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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]

Article Type

Research Article

Publication history

Received: June 31, 2021
Accepted: June 07, 2021
Published: June 10, 2021

Citation

Huang L (2021) Chemical Composition Similarity Relationships among the Various Organs of the Ilex Cornuta Lindl. & Paxton Based on the Analysis of Hydrophilic Volatile Compounds. Cancer Stud Ther J Volume 6(2): 1–10.

Corresponding author

Luosheng Huang
School of Traditional Chinese Pharmacy
China Pharmaceutical University
Nanjing 211198
P.R.China