem skin	9	36.0%	3.240	1.929	2.585
	Flower	11	44.0%	4.840	1.891	3.366
	Fruit	5	20.0%	1.000	0.962	0.981
Flower	Root	7	10.9%	0.766	1.361	1.064	1.844
	Stem	10	15.6%	1.563	2.174	1.859
	Stem skin	10	14.1%	1.266	1.929	1.598
	Leaf	11	17.2%	1.891	4.840	3.366
	Fruit	7	10.9%	0.766	1.885	1.326
Fruit	Root	5	19.2%	0.962	0.694	0.828	1.090
	Stem	6	23.1%	1.385	1.000	1.193
	Stem skin	6	23.1%	1.385	0.857	1.121
	Leaf	5	19.2%	0.962	1.000	0.981
	Flower	7	26.9%	1.885	0.766	1.326

Conclusion

The root, stem, stem skin, leaf, flower and fruit of the Ilex cornuta Lindl. & Paxton contain hydrophilic volatile compounds that are evenly distributed in the water phase of the various organs of the plant and can volatilize with water vapor. The number and type of hydrophilic volatile compounds vary from organ to organ. There is only a small number of common compounds among the six organs and the number of overlapping compounds between each of the two organs is also relatively small. In addition, there are large number of exclusive compounds from each organ. Therefore, it is possible to identify the plant through the assessment of the hydrophilic volatile compounds isolated from each individual organ.

In conclusion, we found that hydrophilic volatile metabolites are a class of natural products that are rarely investigated but constitute a significant part of the plant chemical composition. Chemical profiling of these secondary metabolites could provide a valuable tool for identification and authentication of the plant samples, as well as resolving taxonomic problems and understanding the chemically mediated biological phenomena.

References

1. Bickford D, Lohman DJ, Sodhi NS, Ng PKL, Meier R, et al. (2007) Cryptic species as a window on diversity and conservation. Trends Ecol Evol 22: 148-155. [crossref]
2. Heinrichs J, Kreier HP, Feldberg K, Schmidt AR, Zhu RL, et al. (2011) Formalizing morphologically cryptic biological entities: New insights from DNA taxonomy, hybridization, and biogeography in the leafy liverwort Porella platyphylla (Jungermanniopsida, Porellales). Am J Bot 98: 1252-1262. [crossref]
3. Ludwiczuk A (2014) Fingerprinting of secondary metabolites of liverworts: chemosystematic approach. J of AOAC Int 97: 1234-1243.
4. Wink M (2003) Evolution of secondary metabolites from an ecological and molecular phylogenetic perspective. Phytochemistry 64: 3-19. [crossref]
5. Liu K, Abdullah AA, Huang M, Nishioka T, Altaf-Ul-Amin M, et al. (2017) Novel Approach to Classify Plants Based on Metabolite-Content Similarity. BioMed Res Int. doi: 10.1155/2017/5296729
6. Ghaste M, Narduzzi L, Carlin S, Vrhovsek U, Shulaev V, et al. (2015) Chemical Composition of Volatile Aroma Metabolites and Their Glycosylated Precursors that Can Uniquely Differentiate Individual Grape Cultivars. Food Chem 188: 309-319. [crossref]
7. Peters K, Treutler H, Doll S, Kindt ASD, Hankemeier T, et al. (2019) Chemical Diversity and Classification of Secondary Metabolites in Nine Bryophyte Species. Metabolites 9: 222. [crossref]
8. Staszek D, Orlowska M, Rzepa J, Wrobel MS, Kowalska T (2014) Fingerprinting of the Volatile Fraction from Selected Thyme Species by Means of Headspace Gas Chromatography with Mass Spectrometric Detection. J of AOAC Int 97: 1250-1258. [crossref]
9. Tundis R, Peruzzi L, Menichini F (2014) Phytochemical and biological studies of Stachy Species in Relation to Chemotaxonomy: A Review. Phytochemistry 102: 7-39. [crossref]
10. Peñuelas J, Llusià J (1999) Seasonal emission of monoterpenes by the Mediterranean tree Quercus ilex in field conditions: Relations with photosynthetic rates, temperature and volatility. Physiol Plant 105: 641-647.
11. Llusia J, Penuelas J (2000) Seasonal patterns of terpene content and emission from seven mediterranean woody species in field conditions. Am J Bot 87: 133-140. [crossref]
12. Ormeo E, Goldstein A, Niinemets ü (2011) Extracting and trapping biogenic volatile organic compounds stored in plant species. TRAC-Trend Anal Chem 30: 978-989.
13. Claudia G, Roberta A, Daniela L, Giacomo T, Laura S, et al. (2018) Salvia verticillata: Linking glandular trichomes, volatiles and pollinators. Phytochemistry 155: 53-60.
14. Wei X, Song M, Chen C, Tong H, Liang G, et al. (2018) Juice volatile composition differences between Valencia orange and its mutant Rohde Red Valencia are associated with carotenoid profile differences. Food Chem 245: 223-232. [crossref]
15. Berlinck RGS, Monteiro AF, Bertonha AF, Bernardi DI, Gubiani JR, et al. (2019) Approaches for the isolation and identification of hydrophilic, light-sensitive, volatile and minor natural products. Nat Prod Rep 36: 981-1004. [crossref]