Antioxidant Activity and Chemical Constituents of Essential Oil And

Food Chemistry 125 (2011) 456–463

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Food Chemistry

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Antioxidant activity and chemical constituents of essential oil and extracts of Rhizoma Homalomenae ⇑ Ling-Bin Zeng a, Zhong-Rong Zhang a, Zhu-Hua Luo b, Ji-Xiao Zhu c, a Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Hong Kong SAR, China b Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, Xiamen 361005, Fujian, China c College of Pharmaceutical Science, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, Jiangxi, China article info abstract

Article history: Antioxidant activity and composition of essential oil and extracts of Rhizoma Homalomenae were deter- Received 25 February 2010 mined. The extracts, especially the ethyl acetate extract (QJ4 fraction) of the aqueous residue after oil dis- Received in revised form 28 July 2010 tillation, had considerable antioxidant potency which was significantly associated with their total Accepted 7 September 2010 phenolic and flavonoid contents, but the essential oil showed only weak or moderate activity. GC–MS analysis of the essential oil (yield: 0.82%, v/w) resulted in the identification of 77 compounds, accounting for 96.5% of the content of the oil. The major components, epi-a-cadinol (14.8%), a-cadinol (14.8%), a-ter- Keywords: pineol (13.8%), linalool (11.1%), terpinen-4-ol (4.92%), and d-cadinene (4.91%) constituted 64.3% of it. LC– Homalomena occulta MS/MS and HPLC analyses showed seven phenolic compounds (protocatechuic acid, vanillic acid, syringic Rhizoma Homalomenae Antioxidant activity acid, caffeic acid, p-coumaric acid, ferulic acid and apigenin) with a great amount in the ethyl acetate Essential oil extract (QJ4 fraction). The strong antioxidant properties of the plant extracts may be attributed to the GC–MS presence of these phenolics. LC–MS/MS Ó 2010 Elsevier Ltd. All rights reserved. HPLC

1. Introduction muscles and bones, relieving stomachache, and relief from rheu- matoid arthritis, healing pain and swelling due to traumatic injury Reactive oxygen species (ROS) include both oxygen radicals, (Zhong Hua Ben Cao Editorial Committee, 1999). Traditionally, it is such as superoxide, hydroxyl, peroxyl, and hydroperoxyl radicals, mostly used as an aqueous decoction, alcoholic beverage or for and some non-radical oxidising agents, such as hydrogen peroxide, external application (Zhong Hua Ben Cao Editorial Committee, hypochlorous acid, and ozone and the non-radical ROS can convert 1999). Its essential oils showed anti-virus and antibiotic activities easily into radicals (Bayr, 2005). Excessive ROS levels damage lip- and the aqueous or alcohol extracts had anti-histamine, anti-coag- ids, proteins and nucleic acids through oxidation and thus are asso- ulant, anti-inﬂammatory and antialgesic activities (Zhong Hua Ben ciated with various diseases, such as atherosclerosis, arthritis, Cao Editorial Committee, 1999). A series of sesquiterpenoids, phe- neurodegenerative disorders, and cancers (Balsano & Alisi, 2009). nolic acids and other compounds (Elbandy, Lerche, Wagner, & Regular supplement of antioxidants can assist the endogenous de- Lacaille-Dubois, 2004; Hu, Yang, Ye, & Cheng, 2003; Hu et al., fence systems to counterbalance the harmful effects of excessive 2008; Hu, Yang, Wang, & Ye, 2009; Tian, Zhao, Yu, & Fang, 2010) ROS (Kaur & Geetha, 2006). But, synthetic antioxidants, such as was obtained from organic extracts of dry rhizome or aerial parts butylated hydroxytoluene (BHT) and butylated hydroxyanisole of H. occulta. The phenolic acids exhibited BACE1 (b-secretase) (BHA) are suspected of being responsible for some severe toxic ef- inhibitory activity (Tian et al., 2010) and four of the sesquiterpe- fects. Thus, natural antioxidants, from medicinal plants, vegetables, noids had a stimulative effect on proliferation and differentiation and fruits, are considered to be better alternatives and receive of cultured osteoblasts (Hu et al., 2008). increasing attention. To the best of our knowledge, no study on the antioxidant activ- Rhizoma Homalomenae is the dry rhizome of Homalomena occ- ity of essential oil and organic or aqueous extracts from Rhizoma ulta (Lour.) Schott. It is a famous traditional Chinese medicine Homalomenae has been reported so far. Thus, in the present study, (called as Qiannianjian in Chinese). Its main medical functions in- the antioxidant activity of the essential oil and various solvent ex- clude invigoration of the kidney and liver, strengthening of the tracts of Rhizoma Homalomenae were evaluated. Total phenolic and ﬂavonoid contents of the extracts, chemical composition of ⇑ Corresponding author. Tel.: +86 791 7118873. the essential oil and the phenolic constituents of the extract with E-mail address: [email protected] (J.-X. Zhu). most potent antioxidant ability were also determined.

2. Materials and methods (10 ll) was mixed with 900 ll of 100 mM Tris–HCl buffer (pH 7.4), 40 ll of methanol and 50 ll of 0.5% (w/w) Tween 20 solution 2.1. Plant materials and chemicals (Bruni et al., 2004). And ABTS radical was generated by mixing 7 mM ABTS and 2.45 mM potassium persulphate via incubation Rhizoma Homalomenae was purchased from Hong Kong in Jan at 23 °C in the dark for 12 h. Then, 0.1 ml of sample solution was 17, 2009 (originally produced in Guangxi Province, China). The mixed with 2.6 ml of diluted ABTS radical solution. After incuba- dry materials were ground to powder and passed through a 20- tion at 23 °C for 6 min, absorbance of the mixture solution was mesh sieve for the preparation of essential oil and extracts. measured at 734 nm in a microplate reader (PowerWave XS, Bio- 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,20-azinobis(3-ethyl- Tek Instruments Inc.). The ABTS radical-scavenging activity (%) benzothiazoline-6-sulphonic acid) (ABTS), potassium persulphate, was calculated by the following equation: scavenging activity 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), (%) = (1 Asample/Acontrol) 100, where Asample is the absorbance nitroblue tetrazolium (NBT), b-nicotinamide adenine dinucleotide in the presence of extracts and Acontrol is the absorbance of control. (NADH), phenazin methosulphate (PMS), b-carotene, linoleic acid, BHT was used as the reference compound. potassium ferricyanide, trichloroacetic acid, ferric chloride, BHT, Folin–Ciocalteau reagent, gallic acid, rutin, protocatechuic acid, vanillic acid, syringic acid, caffeic acid, p-coumaric acid, ferulic 2.5. Antioxidant assay using the b-carotene bleaching method acid, and apigenin were purchased from Sigma–Aldrich (St. Louis, MO). All other reagents were of analytical or HPLC grades. The antioxidant activity of the extracts and essential oil were also evaluated by the b-carotene bleaching test (Conforti, Statti, b 2.2. Preparation of essential oil and extracts Uzunov, & Menichini, 2006). Briefly, 1 ml of -carotene solution (0.2 mg/ml in chloroform) was added to 0.02 ml of linoleic acid The sieved powder was subjected to water distillation for 5 h and 0.2 ml of 100% Tween 20. The mixture was then evaporated at 40 C for 10 min in a rotary evaporator to remove chloroform. using a distillation apparatus. The essential oil obtained was dried ° After evaporation, 60 ml of distilled water were slowly added to over anhydrous sodium sulphate and stored at 4 °C in the dark un- the mixture and agitated vigorously to form an emulsion. 0.2 ml til used. of sample solution was transferred to the emulsion (5 ml) and The aqueous phase, after the essential oil distillation, was 0.2 ml of solvent instead of sample solution in 5 ml of the above extracted exhaustively by dichloromethane, ethyl acetate, and emulsion was used as a control. BHT was used as the reference n-butanol, successively, at a ratio of 1:1 (v/v, aqueous phase/ compound. The mixture obtained was then gently shaken and solvent) and the extracts obtained were numbered as QJ3, QJ4, placed at 50 C for 60 min. The absorbance of the mixture was and QJ5, respectively. After each extraction, the organic solvents ° measured at 470 nm, using a microplate reader (PowerWave XS, were removed under vacuum at 50 °C with a rotary evaporator. BioTek Instruments Inc.) against a blank, consisting of an emulsion One part of the aqueous phase after the solvent extraction above without b-carotene. The antioxidant activity was measured using was lyophilised and numbered as QJ1, while the other part was the following equation: antioxidant activity = [1 (S S )/ treated with ethanol to precipitate carbohydrates and the superna- 0 t (C C )] 100, where S and C are the absorbances measured tant after the removal of ethanol with a rotary evaporator was 0 t 0 0 at the initial incubation time for samples and control, respectively, lyophilised and numbered as QJ2. The solid residue after the essen- while S and C are the absorbances measured in the samples and tial oil distillation was lyophilised and then extracted exhaustively t t control at t = 60 min, respectively. with ethyl acetate and ethanol, successively, at a ratio of 1:20 (w/v, lyophilised solid residue/solvent) and the extracts obtained were numbered as QJ6 and QJ7, respectively. After each extraction, the 2.6. Reducing power assay organic solvents were removed. All the extracts were lyophilised and kept in the dark at 4 °C until used. The preparation procedure Reducing power of the extracts was also determined (Lee et al., of the essential oil and extracts is schematically represented in 2007). Extract solution (80 l), sodium phosphate buffer (200 l, Fig. 1. l l 0.2 M, pH 6.6) and potassium ferricyanide (200 ll, 10 mg/ml) were mixed and incubated at 50 °C for 20 min. Then, trichloroacetic acid 2.3. DPPH radical-scavenging assay (200 ll, 100 mg/ml) was added and incubated for 5 min to stop the reaction. Afterwards, the mixture (680 ll) was mixed with 680 ll DPPH radical-scavenging effect of the extracts and essential oil of distilled water and 68 ll of ferric chloride (10 mg/ml). Finally, was estimated by the method described by Brand-Williams, Cuve- absorbance of the resulting mixture was measured in a microplate lier, and Berset (1995) with a little modification. In brief, 0.1 ml of reader (PowerWave XS, BioTek Instruments Inc.). Increased absor- extract or essential oil solution was mixed with 2 ml of DPPH solu- bance at 700 nm, of the reaction mixture, indicates increased tion with absorbance of 0.700 ± 0.005 at 517 nm. The mixture was reducing power. BHT was used as the reference compound. incubated for 30 min at 23 °C. The absorbance of the resulting solution was measured in a microplate reader (PowerWave XS, BioTek Instruments Inc.) at 517 nm. The DPPH radical-scavenging activity 2.7. Determination of total phenolic content (TPC) (%) was calculated by the following equation: scavenging activity (%) = (1 Asample/Acontrol) 100, where Asample is the absorbance TPC of the extracts was measured by the Folin–Ciocalteu meth- in the presence of extracts and Acontrol is the absorbance of control. od (Singleton & Rossi, 1965). Briefly, 1 ml of extract solution was BHT was used as the reference compound. mixed with 5 ml of 0.2 N Folin–Ciocalteu reagent. After 6 min, 4 ml of sodium carbonate (75 g/l) were added. Absorbance of the 2.4. ABTS radical-scavenging assay resulting solution was measured at 760 nm in a microplate reader (PowerWave XS, BioTek Instruments Inc.) after incubation at 23 °C The improved ABTS method was used to measure the ABTS rad- for 2 h with intermittent shaking. A standard curve was plotted, ical-scavenging activity of the extracts and essential oil (Re et al., using gallic acid. The amount of phenolic content was calculated 1999). Briefly, an aliquot of extract or essential oil solution as gallic acid equivalent (mg GAE/g dry extract). 458 L.-B. Zeng et al. / Food Chemistry 125 (2011) 456–463

Fig. 1. Preparation scheme of essential oil and extracts from Rhizoma Homalomenae.

2.8. Determination of total flavonoid content (TFC) out using an Agilent 1200 series liquid chromatograph (Agilent, San- ta Clara, California, USA) and an Applied Biosystem API 2000 mass TFCs in the extracts were determined spectrophotometrically spectrometer instrument (Life Technologies Corporation, Carlsbad, according to the method described by Liu et al., 2007). 1 ml of sam- California, USA) equipped with an electrospray ionisation (ESI) ple solution was mixed with 1 ml of 2% aluminium chloride meth- interface. The LC system includes a binary pump and an in-line deg- anolic solution. After incubation at 23 °C for 15 min, the asser. The HPLC separation was completed on a reverse-phase Zor- absorbance of the reaction mixture was measured at 430 nm with bax C18 column (250 mm i.d. 3.6 mm, 5 lm, Agilent, Santa a microplate reader (PowerWave XS, BioTek Instruments Inc.). Ru- Clara, California, USA). Analyst software (Life Technologies Corpora- tin was used as a standard to make the calibration curve and the tion, Carlsbad, California, USA) was used for instrument control and flavonoid content was expressed as rutin equivalent (mg RE/g data processing. Acidified water (0.1% acetic acid, v/v) and methanol dry extract). were used as the mobile phases A and B, respectively. The gradient elution was programmed as follows: from 5% to 10% B in 15 min; from 10% to 30% B in 5 min; from30% to 50% B in 15 min; from 50% 2.9. Constituents of essential oil analysed by GC–MS B to 60% B in 12 min; from 60% to 100% B in 5 min and held at 100% for 10 min. The flow rate was 1 ml/min and the injection vol- The GC–MS analysis of essential oil was performed in an Agilent ume was 10 ll. The effluent from the HPLC column was split using 7890A GC system (Agilent, Palo Alto, California, USA) coupled with a T-type phase separator before being introduced into the mass an Agilent 5975C mass spectrometer in EI (electron impact) mode spectrometer. Conditions for MS analysis included the IonSpray with electron energy set at 70 eV, and mass range at m/z 25–500. voltage of 4500 V, the nebulizer gas pressure of 60 psi, the drying The column used was an HP-5MS capillary column temperature of 550 °C, the heater gas pressure of 45 psi, the curtain (30 m 0.25 mm 0.25 lm). gas of 20 psi and the interface heater was on. The declustering po- GC injector temperature was set at 280 °C and the temperature tential, focusing potential and entrance potential, were 25, 250 of the MS source was 230 °C. Helium was carrier gas at a flow rate and 10 V, respectively. The collision energy for each compound of 1.5 ml/min. Oven temperature was programmed from 60 °C, held was optimised. The nebulising and collision gas used in the MS anal- for 2 min, to 90 °C at a rate of 15 °C/min, held for 3 min, then to ysis was nitrogen. The phenolic compounds in the QJ4 fraction were 125 °Cat5°C/min, held isothermal for 8 min, then to 140 °Cat characterised and identified by MS/MS by comparison with MS data 5 °C/min, held for 7 min; finally it was raised to 220 °Cat16°C/ and LC retention times of the standard compounds. min and held for 2 min. Two microliter of diluted essential oil (1:49 in acetone, v/v) were injected automatically in split mode with a ratio of 30:1. 2.11. Phenolic content determined by HPLC analysis The essential oil constituents were identified by comparison of their linear retention indices (relative to C8-C40 n-alkanes on the The content analysis of phenolic compounds in the QJ4 fraction HP-5MS column) with literature values and their mass spectra was conducted using a HPLC system consisting of a Waters 600 with those from the NIST05 library. pump, a Waters 600 controller, a Waters 2487 UV detector, a Waters in-line degasser (Waters Corporation, Massachusetts, 2.10. Phenolic components in QJ4 fraction analysed by LC–MS/MS USA) and a Zorbax C18 column (250 mm i.d. 3.6 mm, 5 lm, Agi- lent, Santa Clara, California, USA). Empower software (Waters Cor- Phenolic analysis of the QJ4 fraction by LC–MS/MS, in the nega- poration, Massachusetts, USA) was used to control the system and tive ion MRM (multiple reaction monitoring) mode, was carried process the data. The LC conditions were the same as in the LC part L.-B. Zeng et al. / Food Chemistry 125 (2011) 456–463 459

in the LC–MS/MS analysis. The quantiﬁcation was carried out using IC50 of 0.10 mg/ml. QJ4, QJ5, and QJ6 had similarly strong activities the external standard method and calibration plots were con- inhibiting b-carotene bleaching (p < 0.050), whose IC50 values were structed by plotting the peak area against concentration of each 0.28, 0.61 and 0.39 mg/ml, respectively. The QJ2, QJ3, and QJ7 frac- phenolic standard. The wavelength used for the analysis was tions had similarly moderate activities (p < 0.035) and their IC50 280 nm. The amount of phenolic compounds was expressed as values were 1.92, 2.44 and 1.94 mg/ml, respectively. The QJ1 frac- microgramme per gramme of dry extract (lg/g). tion showed the weakest antioxidant activity in the b-carotene /

linoleic acid test with an IC50 of 6.54 mg/ml in the extracts. The 2.12. Statistical analysis essential oil (IC50 = 3.62 mg/ml) also had moderate antioxidant activity in the test. Fig. 2 suggests that the antioxidant activity of All the data were presented as means ± SD. Differences at all the extracts and the essential oil was dose-dependent in the DPPH, ABTS and b-carotene/linoleic acid tests. p < 0.05 were considered statistically significant. The IC50 values were calculated from linear regression analysis. All the statistical analyses were performed with Origin 6.0 (Microcal Software Inc., 3.4. Reducing power Northampton, USA). Ferric ion (Fe3+) reduction is often used as an indicator of elec- 3. Results and discussion tron-donating activity, which is an important mechanism of phenolic antioxidant action, and can be strongly correlated with 3.1. DPPH radical-scavenging activity other antioxidant properties. In the reducing power test, increased absorbance of the reaction mixture indicates increased reducing The DPPH radical is a stable free radical, commonly used as a power. As shown in Fig. 2D, the QJ4 fraction exhibited the highest substrate, to evaluate in vitro antioxidant activity of extracts of reducing power among the extracts, while the QJ3, QJ5, and QJ6 fruits, vegetables and medicinal plants. Antioxidants can scavenge fractions had similarly moderate reducing powers (p < 0.070), the radical by hydrogen donation, which causes a decrease of DPPH being more active than the QJ7 fraction. The QJ1 and QJ2 fractions absorbance at 517 nm. Fig. 2A shows the DPPH radical-scavenging had only low reducing powers. The reducing power of the extracts activity of the different extracts and the essential oil compared was also dose-dependent. with that of BHT. The QJ4 fraction had the strongest ability to scavenge DPPH radicals. The QJ3, QJ5 and QJ6 fractions also had strong 3.5. Total phenolic and flavonoid contents antioxidant activity and the QJ7 fractions had moderate activity. The QJ1and QJ2 fractions, especially the former, exhibited only Naturally occurring phenolic compounds, including phenolic weak ability to scavenge the radicals. From Fig. 2A, it was found acids, flavonoids, tannins, stilbenes, curcuminoids, coumarins, lign- that the order of DPPH radical-scavenging ability of these samples ans, quinones and others (Huang, Cai, & Zhang, 2010), have been re- was BHT > QJ4 > QJ3 > QJ5 > QJ6 > QJ7 > QJ2 > QJ1. The concentra- ported to be significantly associated with the antioxidant activity of tion of sample at which the inhibition percentage reaches 50% is plant and food extracts, mainly because of their redox properties, defined as the IC50 value. Thus, IC50 value is negatively related to allowing them to act as reducing agents, hydrogen donors, singlet the antioxidant activity, the lower the IC50 value, higher is the anti- oxygen quenchers, hydroxyl radical quenchers, and metal chelators oxidant activity of the tested sample. The IC50 values of BHT, QJ3, (Gupta & Prakash, 2009). In the present study, the Folin–Ciocalteu QJ4, QJ5, QJ6 and QJ7 fractions were, 0.11, 1.73, 0.80, 2.17, 3.12, reagent was used to obtain a crude estimate of the amount of pheno- and 5.97 mg/ml, respectively. These values are significantly differ- lic compounds present in the extracts. As results, TPCs of the extracts ent (p < 0.05). The IC50 values confirmed the activity order of the decreased in the following order, as shown in Table 1: samples discussed above. The essential oil had only very weak QJ4 > QJ3 > QJ5 P QJ6 > QJ7 > QJ2 > QJ1. The TPC of the QJ4 at DPPH radical-scavenging activity with an IC50 of 120 mg/ml. 141 ± 1.21 mg GAE/g dry extract was much higher than those of all other extracts. The flavonoid content in the extracts was determined 3.2. ABTS radical-scavenging activity spectrophotometrically, based on the formation of flavonoid-aluminium complexes, having the absorptivity maximum at 430 nm. The ABTS radical is also commonly used to evaluate the in vitro As shown in Table 1, the total flavonoid content of the extracts varied antioxidant activity of different substrates. As shown in Fig. 2B, the from 1.26 ± 0.02 to 13.5 ± 0.14 mg RE/g dry extract and the QJ4 frac- concentration–response curves of extracts and BHT to ABTS radical tion had the highest level of flavonoids, followed by assay were quite similar to the curve obtained in the DPPH assay. QJ6 > QJ5 > QJ3 > QJ7 > QJ2 in that decreasing order. Due to the for- The activities of the extracts scavenging the ABTS radicals were in mation of a cotton-like precipitate in the process of measurement, the descending order: BHT > QJ4 > QJ3 > QJ5 > QJ6 > QJ7 > QJ2 and the total flavonoid content of the QJ1 fraction could not be obtained. their IC50 values were 0.10, 0.53, 1.08, 1.49, 1.86, 4.60, and 8.88 mg/ml, respectively. The differences of these IC50 were signif- 3.6. Correlations of the assays determining antioxidant activity of the icant (p < 0.05). The QJ1 fraction showed only 25.7 ± 0.23% ABTS extracts radical-scavenging activity, even at the concentration of 10 mg/ ml. The essential oil only had weak ABTS radical-scavenging activ- As shown in Table 2, the mutual correlations among the four ity, with an IC50 of 11.5 mg/ml/. methods were determined by linear regression analysis. The highly positive linear correlations (R > 0.67), among all the methods, dem- 3.3. b-carotene bleaching inhibition activity onstrated that the four methods, especially the DPPH, ABTS and reducing power tests, have similar predictive capacities for antiox- In the b-carotene/linoleic acid test, the oxidation of linoleic acid idant activity of Rhizoma Homalomenae extracts. In addition, the generates peroxyl free radicals which will then oxidise the highly strong linear correlation (R > 0.89) between the reciprocal values unsaturated b-carotene. The presence of antioxidants will mini- of the three kinds of IC50 and reducing power values suggested that mise the oxidation of b-carotene. The b-carotene bleaching inhibi- the components present in the extracts capable of scavenging tion effect of BHT, the extracts and the essential oil are shown in DPPH radicals, ABTS radicals and peroxyl radicals are also able to Fig. 2C. BHT had strong antioxidant activity in the test with an reduce ferric ions and that the reducing ability may be an impor- 460 L.-B. Zeng et al. / Food Chemistry 125 (2011) 456–463

100 A 70 90

80 60

70 50 60

50 40

40 BHT QJ1 30 30 QJ2 QJ3 20 QJ4 20 QJ5 DPPH radical scavenging activity (%) activity scavenging DPPH radical DPPH radical scavenging activity (%) 10 QJ6 QJ7 10 0 01234567891011 0 20 40 60 80 100 120 140 160 180 200 Concentration (mg/ml) of extracts of Rhizoma Homalomenae and BHT Concentration (mg/ml) of essential oil of Rhizoma Homalomenae

B 100 100 90 90

80 80

70 70

60 60

nging activity (%) 50 50 40 40 BHT 30 QJ2 QJ3 30 20 QJ4 QJ5 20

10 QJ6 radicalABTS scavengingactivity (%)

ABTS radical scave QJ7 10 0 01234567891011 246810121416182022 Concentration (mg/ml) of extracts of Rhizoma Homalomenae and BHT Concentration (mg/ml) of essential oil of Rhizoma Homalomenae

C 100 60 90

80 50 70

60 40

50 30 40 BHT QJ1 30 QJ2 20 QJ3 20 QJ4 QJ5 10 10 QJ6 -carotene bleaching inhibition activity (%) QJ7 -carotene bleaching inhibition activity (%) β β 0 0 01234567891011 012345 Concentration (mg/ml) of extracts of Rhizoma Homalomenae and BHT Concentration (mg/ml) of essential oil of Rhizoma Homalomenae

0.65 BHT 1.6 D QJ3 0.60 QJ1 QJ4 QJ2 1.4 QJ5 0.55 QJ6 0.50 QJ7 1.2 0.45 0.40 1.0 0.35 0.8 0.30 0.25 0.6 0.20 Absorbance in 700 nm 0.4 Absorbance in 700 nm 0.15 0.10 0.2 0.05 0.0 0.00 0.00.20.40.60.81.01.21.41.6 0246810 Concentration (mg/ml) of extracts of Rhizoma Homalomenae and BHT Concentration (mg/mL) of extracts of Rhizoma Homalomenae

Fig. 2. Antioxidant activity of extracts and essential oil of Rhizoma Homalomenae: (A) DPPH radical-scavenging activity; (B) ABTS radical-scavenging activity; (C) b-carotene bleaching inhibition activity; (D) reducing power. Values are means of three determinations ± SD. L.-B. Zeng et al. / Food Chemistry 125 (2011) 456–463 461

Table 1 Total phenolic content (GAE, mg/g) and total ﬂavonoid content (RE, mg/g) of Rhizoma Homalomenae extracts (n = 3).

QJ1 QJ2 QJ3 QJ4 QJ5 QJ6 QJ7 Total phenolic content 5.14 ± 0.40a 15.1 ± 0.39b 75.5 ± 0.92e 141 ± 1.21f 65.2 ± 0.41d 64.2 ± 2.50d 27.5 ± 0.51c Total ﬂavonoid content ND1 1.26 ± 0.02a 6.35 ± 0.06c 13.5 ± 0.14f 9.91 ± 0.06d 12.0 ± 0.25e 5.43 ± 0.06b

Different letters within a row indicate signiﬁcant difference at p < 0.05. 1 ND = not determined.

Table 2 Linear correlation coefﬁcients, R, for relationships between the assays for the extracts of Rhizoma Homalomenae.

ABTS (1/IC50) Reducing power(at 1.25 g/l) b-carotene(1/IC50) Total phenolic content Total ﬂavonoid content

DPPH(1/IC50) 0.9977 0.9659 0.6771 0.9817 0.6259

ABTS(1/IC50) 0.9724 0.7334 0.9873 0.7256

Reducing power(at 1.25 g/l) 0.8940 0.9876 0.8316

D-carotene(1/IC50) 0.8347 0.8967 Total phenolic content 0.8211

tant factor dictating free radical-scavenging capacity of the Tepe, Polissiou, & Sokmen, 2007; Ebrahimabadi et al., 2010). The extracts. weak antioxidant activity of the essential oil may be attributed to the absence of significant antioxidants. The results imply that the major functions, such as the healing of pain and swelling due 3.7. Correlations of antioxidant activity with the total phenolic and to traumatic injury, of the essential oil of Rhizoma Homalomenae flavonoid contents had no evident association with its antioxidant potency. As shown in Table 2, positive linear correlations were observed between total phenolic content and antioxidant activity of the ex- 3.9. Phenolic components in the QJ4 fraction tracts in the DPPH (R = 0.9817), ABTS (R = 0.9873), b-carotene/linoleic acid (R = 0.8347) and reducing power (R = 0.9876) tests, The QJ4 fraction had the highest activity in all the antioxidant respectively. The results suggest that the total phenolic content assays, so it was chosen for the component analysis by LC–MS/ is strongly linearly correlated with antioxidant activity of the MS. The presence of phenolic compounds in the QJ4 fraction was Rhizoma Homalomenaes extracts. Similarly, the positive linear cor- determined by the negative MRM method. The phenolic com- relations between total flavonoid content and antioxidant activity pounds were characterised and identified by comparison with of the extracts in the DPPH (R = 0.6259), ABTS (R = 0.7256), b-caro- MS data and LC retention times of the phenolic standards. In total, tene/linoleic acid (R = 0.8967) and reducing power tests seven phenolic compounds were identified. They include three (R = 0.8317), respectively, indicated that flavonoids were largely hydroxybenzoic acids (protocatechuic acid, vanillic acid, and syrin- responsible for the antioxidant activity of the extracts. gic acid), three hydroxycinnamic acids (caffeic acid, p-coumaric In addition, a highly positive linear correlation (R = 0.8211) be- acid, and ferulic acid) and one flavanone, namely apigenin. The tween total phenolic content and total flavonoid content was ob- contents of the phenolic compounds identified by LC–MS/MS were served in the study, which implied that the flavonoids seem to determined by HPLC by the external standard method. The content be the main phenolic components responsible for the antioxidant of each of the phenolic components was higher than 100 lg/g dry activity of Rhizoma Homalomenae extracts. extract, especially protocatechuic acid with content higher than 11 mg/g dry extract. The seven phenolic compounds, their MS/MS 3.8. Chemical constituents of the essential oil data for the fragmentation and their contents are listed in Table 4. Phenolic acids are the main phenols consumed by humans. The The essential oil obtained (yield: 0.82%, v/w) was yellow in col- six phenolic acids identified in this study are all prominent and nat- our with a density of 0.95 g/ml at 23 °C. The GC–MS analysis re- urally occurring. All of them individually possess potent antioxi- sulted in the identification of 77 constituents eluted between 3.5 dant activity (Natella, Nardini, Felice, & Scaccini, 1999; Robbins, and 38.5 min, representing 96.5% of the essential oil (shown in Ta- 2003). Apigenin is a common dietary flavonoid widely distributed ble 3). The essential oil consisted mostly of monoterpenoids and in fruits, vegetables and red wine. It is used as a health food supple- sesquiterpenoids. Epi-a-cadinol (14.8%), a-cadinol (14.8%), a-ter- ment and has potent antioxidant properties (Shukla & Gupta, 2010). pineol (13.8%), linalool (11.1%), terpinen-4-ol (4.92%), and d-cadin- The total content of phenolics identified was 26,979 lg/g dry ex- ene (4.91%) were the major constituents, which account for 64.3% tract in the QJ4 fraction. The high content of phenolic components of the total oil content. The identified constituents and the percent- with strong antioxidant activity contributed to the strong antioxi- age composition of some major constituents, such as linalool, a- dant potency of the fraction. In addition, apigenin has also been terpineol, terpinen-4-ol, in the essential oil in the present study demonstrated to help improve cardiovascular conditions, stimulate are considerably different from a previous report (Zhou, Yao, Sun, the immune system and provide some protection against cancers Qiu, & Cui, 1991). Several factors, such as geographical, climatic, (Shukla and Gupta, 2010). All of these suggest a great potential of seasonal and experimental conditions, normally influencing the the plant to be exploited for functional foods or beverages. constituents of the essential oils of plants, possibly contribute to Considerable antioxidant activity was observed in the extracts, the difference. Few studies on the antioxidant activity of the major which implies that some functions of Rhizoma Homalomenae, used constituents in the oil have been reported. The major components as traditional Chinese medicine, are possibly attributable to its of the essential oil, such as linalool, a-terpineol and terpinen-4-ol, antioxidant ability. The antioxidant activity has significant associ- had no notable antioxidant activity (Dorman, Figueiredo, Barroso, ation with the phenolic and flavonoid contents. The high content of & Deans, 2000; Koroch, Juliani, & Zygadlo, 2007; Tepe, Daferera, phenolics suggests that the plant is a potential source of natural 462 L.-B. Zeng et al. / Food Chemistry 125 (2011) 456–463

Table 3 Chemical composition of Rhizoma Homalomenae essential oil by GC–MS analysis.

Peaka Rtb (min) RCc (%) RId Compound CAS 1 3.522 0.04 901 2-Heptanol 543-49-7 2 4.102 0.01 944 Alpha-Pyronene 514-94-3 3 4.167 0.03 949 Camphene 79-92-5 4 4.278 0.01 957 Fenchene 471-84-1 5 4.461 1.25 971 2,2,6-Trimethyl-6-vinyltetrahydropyran 7392-19-0 6 4.735 0.23 991 Beta-Myrcene 123-35-3 7 4.820 0.07 998 2-Carene 554-61-0 8 4.983 0.03 1006 Alpha-Phellandrene 99-83-2 9 5.08 0.10 1011 (+)-3-Carene 498-15-7 10 5.152 0.53 1014 1,4-Cineole 470-67-7 11 5.191 0.09 1016 (+)-4-Carene 29050-33-7 12 5.283 0.01 1020 o-Cymene 527-84-4 13 5.341 0.95 1023 p-Cymene 99-87-6 14 5.42 1.01 1027 Limonene 138-86-3 15 5.465 0.23 1029 Eucalyptol 470-82-6 16 5.557 0.16 1033 p-Ocimene 13877-91-3 17 5.772 0.42 1043 cis-beta-Ocimene 3338-55-4 18 6.026 0.16 1055 Gamma-Terpinene 99-85-4 19 6.339 0.43 1070 cis-Linalool oxide 5989-33-3 20 6.731 0.91 1089 Terpinolene 586-62-9 21 6.776 0.49 1091 Cymenene 1195-32-0 22 7.141 11.08 1106 Linalool 78-70-6 23 7.207 0.23 1108 Hotrienol 29957-43-5 24 7.481 0.06 1118 Fenchol 1632-73-1 25 7.611 0.33 1122 Sylvestrene 1461-27-4 26 8.022 0.52 1137 1-Terpinenol 586-82-3 27 8.335 0.65 1147 Beta-Terpineol 138-87-4 28 9.065 0.10 1173 Epoxylinalol 14049-11-7 29 9.326 4.92 1182 Terpinen-4-ol 562-74-3 30 9.548 0.37 1190 Cryptone 500-02-7 31 9.809 13.77 1199 Alpha-Terpineol 98-55-5 32 9.9 1.04 1202 Gamma-Terpineol 586-81-2 33 10.768 0.14 1232 (Z)-Geraniol 106-25-2 34 11.094 0.16 1243 Cuminal 122-03-2 35 11.302 0.07 1251 Carvotanacetone 499-71-8 36 11.648 2.35 1263 (E)-Geraniol 106-24-1 37 11.955 0.10 1273 Geranial 141-27-5 38 12.079 0.14 1278 Phellandral 21391-98-0 39 12.333 0.07 1287 2-Caren-10-al 100015-18-5 40 12.561 0.23 1295 Cuminol 536-60-7 41 12.692 0.07 1299 Thymol 89-83-8 42 12.952 0.14 1308 Carvacrol 499-75-2 43 13.416 0.27 1324 Methyl geranate 2349-14-6 44 14.224 0.06 1352 Citronellyl acetate 150-84-5 45 14.857 0.16 1374 Copaene 3856-25-5 46 15.137 0.65 1383 Geranyl acetate 105-87-3 47 15.392 0.30 1392 Beta-Elemene 515-13-9 48 15.503 0.04 1396 Longifolene 475-20-7 49 15.861 0.36 1405 Isocaryophyllene 118-65-0 50 15.94 0.03 1407 Alpha-Gurjunene 489-40-7 51 16.292 0.10 1415 Beta-Caryophyllene 87-44-5 52 17.029 0.36 1433 (+)-Aromadendrene 489-39-4 53 17.635 0.95 1447 Alpha-Caryophyllene 6753-98-6 54 17.916 0.14 1453 ()-Alloaromadendrene 25246-27-9 55 18.131 0.01 1458 Beta-Humulene 116-04-1 56 18.679 0.69 1471 gamma-Muurolene 30021-74-0 57 19.181 0.30 1482 Beta-Selinene 17066-67-0 58 19.572 0.92 1492 Delta-Selinene 28624-23-9 59 19.911 1.77 1499 Alpha-Muurolene 31983-22-9 60 20.127 0.09 1503 Alpha-Selinene 473-13-2 61 20.394 0.12 1508 Beta-Bisabolene 495-61-4 62 20.635 2.20 1513 Gamma-Cadinene 39029-41-9 63 20.772 0.07 1515 Cuparene 16982-00-6 64 21.255 4.91 1524 Delta-Cadinene 483-76-1 65 21.659 0.09 1531 Cadine-1,4-diene 16728-99-7 66 21.979 0.40 1537 Alpha-Cadinene 24406-05-1 67 22.318 1.05 1543 Alpha-Calacorene 21391-99-1 68 23.701 0.37 1569 Nerolidol 7212-44-4 69 24.118 2.32 1576 Spathulenol 6750-60-3 70 24.757 0.07 1588 Caryophyllene oxide 1139-30-6 71 27.366 14.81 1633 Epi-alpha-Cadinol 5937-11-1 72 27.679 2.69 1639 Delta-Cadinol 36564-42-8 73 28.286 14.77 1649 Alpha-Cadinol 481-34-5 74 29.186 1.41 1665 Cadalene 483-78-3 L.-B. Zeng et al. / Food Chemistry 125 (2011) 456–463 463

Table 3 (continued)

Peaka Rtb (min) RCc (%) RId Compound CAS 75 36.934 0.16 1959 Cembrene 1898-13-1 76 38.362 0.09 2096 Linoleic acid, methyl ester 112-63-0 77 38.427 0.06 2102 Oleic acid, methyl ester 112-62-9 Total 96.52

a Compounds listed in order of elution from HP-5MS column. b Retention time (in min) of elution from HP-5MS column. c Relative composition of the compounds in the essential oil. d Retention indices as determined on HP-5MS column using a homologous series of n-alkanes.

Table 4 Phenolics identiﬁed by LC–ESI–MS/MS and their content (lg/g dried extract) determined by HPLC in QJ4 fraction.

No Compound Rt (min)a MS (m/z) [M–H] MS/MS (m/z) Content (lg/g)b 1 Protocatechuic acid 15.37 153 109, 108, 91 11646 ± 20.1 2 Vanillic acid 25.27 167 108, 123, 152 7218 ± 14.2 3 Caffeic acid 25.46 179 135 686 ± 17.2 4 Syringic acid 26.48 197 182, 167, 123 3723 ± 14.7 5 p-Coumaric acid 29.89 163 119 131 ± 5.33 6 Ferulic acid 30.97 193 134, 178 602 ± 6.78 7 Apigenin 48.93 269 117, 149, 151 2973 ± 15.2 Total 26979

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