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. 2015 Aug 28;20(9):15781–15796. doi: 10.3390/molecules200915781

Chemical Composition, Antioxidative and Anticancer Activities of the Essential Oil: Curcumae Rhizoma–Sparganii Rhizoma, a Traditional Herb Pair

Guan-Ling Xu 1, Di Geng 1, Meng Xie 1, Kai-Yue Teng 1, Yu-Xin Tian 1, Zi-Zhen Liu 1, Cheng Yan 1, Yan Wang 1, Xia Zhang 1, Yan Song 2, Yue Yang 1, Gai-Mei She 1,*
Editor: Derek J McPhee
PMCID: PMC6332236  PMID: 26343630

Abstract

As a classical herb pair in clinics of traditional Chinese medicine, Curcumae Rhizoma–Sparganii Rhizoma (HP CR–SR) is used for activating blood circulation to remove blood stasis. The essential components in HP CR–SR and its single herbs were comparatively analyzed using gas chromatography-mass spectrometry data. 66, 22, and 54 components in volatile oils of Curcumae Rhizoma, Sparganii Rhizoma, and HP CR–SR were identified, and total contents accounted for 75.416%, 91.857%, and 79.553% respectively. The thirty-eight components were found in HP CR–SR, and not detected in single herbs Curcumae Rhizoma and Sparganii Rhizoma. The highest radical trapping action was seen by an essential oil of HP CR–SR (IC50 = 0.59 ± 0.04 mg/mL). Furthermore, the HP CR–SR essential oil showed more remarkable cytotoxicity on tumor cell lines than that of the single herbs Curcumae Rhizoma and Sparganii Rhizoma in a dose-dependent manner: IC50 values showing 32.32 ± 5.31 μg/mL (HeLa), 34.76 ± 1.82 μg/mL (BGC823), 74.84 ± 1.66 μg/mL (MCF-7), 66.12 ± 11.23 μg/mL (SKOV3), and 708.24 ± 943.91 μg/mL (A549), respectively. In summary, the essential oil of HP CR–SR is different from any one of Curcumae Rhizoma and Sparganii Rhizoma, nor simply their superposition, and HP CR–SR oil presented more remarkable anticancer and antioxidant activities compared with Curcumae Rhizoma and Sparganii Rhizoma oils.

Keywords: Curcumae Rhizoma, Sparganii Rhizoma, Curcumae Rhizoma–Sparganii Rhizoma, herb pair, essential oils, antioxidant activity, anti-cancer activity

1. Introduction

Herbal pair (HP), as a basic unit in compatibility of traditional Chinese medicine (TCM), has been widely applied in folk medicine for thousands of years [1,2]. Based on the compatibility mechanism, HP was composed of two relatively fixed herbs in the clinic. When the two fixed herbs are used together, interactions will occur so as to enhance the effect, reduce the toxicity or even remove the side effects compared with using a single herb alone [3,4,5]. It has been proved that HP played an important role in the development of TCM [2]. Compared with TCM compatibility, HP is not only based on the basic features and principles of TCM compatibility, but also easier to study due to their simpler chemical compositions [2]. Therefore, the studies to HP chemistry will provide a different and relatively simple way for the study of prescription chemistry in TCM.

Curcumae Rhizoma–Sparganii Rhizoma (CR–SR) is a classical herbal pair in clinics. It is used for activating blood circulation to remove blood stasis. CR is good at breaking the Qi and dispersing the blood accumulation; SR is expert in removing blood stasis and dredging meridians [6]. HP CR–SR was first recorded as Sanleng Wan in “Jingyan Liangfang” during the Qing dynasty, and has long been applied for the treatment of blood stasis and dysmenorrheal from the TCM perspective [7,8]. So far, HP CR–SR has been involved as a TCM prescription preparation in Chinese pharmacopoeia, such as Fuke Tongjing Wan, Tongjingbao Keli, commonly being used to cure dysmenorrheal [9]. Moreover, HP CR–SR was usually used to treat tumors in the TCM clinic, especially with respect to the gynecology aspect [10,11,12].

At present, modern pharmacological research and clinical practices mostly focus on nonvolatile fractions of HP CR–SR, and it possesses extensive activities, including anti-cancer activity [13,14,15,16], anti-inflammatory activity, and anti-thrombotic activity [17]. The pharmacological activities of HP CR–SR essential oil are rarely reported and there is only one paper on the chemical components of HP CR–SR oil [18]. It is noticed that CR essential oil has been recorded in the Pharmacopoeia of People’s Republic of China (2010 edition), to treat gynecological oncology [9,19,20,21]. The potential biological damage caused by free radicals is a leading contributor to chronic diseases, including cancer, diabetes mellitus, aging, and neurodegenerative disorders [22,23] and the essential oil of CR was also good at antioxidant activity [24,25]. The difference between the HP oil and CR oil has not been described from the perspective of chemical compositions and pharmacological activities to the best of our knowledge.

In this paper, the essential oils from CR, SR, and HP CR–SR have been extracted, respectively, and the components have been identified by gas chromatography-mass spectrometry (GC-MS). Meanwhile, their antioxidant activity was assessed by scavenging free radical ability, and antitumor activity, in vitro, was also evaluated with several cell models by MTT assay. The aim of this work was to clarify the compatibility of HP CR–SR in the TCM clinic from the view of chemical compositions and the pharmacological activities.

2. Results and Discussion

Zedoary turmeric oil, the essential oil extracted from CR, is documented in the Chinese Pharmacopoeia (2010) that it has exhibited many potential medical characteristics in the aspects of anticancer and antivirus [26,27]. In records of Chinese medical formula books or traditional application, CR is always combined with SR [7,8]. The difference between HP oil and CR oil has been analyzed by GC-MS.

2.1. The Volatile Components of CR, SR and HP CR–SR

The essential oils of CR, SR, and HP CR–SR were extracted by hydrodistillation, and the yield was 6.0 mL/kg, 0.1 mL/kg, and 2.8 mL/kg, respectively. By means of the overall volume integration method, quantitative results of each component in pure chromatographic profiles of HP CR–SR, single herbs CR and SR can be obtained, as shown in Table 1. In total, 66, 22, and 54 volatile components in CR, SR, and HP CR–SR were determined quantitatively, accounting for 75.416%, 91.857%, and 79.553%, respectively. The volatile chemical components in HP CR–SR are notably different from those of single herb CR or SR.

Table 1.

Main chemical components of volatile oils from CR, SR, and HP CR–SR.

No. Name of Components RI CAS Identification Number CR SR HP (CR–SR)
φ/% t/min φ/% t/min φ/% t/min
1 4-Hydroxy-4-methyl-2-pentanone 834 123-42-2 - - 5.691 4.487 4.343 4.490
2 Isoborneol 1163 124-76-5 0.098 13.087 - - - -
3 Borneol 1172 507-70-0 0.229 13.327 - - - -
4 α-Terpineol 1194 98-55-5 0.263 13.970 - - - -
5 (2E,4E)-Decadienol 1300 18409-21-7 0.023 16.927 - - - -
6 Dimethoxy-(Z)-Citral 1310 75128-97-1 0.042 17.168 - - - -
7 Geranyl acclate 1355 105-87-3 0.025 18.379 - - - -
8 neiso-Dihydro carveol acetate 1360 256332-36-2 0.021 18.495 - - - -
9 (Z)-Trimenal 1371 300733-87-3 0.012 18.794 - - - -
10 trans-Menth-6-en-2,8-diol 1379 42370-41-2 0.008 19.014 - - - -
11 Orcinol 1382 504-15-4 - - 0.008 19.090 - -
12 β-Elemene 1389 515-13-9 0.089 19.280 - - - -
13 Myltayl-(12)-ene 1455 79562-97-3 0.286 20.937 - - - -
14 Ethyl-(2E,4Z)-decadienoate 1466 3025-30-7 0.760 21.285 - - - -
15 β-Chamigrene 1473 18431-82-8 0.406 21.405 - - - -
16 α-Amorphen 1479 20085-19-2 0.278 21.550 - - - -
17 α-Cuprenene 1480 29621-78-1 - - 0.011 21.576 - -
18 Isomethyl-α-(E)-Ionone 1488 55093-42-0 0.169 21.708 - - - -
19 Premnaspirodiene 1490 82189-85-3 0.896 21.813 - - - -
20 cis-β-Gualene 1493 88-84-6 0.096 21.896 - - - -
21 α-Sclinene 1497 473-13-2 0.737 21.998 - - - -
22 Lilial 1503 80-54-6 - - 0.025 22.148 - -
23 γ-Cadinene 1521 39029-41-9 0.265 22.561 - - - -
24 * Silphiperfol-5-en-3-one B 1545 199014-11-4 - - - - 0.113 23.148
25 * Elemd 1548 633-99-6 - - - - 0.117 23.204
26 Hedycaryol 1548 21657-90-9 0.483 23.204 - - - -
27 trans-Dauca-4(11),7-diene 1556 000-00-0 0.165 23.405 - - 0.024 23.321
28 * Germacrene B 1556 15423-57-1 - - - - 0.105 23.400
29 Dodecanoic 1556 143-07-7 - - 0.049 23.415 - -
30 (E)-Nerolidol 1559 40716-66-3 - - 0.014 23.486 - -
31 * Eremophila ketone 1561 158930-41-7 - - - - 0.177 23.523
32 Longicamphenylone 1563 58560-59-1 0.256 23.526 - - - -
33 Germacrene D-4-ol 1572 74841-87-5 0.125 23.787 - - - -
34 Caryophyllenyl alcohol 1567 000-00-0 0.436 23.882 - - - -
35 * Caryolan-8-ol 1576 178737-45-6 - - - - 0.144 23.890
36 * Thujopsan-2-β-ol 1588 150737-93-2 - - - - 0.132 24.033
37 Fokiend 1588 33440-00-5 0.586 24.145 - - - -
38 * Globulol 1590 51371-47-2 - - - - 0.529 24.208
39 * trans-β-Elemenone 1593 30824-86-3 - - - - 0.135 24.276
40 Curzerenone 1593 20493-56-5 26.078 24.481 - - 39.937 24.481
41 * β-Atlantol 1607 38142-56-2 - - - - 0.892 24.606
42 Citronellyl pentanoata 1607 7540-53-6 1.501 24.616 - - - -
43 Syringaldehyde 1601 134-96-3 - - 0.035 24.843 - -
44 * epi-Cedrol 1619 199003-73-2 - - - - 1.009 24.871
45 Khusimone 1619 30557-76-7 0.356 24.876 - - - -
46 * Isolongifolan-7-α-ol 1622 57566-26-4 - - - - 0.831 24.949
47 Isolongifolanone 1629 26839-51-0 0.600 24.962 - - - -
48 Torreyol (α-Muurolol) 1627 19435-97-3 0.453 25.058 - - 0.316 25.436
49 * 10-epi-γ-Eudesmol 1624 15051-81-7 - - - - 1.194 25.064
50 γ-Eudesmol 1632 1209-71-8 3.948 25.171 - - 2.484 25.189
51 2-Hydroxydiphenyl methane 1632 28994-41-4 - - 0.102 25.192 - -
52 * allo-Aromadendrene epoxide 1638 85160-81-2 - - - - 0.341 25.305
53 * Hinesol 1640 23811-08-7 - - - - 0.449 25.367
54 Cubenol 1641 21284-22-0 0.443 25.374 - - - -
55 α-Muurold 1643 19435-97-3 1.037 25.434 - - - -
56 Desmethoxy encecalin 1648 (CR), 1649 (HP) 10913-07-1 0.817 25.544 - - 0.749 25.549
57 β-Eudesmol 1655 (SR), 1656 (HP) 473-15-4 - - 0.091 25.704 3.402 25.709
58 α-Eudesmol 1656 473-16-5 3.310 25.717 - - - -
59 * Selin-11-en-4-α-ol 1659 16641-47-7 - - - - 1.675 25.784
60 neo-Intermeded 1659 5945-72-2 0.478 25.788 - - - -
61 ar-Turmerone 1661 532-65-0 - - 0.099 25.836 - -
62 * Germacra-4(15),5,10(14)-trien-1-α-ol 1668 81968-62-9 - - - - 0.488 25.909
63 5-Hydroxy isobornyl isobutanoata 1664 107783-33-5 0.493 25.909 - - - -
64 * 14-Hydoxy-9-epi-(E)-Caryophyllene 1669 79768-23-5 - - - - 0.679 26.002
65 Cadalene 1672 483-78-3 0.639 26.078 - - - -
66 Germacrone 1693 6902-91-6 2.778 26.560 - - 4.822 26.563
67 * Zizanal 1689 82509-29-3 - - - - 0.235 26.665
68 Sinensal 1699 60066-88-8 0.382 26.686 - - - -
69 (Z)-β-Santalol 1711 77-42-9 2.550 26.996 - - - -
70 * Mayurone 1711 4677-90-1 - - - - 0.560 26.961
71 Curcumenol 1720 18431-84-6 0.915 27.142 - - - -
72 * Longifold 1722 469-27-2 - - - - 0.498 27.183
73 Isobicyclogermacrenal 1723 73256-82-3 0.438 27.196 - - - -
74 Cryptomerione 1729 5988-72-7 1.730 27.422 - - - -
75 Ambroxide 1740 (CR), 1733 (HP) 6970-58-5 1.597 27.575 - - 0.374 27.415
76 * Cyclocolorenone 1740 489-45-2 - - - - 0.354 27.568
77 β-Acoradienol 1761 149496-35-5 0.991 28.015 - - - -
78 * Squamuloseone 1771 34413-94-0 - - - - 0.856 28.396
79 * 14-Hydroxy-α-Muurolene 1781 135118-51-3 - - - - 0.856 28.396
80 (2E,6E)-Methyl farnesoate 1783 3675-00-1 1.643 28.498 - - - -
81 * Vetivenic acid 1792 16203-25-1 - - - - 1.287 28.687
82 8-Cedren-13-ol accetate 1792 18319-41-0 2.781 28.695 - - - -
83 1-Octadecene 1794 112-88-9 - - 0.028 28.727 - -
84 14-Hydroxy-δ-Cadinene 1802 135118-52-4 0.509 28.925 - - 1.328 28.84
85 * 2-Ethylhexyl-Salicyate 1810 118-60-5 - - - - 0.314 29.073
86 iso-Acorone 1811 6168-64-5 0.396 29.083 - - - -
87 Acorone 1816 10121-28-5 0.405 29.210 - - - -
88 * Eudesm-7(11)-en-4-ol acetate 1831 67987-89-7 - - - - 0.417 29.499
89 Cyclopentadecanolide 1834 106-02-5 - - 0.348 29.556 - -
90 2,7(14),10-Bisabolatrien-1-ol-4-one 1837 216372-16-6 1.090 29.628 - - - -
91 * Phenyl ethyl octanoate 1838 5457-70-5 - - - - 0.658 29.637
92 n-Hexadecanol 1840 36653-82-4 0.476 29.678 - -
93 (E)-Nerolidy isobutyrate 1844 000-00-0 0.504 29.772 - - - -
94 1,10-β-epoxy-6-Oxofuranoeyemophilane 1850 5974-12-0 1.224 29.887 - - 0.962 29.880
95 Cubitene 1874 (CR), 1864 (HP) 66723-19-1 0.396 30.380 - - 0.451 30.181
96 cis-Thujopsenic acid 1865 546-53-2 1.015 30.199 - - - -
97 * Dihydro-Columellarin 1905 66873-38-9 - - - - 0.652 30.940
98 Totarene 1903 000-00-0 0.881 30.952 - - - -
99 Isopimara-9(11),15-dien 1906 39702-28-8 0.432 31.021 - - - -
100 * 11,12-dihydroxy Valencene 1919 000-00-0 - - - - 0.155 31.281
101 Methyl hexadecanoate 1923 112-39-0 - - 0.287 31.351 - -
102 * (3E)-Cembrene A 1929 31570-39-5 - - - - 0.121 31.481
103 Cyclohexadecanolide 1937 109-29-5 - - 1.208 31.636 - -
104 * Columellarin 1949 66873-37-8 - - - - 0.435 31.860
105 Hexadecanoic acid 1967 (SR), 1955 (HP) 57-10-3 - - 38.634 32.214 0.266 31.990
106 (3Z)-Cembrene A 1964 71213-92-8 1.026 32.162 - - - -
107 Nootkatin 1985 4431-03-2 1.775 32.573 - - - -
108 * epi-Catalponol 1988 70368-23-9 - - - - 1.348 32.625
109 Ethyl hexadecanoate 1991 628-97-7 - - 1.828 32.687 - -
110 Polygodial 2014 6754-20-7 0.285 33.117 - - 0.523 32.851
111 Dolabradiene 2000 134507-28-1 0.747 32.858 - - - -
112 * Phyllocladene 2015 20070-61-5 - - - - 0.254 33.162
113 Warburganal 2016 62994-47-2 0.296 33.354 - - - -
114 Kaurene 2035 34424-57-2 0.714 33.526 - - - -
115 * Osthole 2040 484-12-8 - - - - 0.551 33.607
116 * Sclaredide 2070 564-20-5 - - - - 0.140 34.185
117 Methyl linoleate 2089 (SR), 2124 (HP) 112-63-0 - - 0.572 34.523 0.099 35.160
118 Nootkatinol 2095 2492-08-2 0.448 34.638 - - 0.359 34.636
119 n-Octadecanol 2102 112-92-5 - - 0.404 34.763 - -
120 * (E)-Isoeugenyl Benzyl ether 2116 92666-21-2 - - - - 0.188 35.010
121 Linoleic acid 2124 (CR), 2285 (SR) 60-33-3 0.561 35.531 37.981 35.329 - -
122 * Oroselone 2131 1760-27-6 - - - - 0.113 35.279
123 * Abienol 2151 1616-86-0 - - - - 0.112 35.662
124 n-Docosene 2163 629-97-0 - - 3.196 35.874 - -
125 1-Docosene 2170 1599-67-3 - - 0.770 35.983 - -
Total 75.416 91.857 79.553
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RI refers to the retention index experimentally calculated using C7–C40 alkanes; φ is relative content; t is retention time; CR: Curcumae Rhizoma; SR: Sparganii Rhizoma; HP: herb pair CR–SR; -: Not detected; * New constituents in herb pair CR–SR.

2.1.1. The Common Volatile Oils of HP and CR

Several characteristics about the volatile chemical components in HP CR–SR can be seen from Table 1. The main volatile chemical compounds in HP were curzerenone (39.937%), 4-hydroxy-4-methyl-2-pentanone (4.343%), germacrone (4.822%), β-eudesmol (3.402%), γ-eudesmol (2.484%), selin-11-en-4-α-ol (1.675%), epi-catalponol (1.348%), 14-hydroxy-δ-cadinene (1.328%), and vetivenic acid (1.287%). Thereinto, curzerenone, γ-eudesmol, and germacrone, as the common volatile oils of HP and CR, were also the higher content components of CR, accounting for 26.078%, 3.948%, and 2.778% respectively.

It is noteworthy that curcumenol has broad-spectrum anticancer activity [21] and its content in HP oil was approximately two times higher than that of CR oil. Clinically, germacrone, as the official quality control marker for Zedoary turmeric oil, exhibited many pharmacological activities including anti-tumor activity, antiviral activity, and so on [28,29]. Comparing with CR oil, the content of germacrone was increased in HP oil. Three common components in volatile oils of HP and SR were detected, including 4-hydroxy-4-methyl-2-pentanone (5.691%) and two other trace amount ingredients. The conclusion from the analysis above is that the volatile chemical components in HP CR–SR were not completely the same as those of single herb CR or SR.

2.1.2. The New Chemical Components of HP Volatile Oil

There are 38 terpene alcohol-type components that were first found in HP, such as epi-cedrol (1.009%), 10-epi-γ-eudesmol (1.194%), selin-11-en-4-α-ol (1.675%), epi-catalponol (1.348%), vetivenic acid (1.287%), and so on. Those new component contents in HP reach up to 19.380%. The reports about their pharmacological activities are rather rare. The following compounds were worth mentioning: epi-cedrol, as a sesquiterpene alcohol, was also detected in Artemisia annua L, which could increase production of artemisinin by genetically-engineered methods [30]. Selin-11-en-4-α-ol was the principle component in the oil of Ononis Sicula Guss., exhibited moderate antioxidant activity [31]. It is the purportedly unique sesquiterpene compound, 10-epi-γ-eudesmol, that has been suggested as a marker for distinguishing the various cultivars and hybrids of Pelargonium graveolens [32]. In addition, vetivenic acid was isolated by column chromatography and identified by NMR and CG-MS from Vetiveria zizanioides root, with promising antimicrobial effects [33]. Interestingly, 2-ethylhexyl-salicyate, a significant sunscreen, was widely used in cosmetics and the medicine industry [34]. Mayurone was first reported from Mayur pankhi and Thujopsis dolabrata, respectively, in 1965, and widely existed in many kinds of plants such as Panax ginseng and Franco Thujaorientalis [35]. Considering the relatively high content and potential activity of those new components, it is inferred that they may impact on the activity and efficacy of HP oil.

Moreover, some volatile chemical compounds of CR, such as α-eudesmol (3.310%, 8-cedren-13-ol accetate (2.781%), (Z)-β-santalol (2.550%), nootratin (1.775%), and cryptomerione (1.730%), have not been detected in HP. In addition, hexadecanoic acid (38.634%), linoleic acid (37.981%), and n-docosene (3.196%), as the main chemical ingredients of SR volatile oil, have not been seen in HP. Possible reasons could be listed as follows: for one thing, some organic acids of SR, like succinic acid or sanleng acid, could play a role of solubilization effect, which have improved the constituents of CR dissolving out during the process of decocting the two single herbs, CR and SR [36]. For another, it has a chance to easily interact among many active organic compounds, such as aldehydes, ketones, and alcohols, during the process of decocting CR and SR [3,18,37,38,39,40]. In addition, the total contents of essential oils in CR and SR were reported to be quite uneven (2% and 0.04%, respectively) [36], thus, it probably means that volatile oil of SR were covered by the volatile oil of CR when the compatibility ratio of two herbs is one to one.

2.2. DPPH Radical Scavenging Activity

Free radicals and oxidants could affect the metabolism of the body [41]. Antioxidants have become important auxiliary products for health. DPPH is a useful reagent for investigating the free radical scavenging ability of compounds. The dose-response curve of DPPH radical scavenging activities of the essential oils from CR, SR, and HP CR–SR was distinct in Figure 1. The essential oil from HP CR–SR possessed the best activity with a rate of 90.6% under 5.0 mg/mL, and the essential oil from SR showed little DPPH radical scavenging activity.

Figure 1.

Figure 1

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The DPPH free radical scavenging activities of essential oil from CR, SR, and HP CR–SR.

Among total essential oils of the four samples, HP CR–SR possessed the highest antioxidant activity (IC50 = 0.59 ± 0.04 mg/mL). Compared with HP CR–SR oil, the volatile oil of CR showed moderate DPPH scavenging ability (IC50 = 1.39 ± 0.06 mg/mL), and the other two samples, SR and CR + SR, their IC50 were 8.39 ± 1.20 mg/mL and 2.20 ± 0.09 mg/mL, respectively. In addition, as a positive control, IC50 of vitamin C was 0.07 mg/mL in this condition.

In daily life, the increased dietary intake of natural antioxidants could reduce cancer mortality [42,43,44]. Moreover, antioxidants not only selectively inhibit the proliferation of cancer cells but also do not harm normal cells [45]. This means that the positive correlation exists between the antioxidant and antitumor activities.

2.3. Anti-Tumor Activity

The essential oils (CR, SR, and HP) were tested in vitro for their potential human tumor cell growth inhibitory effect on five human tumor cell lines (BGC823, A549, MCF-7, HeLa, and SKOV3), using MTT assay. The five human tumor cell lines are the most conventional tumor cell lines for the screening of antitumor activity, which are deemed to be high in veracity and reliability.

The concentration that inhibited cell vitality by 50% (IC50) is presented in Table 2. Apparently, HP essential oil was the most effective in all tested cell lines, and presented IC50 values ranging from 32.32–74.84 μg/mL, except for human lung cancer cell lines (A549). The CR oil had less activity compared with HP oil, IC50 in the range between 31.57 and 162.87 μg/mL. The oil of SR had the lowest antitumor activity, and it showed nearly no efficacy in inhibiting, instead promoting five human tumors cell lines. On the other hand, the HeLa cell line was the most susceptible to the oils of CR and HP, while, among the tumor cell lines, the A549 was the most resistant one (Figure 2).

Table 2.

In vitro cytotoxic activities of the essential oils.

Treatments MCF-7 IC50 (μg/mL) a HeLa SKOV3 BGC823 A549
HP 74.84 ± 1.66 32.32 ± 5.31 66.12 ± 11.23 34.76 ± 1.82 708.24 ± 943.91
CR + SR 184.51 ± 59.09 80.88 ± 10.06 131.02 ± 35.83 48.83 ± 2.76 >1000
SR >1000 185.19 ± 187.88 >1000 >1000 >1000
CR 82.30 ± 5.36 31.57 ± 8.13 72.23 ± 5.20 44.11 ± 1.95 162.87 ± 43.36
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a Results are expressed as IC50 values (μg/mL) from three independent experiments performed in quadruplicate, measured by MTT assay after 24 h of incubation. The IC50 value, relative to untreated control, represents the concentration that inhibited cell vitality by 50%.

Figure 2.

Figure 2

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Effect of the HP essential oil on cell viability of five cancer cell lines. Cells were treated with vehicle alone (0.5% DMSO) and specified concentrations of the essential oil in 0.5% DMSO for 24 h; cell viability was determined by the MTT assay as detailed in Section 2. The values are represented as the percent of viable cells, with vehicle-treated cells regarded as 100% viable. Each point represents the mean of the values obtained from three independent experiments.

In particular, in order to further compare the activity differences between HP and two single herbs (CR and SR), group (CR + SR) is also set in the test. Different from group HP, the group (CR + SR) was made of the oils of CR and SR with 1:1 input quantity. Seeing from Table 2, IC50 values of group HP are all lower than that of group (CR + SR) on human tumor cell growth inhibitory effect. Based on these findings, it can be speculated that the HP oil was remarkably better than the oil of any single one on anticancer activity.

In the inhibitory effect assays on five tested human tumor cell lines, the essential oil of HP, in a dose-dependent manner, was the most effective compared to CR and SR oils. In other words, with the SR being put into HP, more effective ingredients in CR were dissolved out. This is highly consistent with chemical research results, especially being related with new components in HP oil. In spite of having found new constituents in HP oil, their pharmacological activities were less reported. Their activities should be focused on in the further studies.

3. Experimental Section

3.1. Materials

Single herb CR and SR, with vinegar processing, were purchased from Hebei Anguo Medical Materials Corporation (Anguo, China), and identified as Curcuma kwangsiensis S.G. Lee et C.F. Liang and Sparganium stoloniferum Buch.-Ham., respectively, by Yuan Zhang from the Beijing University of Chinese Medicine, China. n-alkane standard solutions of C7–C40 (No. 49452-U) was purchased from SUPELCO (Bellefonte, PA, USA).

3.2. Extraction of the Essential Oils

A total of 100 g of CR and 100 g of SR single herbs were mixed and soaked with 1000 mL deionized water in a standard extractor for extracting volatile oil at room temperature for 12 h. Then, the essential oil was prepared by using the standard steam distillation method, according to the Chinese Pharmacopeia [9]. The essential oil was collected and stored in a refrigerator at 4 °C before use. The essential oils of single herb CR or SR was extracted in the same method.

3.3. GC-MS Analysis

Volatile oils were analyzed by an Agilent Technologies 5975C gas chromatograph (GC) equipped with a HP-5 MS capillary column (5% phenyl methyl Siloxane, 30 m × 0.25 mm i.d., 0.25 μm film thickness) and an HP 5973 mass selective detector (Agilent Technologies, Palo Alto, CA, USA) in the electron impact ionization mode (70 eV) under the following operating conditions: split ratio, 1:40; injection volume, 1.0 µL (TBME solution); inlet temperature, 260 °C; detector temperature, 260 °C; The oven temperature was programmed to start at 50 °C, increased at 5 °C/min to 100 °C, 100 °C increased at 3 °C/min to 150 °C, 150 °C increased at 5 °C/min to 250 °C, then held at 250 °C for 30 min. The helium (99.999%) carrier gas was kept at a constant flow of 1.2 mL/min.

3.4. Identification of Compounds

The chemical ingredients of volatile oils were identified by the retention indices (RI), relative to a series of alkanes (C7–C40) at the same chromatographic conditions, consulting the Van Den Dool method [46]. The data were analyzed by the Xcalibur 1.1 software (Thermo Fisher Scientific, Waltham, MA, USA, version 1.1), compared with the NIST/EPA/NIH database (2005, version 2.0d, Scientific Instrument Services, Inc., Ringoes, NJ, USA) and several references [47,48,49,50]. The book “Identification of Essential Oils Components by Gas Chromatogrtaphy/Mass Spectrometry, 4th edition” [51] played an equally vital role in identifying the individual compounds.

3.5. Anti-Tumor Activity

Human gastric cancer cell lines (BGC823), human lung cancer cell lines (A549), human breast carcinoma cell lines (MCF-7), human cervix carcinoma cell lines (HeLa), and human ovary carcinoma cell lines (SKOV3) were purchased from Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. These cell lines were maintained in RPMI 1640 culture medium plus 10% calf serum and 100 U/mL penicillin, 75 U/mL streptomycin, in a 37 °C incubator supplied with 95% room air and 5% CO2.

The growth inhibitory effect of essential oils of HP, CR, and SR were measured using the standard MTT assay [52,53]. Briefly, 100 μL samples of cell suspension (6 × 104 cells/well) were cultivated in 96-cell plates for 18 h, as described. The samples of HP, CR, and SR oil were well dissolved in DMSO, respectively, followed ultrasonic vibration. Then, cells were incubated for 24 h with varying concentrations of the oil. After adding 100 μL MTT (1 mg/mL) solution to each well, the cells were further incubated at 37 °C for15 min, in a humidified atmosphere of 95% air/5% CO2. For MTT assay, the supernatant was thrown away and 200 μL DMSO was mixed into, the 96 wells plate was oscillated on micro-vibrator for an additional 10 min, the absorbency of each well was determined at λ 560 nm by an enzyme-immunoassay instrument (Beijing Perlong New Technology Co., Ltd., Beijing, China). Anti-tumor activity was expressed as the concentration of the drug inhibiting cell growth by 50% (IC50 value).

3.6. DPPH Radical Scavenging Activity

The DPPH radical scavenging activity was determined according to the method reported by Sharififar and others with some modifications, and compared with the activity of vitamin C as a positive control [54]. An equal amount of (1 mL of 0.05 mg) DPPH solution in ethanol was mixed with 0.1 mL essential oil from CR, SR, and HP CR–SR in ethanol at various concentrations. After mixing, the solution was allowed to reach a steady state at room temperature for about 30 min. The DPPH radical scavenging activity was determined by the absorbance at λ 517 nm with the DNM9602A microplate reader (Beijing Perlong New Technology Co., Ltd., Beijing, China) and calculated by the following equation:

DPPH radical scavenging activity = (A0 − A1)/A0 × 100%

where A0 is the absorbance of the control (blank, without extract) and A1 is the absorbance of the mixture with the essential oil.

4. Conclusions

In the present study, it is first reported that the essential oils of CR, SR, and HP CR–SR were comparatively explored from the aspects of the chemical compositions, antioxidant, and anticancer activities. The essential oil of HP is different from any, one of CR and SR, nor simply their superposition. Thirty-eight new components were detected in HP, and the relative contents of those new components reach up to 19.380%. Moreover, HP oil showed significant antioxidant and anticancer activities and the essential oils from four groups on antioxidant and anticancer activities were consistent, in order: SR < CR + SR < CR < HP. It is well known that the essential oil of CR reveals prominent activities in the clinic, and it has been documented in different versions of Chinese Pharmacopoeia since 1977. Compared with CR oil, HP oil possessed the stronger antioxidant and anticancer activities. Thus, the volatile oil of HP has the better medicinal application prospect and deserves more attention in the pharmaceutical industry.

HP CR–SR belongs to a mutual reinforcement relationship according to seven compatibilities in TCM theory. Comparison and analysis of the essential oils of CR, SR, and HP CR–SR from chemical compositions and pharmacological activities, we can infer that CR may play a predominant role in HP, namely sovereign drug according to TCM theory. In spite of the low concentration of SR oil, it also plays an important part in HP through improving the dissolution of effective constituents and helping interact to produce new ingredients. In order to further illustrate the role of SR in HP, more focus can be concentrated on the change of HP essential oils (produced from different proportion compatibilities of CR and SR) on the chemical composition and pharmacological activities. Furthermore, it can be concluded that the chemical composition in HP CR–SR is responsible for its potent anticancer and antioxidant activities. Further investigations are in progress in our laboratory to identity the exact active principle and their action mechanism involved in anticancer and antioxidant activity.

Recently, the studies about herb pairs mostly concentrated on the content change of some main components and traced the origin of chemical constituents [55,56,57,58]. Based on the integrity of TCM theory, it is suggested to be more focused on the content change of common components and the appearance of new constituents in herb pairs with different proportion compatibilities. Such experimental design and research approaches are encouraged to be applied in the further study on HP CR–SR decoction, in order to clarify the compatibility essence of HP.

Acknowledgments

This project was supported by grants from Beijing Nova Program (No. 2011070), Excellent Talents of Beijing (NO. 2012B009999000003), Self-selected Topic of Beijing University of Chinese Medicine (No. 2015-JYB-XS110) and National Training Programs of Innovation and Entrepreneurship for Undergraduates (No. 201510026047 and 201510026057).

Author Contributions

Guan-Ling Xu, Di Geng, Meng Xie and Gai-Mei She designed reseach, Kai-Yue Teng, Yu-Xin Tian, Cheng Yan, Zi-Zhen Liu, Yan Wang, Yan Song, Xia Zhang and Yue Yang performed reseach and analyzed the data; Guan-Ling Xu, Di Geng, Meng Xie and Gai-Mei She wrote the paper. All authors read and approved the final manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

Sample Availability: Samples of essential oils from CR, SR and HP CR–SR are available from the authors.

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