Chemical composition and synergistic antioxidant activities of essential oils from Atractylodes macrocephala and Astragalus membranaceus

NPC

Natural Product Communications

EDITOR-IN-CHIEF DR. PAWAN K AGRAWAL Natural Product Inc. 7963, Anderson Park Lane, Westerville, Ohio 43081, USA

[email protected] EDITORS PROFESSOR ALEJANDRO F. BARRERO Department of Organic Chemistry, University of Granada, Campus de Fuente Nueva, s/n, 18071, Granada, Spain [email protected] PROFESSOR ALESSANDRA BRACA Dipartimento di Chimica Bioorganicae Biofarmacia, Universita di Pisa, via Bonanno 33, 56126 Pisa, Italy [email protected] PROFESSOR DEAN GUO State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100083, China [email protected] PROFESSOR YOSHIHIRO MIMAKI School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Horinouchi 1432-1, Hachioji, Tokyo 192-0392, Japan [email protected] PROFESSOR STEPHEN G. PYNE Department of Chemistry University of Wollongong Wollongong, New South Wales, 2522, Australia [email protected] PROFESSOR MANFRED G. REINECKE Department of Chemistry, Texas Christian University, Forts Worth, TX 76129, USA [email protected] PROFESSOR WILLIAM N. SETZER Department of Chemistry The University of Alabama in Huntsville Huntsville, AL 35809, USA [email protected] PROFESSOR YASUHIRO TEZUKA Institute of Natural Medicine Institute of Natural Medicine, University of Toyama, 2630-Sugitani, Toyama 930-0194, Japan [email protected] PROFESSOR DAVID E. THURSTON Department of Pharmaceutical and Biological Chemistry, The School of Pharmacy, University of London, 29-39 Brunswick Square, London WC1N 1AX, UK [email protected]

HONORARY EDITOR PROFESSOR GERALD BLUNDEN The School of Pharmacy & Biomedical Sciences, University of Portsmouth, Portsmouth, PO1 2DT U.K. [email protected]

ADVISORY BOARD Prof. Berhanu M. Abegaz Gaborone, Botswana Prof. Viqar Uddin Ahmad Karachi, Pakistan Prof. Øyvind M. Andersen Bergen, Norway Prof. Giovanni Appendino Novara, Italy Prof. Yoshinori Asakawa Tokushima, Japan Prof. Lee Banting Portsmouth, U.K. Prof. Julie Banerji Kolkata, India Prof. Anna R. Bilia Florence, Italy Prof. Maurizio Bruno Palermo, Italy Prof. César A. N. Catalán Tucumán, Argentina Prof. Josep Coll Barcelona, Spain Prof. Geoffrey Cordell Chicago, IL, USA Prof. Ana Cristina Figueiredo Lisbon, Portugal Prof. Cristina Gracia-Viguera Murcia, Spain Prof. Duvvuru Gunasekar Tirupati, India Prof. Kurt Hostettmann Lausanne, Switzerland Prof. Martin A. Iglesias Arteaga Mexico, D. F, Mexico Prof. Leopold Jirovetz Vienna, Austria Prof. Vladimir I Kalinin Vladivostok, Russia Prof. Niel A. Koorbanally Durban, South Africa

Prof. Karsten Krohn Paderborn, Germany Prof. Chiaki Kuroda Tokyo, Japan Prof. Hartmut Laatsch Gottingen, Germany Prof. Marie Lacaille-Dubois Dijon, France Prof. Shoei-Sheng Lee Taipei, Taiwan Prof. Francisco Macias Cadiz, Spain Prof. Imre Mathe Szeged, Hungary Prof. Ermino Murano Trieste, Italy Prof. M. Soledade C. Pedras Saskatoon, Canada Prof. Luc Pieters Antwerp, Belgium Prof. Peter Proksch Düsseldorf, Germany Prof. Phila Raharivelomanana Tahiti, French Polynesia Prof. Luca Rastrelli Fisciano, Italy Prof. Monique Simmonds Richmond, UK Dr. Bikram Singh Palampur, India Prof. John L. Sorensen Manitoba, Canada Prof. Valentin Stonik Vladivostok, Russia Prof. Winston F. Tinto Barbados, West Indies Prof. Sylvia Urban Melbourne, Australia Prof. Karen Valant-Vetschera Vienna, Austria

INFORMATION FOR AUTHORS Full details of how to submit a manuscript for publication in Natural Product Communications are given in Information for Authors on our Web site http://www.naturalproduct.us. Authors may reproduce/republish portions of their published contribution without seeking permission from NPC, provided that any such republication is accompanied by an acknowledgment (original citation)-Reproduced by permission of Natural Product Communications. Any unauthorized reproduction, transmission or storage may result in either civil or criminal liability. The publication of each of the articles contained herein is protected by copyright. Except as allowed under national “fair use” laws, copying is not permitted by any means or for any purpose, such as for distribution to any third party (whether by sale, loan, gift, or otherwise); as agent (express or implied) of any third party; for purposes of advertising or promotion; or to create collective or derivative works. Such permission requests, or other inquiries, should be addressed to the Natural Product Inc. (NPI). A photocopy license is available from the NPI for institutional subscribers that need to make multiple copies of single articles for internal study or research purposes. To Subscribe: Natural Product Communications is a journal published monthly. 2013 subscription price: US$2,395 (Print, ISSN# 1934-578X); US$2,395 (Web edition, ISSN# 1555-9475); US$2,795 (Print + single site online); US$595 (Personal online). Orders should be addressed to Subscription Department, Natural Product Communications, Natural Product Inc., 7963 Anderson Park Lane, Westerville, Ohio 43081, USA. Subscriptions are renewed on an annual basis. Claims for nonreceipt of issues will be honored if made within three months of publication of the issue. All issues are dispatched by airmail throughout the world, excluding the USA and Canada.

NPC

Natural Product Communications

Chemical Composition and Synergistic Antioxidant Activities of Essential Oils from Atractylodes macrocephala and Astragalus membranaceus

2013 Vol. 8 No. 9 1321 - 1324

Jinkui Li1, Feng Li1, Yan Xu, Wenjian Yang, Lili Qu, Qian Xiang, Cong Liu and Dapeng Li* College of Food Science and Engineering, Shandong Agricultural University, Taian 271018, PR China 1

These authors contributed equally to this work.

[email protected] Received: March 28th, 2013; Accepted: June 17th, 2013

Chemical composition of the essential oils derived from Atractylodes macrocephala (AMA), Astragalus membranaceus (AME) and AMA-AME herb pair was investigated using gas chromatography-mass spectrometry (GC-MS). Antioxidant activities were evaluated by 1,1-diphenyl-2-picyrlhydrazyl (DPPH) radicalscavenging and Trolox equivalent antioxidant capacity (TEAC) assays. Forty-five, ten and forty-three components were identified in AMA, AME and AMA-AME essential oils, respectively. AMA-AME essential oil exhibited a significantly higher radical scavenging capacity than the theoretical sum of those of the respective herb essential oils (P < 0.05). Principal component analysis showed that twenty-three components contributed to the scavenging activities against DPPH and ABTS·+ radicals. Moreover, the concentrations of these major components exhibited various increases to some extent when compared with the theoretical sum of the respective herb essential oils. These findings suggest that combination of two or more herbs might be used as a promising source of natural antioxidants in the pharmaceutical and food industries. Keywords: Chemical composition, Essential oils, Synergistic antioxidant, GC-MS, Atractylodes macrocephala, Astragalus membranaceus.

Reactive oxygen species (ROS), including hydrogen peroxide, superoxide anion and hydroxyl radicals, play an important role in the pathological processes of many serious diseases, such as cardiovascular diseases, atherosclerosis, inflammation, neurodegenerative disorders and cancer [1,2]. Atractylodes macrocephala has been widely used for over 2,000 years in traditional Chinese medicine (TCM) to treat tumors, inflammationrelated diseases and oxidative damage [3]. Nowadays, various A. macrocephala products are increasingly used not only as efficacious medicinal prescriptions, but also as health-promoting food supplements. Its essential oil has a distinct phytochemical profile, with atractylone, elemene, aromadendrene and eudesma-4(14),11diene as the main components [4]. Astragalus membranaceus is another widely used medicinal plant that is well-known for its vital-energy tonifying, skin reinforcing, diuretic, abscess -draining and tissue generative actions [5]. It is used in China in stewing fish, stewing chicken, for making tea as a flavoring agent, and as a food additive [6]. Recent studies revealed that A. membranaceus extracts have strong antioxidant activities [7]. Traditional Chinese herbs are generally applied in the form of multi-herb formulas in medical treatments and as dietary supplements. Atractylodes macrocephala and Astragalus membranaceus are one of the most common herb pairs used in clinics in order to obtain a synergistic effect [8]. However, to the best of our knowledge, there appears to be limited investigation focused on the synergistic effect of Atractylodes macrocephala and Astragalus membranaceus. Therefore, as a part of our ongoing screening program to evaluate in vitro antioxidant potentials of common herbal plants, we performed hydrodistillation to prepare essential oils from Atractylodes macrocephala (AMA), Astragalus membranaceus (AME) and the combination of AMA-AME. The essential oils were then subjected to GC-MS to elucidate their

chemical composition. Furthermore, in vitro antioxidant activity of the essential oils was evaluated using DPPH radical-scavenging and Trolox equivalent antioxidant capacity assays. Principal component analysis (PCA) was also performed to identify potential bioactive components from the essential oils. The essential oils extracted from AMA, AME, and AMA-AME combination were qualitatively and quantitatively analyzed by GC and GC-MS. Forty-five compounds were identified in AMA essential oil, accounting for 86.1% of the total peak areas. The most abundant compounds were atractylone (83.1 ± 0.1 μg/mL), β-eudesmol (10.0 ± 0.0 μg/mL), 8,9-dehydro-9-formyl-cycloisolongifolene (6.3 ± 0.2 μg/mL) and n-hexadecanoic acid (5.0 ± 0.1 μg/mL). Similar results were reported by other authors. Li et al. [9] identified 36 constituents in the AMA oil and among them, atractylone (40.1%), γ-elemene (14.7%), aromadendrene (13.1%), and eudesma4(14),11-diene (5.5%) were major constituents. The changes in chemical composition of the essential oils could be attributed to several differences such as geographic origin, environmental factors, extraction and analysis methods [10]. Ten compounds were identified in AME oil, representing 59.7% of the total peak areas, and the principal components were n-hexadecanoic acid (99.4 ± 0.2 μg/mL) and 1-hexadecanol (6.8 ± 0.0 μg/mL). As for AMA-AME oil, GC-MS analysis led to identification of 43 compounds, representing 87.4% of the total oils, where atractylone (55.9 ± 0.2 μg/mL), 9,10-dehydrofukinone (21.1 ± 0.1 μg/mL), eudesma3,7(11)-diene (8.5 ± 0.0 μg/mL), 4-carene (8.0 ± 0.0 μg/mL) and n-hexadecanoic acid (7.8 ± 0.0 μg/mL) were the predominant constituents. It can be seen that among the AMA-AME oil components, 35 were identified in AMA essential oil and 8 in AME essential oil. However, 10 detected in either AMA or AME essential oils were not detected in the mixture. The concentration of these components was less than 1.0 µg/mL in the respective herb oil, with the exception of 8,9-dehydro-cycloisolongifolene (4.7 ± 0.1 µg/mL),

1322 Natural Product Communications Vol. 8 (9) 2013

Li et al.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55

b

Content (μg/mL) AME AMA-AME 6.6 ± 0.1 2.0 ± 0.0 — — — 0.2 ± 0.0 — 0.7 ± 0.0 — 0.5 ± 0.0

Compounds

RI a

2-Methyl-2-butanol 6-(p-Tolyl)-2-methyl-2-heptenol β-Phellandrene α-Pinene 1,2,3,6-Tetramethylbicyclo[2.2.2] octa-2,5-diene 8,9-Dehydro-cycloisolongifolene Caryophyllene γ-Elemene Copaene 2-Pentyl-furan Longifolene-(V4) Eudesma-4(14),11-diene Tetradecane Cyperene δ-Selinene 4-Carene Aromadendrene Cedrene α-Caryophyllene Caryophyllene oxide 2-Hydroxycyperol 1-methyl-4-methylidene-7-(prop1-en-2-yl)decahydroazulene Ledene alcohol Neoclovene oxide 8,9-Dehydro-9-formylcycloisolongifolene Patchoulene Guaia-1(10),11-diene Eudesma-3,7(11)-diene α-Eudesmol γ-Costol 4,11-Selinadiene Humulene-1,2-epoxide 9,10-Dehydrofukinone 4a,8b,10b,11a-Tetramethylbicyclo [6.3.0]undec-1-en-5-one β-Eudesmol α-Bulnesene Hinesol β-Vatirenene Eudesma-4,11-dien-2-ol 1,5-Dimethyl-3-hydroxy-8-(1methylene-2-hydroxyethyl-1)bicyclo[4.4.0]dec-5-ene Atractylone Atractylenolide II Atractylenolide I Juniper camphor 2-methyl-1-hexadecanol Pentadecanoic acid Oxacyclohexadecan-2-one Thymol 1-Hexadecanol 8,9-Dehydro-9-vinylcycloisolongifolene n-Hexadecanoic acid 4,4'-dimethyl-2,2'-dimethylenebicyclohexyl-3,3'-diene 7-epi-β-Eudesmol Hexadecane Oleic acid

658 964 985 987 1161

AMA —c 0.2 ± 0.0 0.2 ± 0.0 1.5 ± 0.0 0.4 ± 0.0

1179 1342 1371 1380 1382 1387 1399 1400 1411 1412 1414 1421 1423 1448 1450 1455 1456

4.7 ± 0.1 2.1 ± 0.0 3.7 ± 0.0 2.8 ± 0.1 — 0.5 ± 0.0 0.2 ± 0.0 — 0.5 ± 0.0 0.3 ± 0.0 0.3 ± 0.0 2.1 ± 0.0 3.0 ± 0.0 0.6 ± 0.0 0.5 ± 0.0 0.4 ± 0.0 1.6 ± 0.0

— — — — 0.5 ± 0.0 — — 0.9 ± 0.0 0.5 ± 0.0 — — — — — — — —

— 4.1 ± 0.0 4.6 ± 0.0 1.4 ± 0.1 0.1 ± 0.0 — 4.8 ± 0.0 — 0.9 ± 0.0 0.4 ± 0.0 8.0 ± 0.0 6.4 ± 0.0 — 0.3 ± 0.0 0.5 ± 0.0 0.4 ± 0.0 1.1 ± 0.0

1459 1476 1479

0.3 ± 0.1 — 6.3 ± 0.2

— — —

0.5 ± 0.0 1.9 ± 0.1 —

1484 1489 1530 1607 1611 1624 1639 1640 1642

0. 9 ± 0.0 1.2 ± 0.0 0.8 ± 0.0 — 1.0 ± 0.1 1.9 ± 0.0 0.3 ± 0.0 3.9 ± 0.1 0.2 ± 0.0

— — — — — — — — —

— 1.0 ± 0.0 8.5 ± 0.0 5.0 ± 0.2 1.2 ± 0.1 2.9 ± 0.0 — 21.1 ± 0.1 0.9 ± 0.0

1645 1651 1690 1703 1710 1710

10.0 ± 0.0 0.7 ± 0.0 2.4 ± 0.0 1.3 ± 0.0 0.5 ± 0.0 0.6 ± 0.0

— — — — — —

4.6 ± 0.0 — 1.7 ± 0.0 1.4 ± 0.0 0.3 ± 0.0 0.9 ± 0.0

1711 1714 1746 1751 1794 1806 1860 1875 1879 1888

83.1 ± 0.1 0.8 ± 0.0 1.5 ± 0.0 2.5 ± 0.0 0.2 ± 0.0 — — 3.2 ± 0.0 — 0.4 ± 0.0

— — — — — 3.7 ± 0.1 1.3 ± 0.0 — 6.8 ± 0.0 —

55.9 ± 0.2 3.4 ± 0.0 — 3.9 ± 0.0 — 7.5 ± 0.0 — 1.2 ± 0.0 7.0 ± 0.0 0.5 ± 0.0

1957 1972

5.0 ± 0.1 1.7 ± 0.1

99.4 ± 0.2 —

7.8 ± 0.0 2.6 ± 0.0

2031 2044 2113

2.5 ± 0.0 — —

— 0.5 ± 0.0 1.7 ± 0.0

1.4 ± 0.0 0.3 ± 0.0 1.5 ± 0.1

a

Retention indices relative to n-alkanes on DB–5ms GC column. The content of each compound was quantified using internal standard method, and 1octanol was used at a concentration of 8.3 μg/mL. c Not detected. b

cedrene (3.0 ± 0.0 µg/mL), oxacyclohexadecan-2-one (1.3 ± 0.0 µg/mL), and 8,9-dehydro-9-formyl-cycloisolongifolene (6.3 ± 0.2 µg/mL). Interestingly, two new compounds, namely neoclovene oxide (1.9 ± 0.1 µg/mL) and α-eudesmol (5.0 ± 0.2 µg/mL), were detected in AMA-AME oil. The antioxidant activity of the test samples depends on the method adopted and the model systems. It is necessary to combine more than one method to evaluate in vitro antioxidant capacity of foodstuffs. Herein, both DPPH radical-scavenging assay and the TEAC method were performed to investigate in vitro antioxidant activity of the essential oils.

100

a

80 60

b

c

40

d

20 0 AMA-AME

AMA

AME

TS (DPPH)

Figure 1: DPPH radical scavenging activity of the essential oils from Atractylodes macrocephala (AMA), Astragalus membranaceus (AME) and their combination (AMA-AME). Histograms marked with different letters indicated that their SC50 values were significantly different at P < 0.05.

As shown in Fig. 1, the lowest SC50 value (27.5 ± 0.6 μg/mL) was observed for AMA essential oil, indicating that it had the highest DPPH radical scavenging efficiency. The DPPH radical-scavenging activity of AMA-AME essential oil was lower than that of AMA essential oil, but significantly higher than AME essential oil, as indicated by its SC50 value of 48.1 ± 1.1 μg/mL (P < 0.05). AME essential oil had the weakest scavenging ability with a highest SC50 value of 85.9 ± 1.5 μg/mL. Moreover, AMA-AME essential oil exhibited a significantly lower SC50 value than the theoretical sum of the respective herb essential oils (48.1 ± 1.1 μg/mL vs. 56.7 ± 1.0 μg/mL, P < 0.05), indicating that a synergistic action might exist between two individual herbs in the DPPH radical-scavenging model. Similar results were also found in our previous study that the ethanolic extract of AMA and AME were able to synergistically scavenge DPPH radicals [11]. On the basis of the SC50 values, CI was calculated and revealed a synergistic interaction between AMA and AME oils at a fixed ratio of 1:1 (CI = 0.9). ABTS scavenging SC50(μg/mL)

No.

DPPH scavenging SC50 (μg/mL)

Table 1: Chemical composition of the essential oils from Atractylodes macrocephala (AMA), Astragalus membranaceus (AME) and the combination of AMA-AME.

30

c

25 20

a

b

d

15 10 5 0 AMA-AME

AMA

AME TS (ABTS· +)

Figure 2: Trolox equivalent antioxidant capacities of the essential oils from Atractylodes macrocephala (AMA), Astragalus membranaceus (AME) and their combination (AMA-AME). Histograms marked with different letters indicated that their SC50 values were significantly different at P < 0.05.

As shown in Fig.2, all essential oils were able to scavenge ABTS•+ radicals within a SC50 range from 16.0 ± 0.1 to 23.2 ± 0.1 μg/mL. Among them, AMA-AME oil had the highest scavenging activity against ABTS•+ (SC50 = 16.0 ± 0.1 μg/mL), followed by AMA oil (SC50 = 20.9 ± 0.9 μg/mL) and AME oil (SC50 = 23.2 ± 0.1 μg/mL). Moreover, AMA-AME oil had a stronger ABTS•+ scavenging effect than the theoretical sum of those of the respective herb oils (P < 0.05). The TEAC values for the essential oils were as follows: AMA-AME (40.2 ± 0.7 µM) > AMA (38.9 ± 1.0 μM) > theoretical sum of two single herbs (35.0 ± 0.6 μM) > AME (31.1 ± 0.3 μM). Previous studies reported that unsaturated carbon-carbon double bonds (C=C) are able to inhibit free radical reactions by attracting single electrons from free radicals to form a stable electron cloud system [12]. Therefore, we calculated the content of the C=C bonds in the AMA-AME oil, and found that it was about 2-fold more than the theoretically sum of those of the respective essential oil (264.1 vs. 117.8), which may be a reason for the synergistic effect of the essential oil’s antioxidant activities.

Antioxidant activity of Atractylodes macrocephala and Astragalus membranaceus Natural Product Communications Vol. 8 (9) 2013 1323

Concentration (μg/mL)

The relationship between the essential oils’ composition and their antioxidant activities was evaluated by PCA. All 55 compounds listed in Table 1 composed a chemical matrix with three rows and fifty-five columns. Based on their correlation coefficient matrix, 55 compounds were combined and subjected to PCA after normalization. The first two principal components (P1 and P2) with more than 85.0% of total variances were extracted for regression. The coefficients for P1 and P2 were –0.6583 and –1.2929 in the ABTS•+ scavenging assay, and –7.0364 and 4.6349 in the DPPH scavenging test, respectively, which implied that there were strong correlations between the two principal components and ABTS•+ radical-scavenging activity, as well as between P1 and DPPH radical-scavenging activity. Finally, 15 major components were selected from the loading matrix of P1. They were β-phellandrene (3), 1,2,3,6-tetramethylbicyclo[2.2.2]octa-2,5-diene (5), caryophyllene (7), γ-elemene (8), δ-selinene (15), caryophyllene oxide (20), 2-hydroxycyperol (21), ledene alcohol (23), γ-costol (30), 4,11-selinadiene (31), β-vatirenene (38), 1,5-dimethyl-3hydroxy-8-(1-methylene-2-hydroxyethyl-1)-bicyclo[4.4.0] dec-5ene (40), juniper camphor (44), 8,9-dehydro-9-vinylcycloisolongifolene (50) and 4,4'-dimethyl-2,2'-dimethylene bicyclohexyl-3,3'-diene (52). Meanwhile, 8 major components were chosen from the loading matrix of P2, which were eudesma4(14),11-diene (12), 4-carene (16), aromadendrene (17), neoclovene oxide (24), α-eudesmol (29), 9,10-dehydrofukinone (33), 4a,8b,10b,11a-tetramethylbicyclo[6.3.0]undec-1-en-5-one (34) and hexadecane (54). 25 20

33

Theoretical concentration Actual concentration

15 10

16

5 0

7 3 5

17 50

8

31 15 20 21 23 30

44 38 40

50

52

12

29 24

34 54

Figure 3: The concentration of potential bioactive components identified in the essential oil from Atractylodes macrocephala and Astragalus membranaceus combination.

The contents of the above major components were quantitatively determined in AMA-AME oil and compared with the theoretical sums of those from the individual herb oils. As shown in Fig. 3, the actual concentration of these major components exhibited various increases to some extent when compared with their respective theoretical sums, which may be responsible for the remarkable antioxidant synergism of the essential oils, since many components identified in this study have reported radical-scavenging activity in previous studies. γ-Elemene (8), β-phellandrene (3) and caryophyllene oxide (20), identified as the main components in Bidens pilosa essential oil, exhibited strong scavenging effect against DPPH radicals [13]. Sroka et al. [14] revealed that the hypothesized bioactive compound (31) was active against free radicals, which was correlated with the structure of phenolic acids. Owing to the structure of unsaturated carbon-carbon double bonds, other major components, including compounds 5, 12, 15–17, 21, 30, 33, 34, 40, 50, and 52, also demonstrated a wide range of antioxidant activities. It is noteworthy that two new compounds, namely α-eudesmol (29) and neoclovene oxide (24), were produced in AMA-AME essential oil with concentrations of 5.0 ± 0.2 µg/mL and 1.9 ± 0.1 µg/mL, respectively. Similar results were also found in previous work, indicating that some new bioactive ingredients might be produced when several medicinal herbs were decocted together [15]. Therefore, the synergistic effects of the essential oils

might be correlated not only with a quantitative addition of the constituents of the individual herbs, but also with the generation of some new active components. In addition, an earlier study on the synergism displayed by α-tocopherol and ascorbic acid showed that ascorbic acid can regenerate α-tocopherol from its radical oxidation product, α-chromanoxyl [16]. A similar synergistic action might occur when neoclovene oxide (24) is regenerated as neoclovene by other antioxidants in the oils. Recent evidence demonstrated that alteration of the acid-base environment, processing method and heating conditions will result in some changes of bioactive components when several herbs are extracted in combination [17]. We measured the pH value of the essential oil extraction solution, and found it was 4.3 ± 0.2 for AMA-AME combination, significantly lower than AMA (4.7 ± 0.2) and AME (5.0 ± 0.1) (P < 0.05), which may be partly responsible for the increased concentration of bioactive components during the extraction of the combination. However, further efforts should be made to elucidate this possible interaction between herb constituents in the future. Experimental Plant materials: The dried roots of Atractylodes macrocephala Koidz (AMA) and Astragalus membranaceus Bge Hsiao (AME) were commercially purchased from Shijiazhuang Pharmaceutical Company (Shijiazhuang, China). Extraction of essential oils: Each sample was comminuted and sieved through a No.40 mesh. Three powdered samples, AMA (100.0 g), AME (100.0 g) and a powdered mixture of AMA-AME (50.0 g each) were subjected to hydrodistillation for 6 h in a Clevenger-type apparatus. The resulting essential oils were dried over anhydrous sodium sulfate and stored at –20°C until further analysis. Analysis of essential oils: The gas chromatography (GC) analysis was performed on a Shimadzu 17-A gas chromatography (Shimadzu Corporation, Kyoto, Japan) with a SUPELCO SPB–5 fused silica capillary column (30 m × 0.32 mm × 0.25 μm) equipped with a flame ionization detector (FID). The oven temperature was held at 40°C for 3 min and then programmed at a rate of 4°C/min to 250°C. The injector and detector temperatures were set at 250°C and 270°C, respectively. Nitrogen was used as the carrier gas at a flow rate of 4.0 mL/min. One µL of sample was injected manually in a splitless mode. Quantitative data were obtained by peak area normalization. An internal standard of 1-octanol was used at a concentration of 8.3 µg/mL. GC-MS analysis was carried out using a Hewlett-Packard 5890 II gas chromatograph equipped with a DB– 5ms capillary column (30 m × 0.25 mm × 0.25 μm) and a Hewlett Packard 5971 mass selective detector (Agilent Technologies, Palo Alto, CA, USA). Separation was achieved under the same conditions as described for GC-FID. The mass spectrometer was operated in the electron impact ionization mode (70 eV). Identification of the essential oil components was based on retention indices (RIs) relative to n-alkanes, and computer matching with the NIST 2.0 library of the GC/MS system, as well as comparisons of the fragmentation pattern of the mass spectra with published data. DPPH free radical scavenging assay: The DPPH radicalscavenging capacity of the essential oils was evaluated according to the previous report with some modifications [18]. Briefly, 0.5 mL samples of various oil concentrations (6.25 to 300 μg/mL) were added to 2.0 mL of 0.05 mM DPPH ethanol solution, and allowed

1324 Natural Product Communications Vol. 8 (9) 2013

to stand for 30 min in the dark at 37°C. Then, the absorbance of the resulting solutions was recorded at 517 nm using an ultravioletvisible spectrophotometer (UNICO UV-2000, Shanghai, China). Percent inhibition of the DPPH radical, and an SC50 value, defined as the concentration required to scavenge 50% of the DPPH, were calculated from the graph. Vitamin E served as a positive control. Isobolographic analysis: To investigate the possible interaction between AMA and AME essential oils in scavenging DPPH radicals, an isobolographic analysis based on the median-effect principle was performed [19]. The isobologram was obtained from the concentration-inhibition curves of the individual oil and their combination at a fixed ratio of 1:1. Combination Index (CI) was calculated; CI < 1, CI = 1 or CI > 1 represent synergistic, additive, and antagonistic effects, respectively.

Li et al.

Trolox equivalent antioxidant capacity (TEAC) assay: Trolox equivalent antioxidant capacity of the essential oils was determined according to the procedures described previously [20]. Statistical analysis: All experiments were performed in triplicate. A one-way analysis of variance (ANOVA) was performed to calculate significant differences (P < 0.05), and multiple comparisons of means were made by the Duncan test using the statistical software SPSS 12.0 (SPSS Inc., Chicago, IL, USA). Principle component analysis (PCA) was performed in order to identify potential bioactive components from the essential oils using the statistical software SAS 8.1 (SAS Institute Inc., Cary, NC, USA). Acknowledgments - This work was supported by the Natural Science Foundation of Shandong Province, China (Grants No. 2009ZRB019PD).

References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20]

Seifried HE, Anderson DE, Fisher EI, Milner JA. (2007) A review of the interaction among dietary antioxidants and reactive oxygen species. Journal of Nutritional Biochemistry, 18, 567-579. Pelicano H, Carney D, Huang P. (2004) ROS stress in cancer cells and therapeutic implications. Drug Resistance Updates, 7, 97-110. Huang HL, Chen CC, Yeh CY, Huang RL. (2005) Reactive oxygen species mediation of baizhu-induced apoptosis in human leukemia cells. Journal of Ethnopharmacology, 97, 21-29. Guo FQ, Huang LF, Zhou SY, Zhang TM, Liang YZ. (2006) Comparison of the volatile compounds of Atractylodes medicinal plants by headspace solid-phase microextraction-gas chromatography-mass spectrometry. Analytica Chimica Acta, 570, 73-78. Cho WCS, Leung KN. (2007) In vitro and in vivo immunomodulating and immunorestorative effects of Astragalus membranaceus. Journal of Ethnopharmacology, 113, 132-141. Yan MM, Liu W, Fu YJ, Zu YG, Chen CY, Luo M. (2010) Optimisation of the microwave-assisted extraction process for four main astragalosides in Radix Astragali. Food Chemistry, 119, 1663-1670. Chen CY, Zu YG, Fu YJ, Luo M, Zhao CJ, Wang W, Zhao BS, Li J, Efferth T. (2011) Preparation and antioxidant activity of Radix Astragali residues extracts rich in calycosin and formononetin. Biochemical Engineering Journal, 56, 84-93. Li X, Wang H. (2005) Chinese herbal medicine in the treatment of chronic kidney disease. Advances in Chronic Kidney Disease, 12, 276-281. Li N, Deng CH, Li Y, Ye H, Zhang XM. (2006) Gas chromatography-mass spectrometry following microwave distillation and headspace solidphase microextraction for fast analysis of essential oil in dry traditional Chinese medicine. Journal of Chromatography A, 1133, 29-34. Quiroga PR, Riveros CG, Zygadlo JA, Grosso NR, Nepote V. (2011) Antioxidant activity of essential oil of oregano species from Argentina in relation to their chemical composition. International Journal of Food Science and Technology, 46, 2648-2655. Yang WJ, Li DP, Li JK, Li MH, Chen YL, Zhang PZ. (2009) Synergistic antioxidant activities of eight traditional Chinese herb pairs. Biological & Pharmaceutical Bulletin, 32, 1021-1026. John M. (2003) Fundamentals of Organic Chemistry. Wadsworth Publishing Company, Belmont, CA, 372-404. Deba F, Xuan TD, Yasuda M, Tawata S. (2008) Chemical composition and antioxidant, antibacterial and antifungal activities of the essential oils from Bidens pilosa Linn. var. radiata. Food Control, 19, 346-352. Sroka Z, Cisowski W. (2003) Hydrogen peroxide scavenging, antioxidant and anti-radical activity of some phenolic acids. Food and Chemical Toxicology, 41, 753-758. Ma XH, Zheng CJ, Han LY, Xie B, Jia J, Cao ZW, Li YX, Chen YZ. (2009) Synergistic therapeutic actions of herbal ingredients and their mechanisms from molecular interaction and network perspectives. Drug Discovery Today, 14, 579-588. Niki E, Tsuchiya J, Tanimura R, Kamiya Y. (1982) Regeneration of Vitamin E from a-chromanoxyl radical by glutathione and vitamin C. Chemistry Letters, 11, 789-792. Li Y, Lu X. (2005) Investigation on the origin of 5-HMF in Shengmaiyin decoction by RP-HPLC method. Journal of Zhejiang University-Science B, 6, 1015-1021. Saint-Cricq De Gaulejac N, Provost C, Vivas N. (1999) Comparative study of polyphenol scavenging activities assessed by different methods. Journal of Agricultural and Food Chemistry, 47, 425-431. Rodea-Palomares I, Petre AL, Boltes K, Leganés F, Perdigón-Melón JA, Rosal R, Fernández-Piñas F. (2010) Application of the combination index (CI)-isobologram equation to study the toxicological interactions of lipid regulators in two aquatic bioluminescent organisms. Water Research, 44, 427-438. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. (1999) Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology & Medicine, 26, 1231-1237.

Natural Product Communications Vol. 8 (9) 2013 Published online (www.naturalproduct.us)

In vitro Anti-diabetic Activity of Sclerocarya birrea and Ziziphus mucronata Nuno M.H. Da Costa Mousinho, Jacob J. van Tonder and Vanessa Steenkamp Secondary Metabolites from the Fungus Emericella nidulans Amer H. Tarawneh, Francisco Leόn, Mohamed M. Radwan, Luiz H. Rosa and Stephen J. Cutler A New Glucuronolactone Glycoside Phoenixoside B from the Seeds of Phoenix dactylifera Sumbul Azmat, Rehana Ifzal, Faryal Vali Mohammad, Viqar Uddin Ahmad and Aqib Zahoor Cancer-Suppressive Potential of Extracts of Endemic Plant Helichrysum zivojinii: Effects on Cell Migration, Invasion and Angiogenesis Ivana Z. Matić, Ivana Aljančić, Vlatka Vajs, Milka Jadranin, Nevenka Gligorijević, Slobodan Milosavljević and Zorica D. Juranić Analysis of Volatile Components, Fatty Acids, and Phytosterols of Abies koreana growing in Poland Anna Wajs-Bonikowska, Karol Olejnik, Radosław Bonikowski and Piotr Banaszczak Cytotoxic Effects of Air Freshener Biocides in Lung Epithelial Cells Jung-Taek Kwon, Mimi Lee, Gun-Baek Seo, Hyun-Mi Kim, Ilseob Shim, Doo-Hee Lee, Taksoo Kim, Jung Kwan Seo, Pilje Kim and Kyunghee Choi GC/GC-MS Analysis, Isolation and Identification of Bioactive Essential Oil Components from the Bhutanese Medicinal Plant, Pleurospermum amabile Phurpa Wangchuk, Paul A. Keller, Stephen G. Pyne, Malai Taweechotipatr and Sumalee Kamchonwongpaisan Antibacterial Activity of the Essential Oil of Heracleum sibiricum Dragoljub L. Miladinović, Budimir S. Ilić, Tatjana M. Mihajilov-Krstev, Dejan M. Nikolić, Olga G. Cvetković, Marija S. Marković and Ljiljana C. Miladinović Assessment of the Chemical Composition and in vitro Antimicrobial Potential of Extracts of the Liverwort Scapania aspera Danka R. Bukvicki, Amit K. Tyagi, Davide G. Gottardi, Milan M. Veljic, Snezana M. Jankovic, Maria E. Guerzoni and Petar D. Marin Essential Oils of Alpinia rafflesiana and Their Antimicrobial Activities Shariha Jusoh, Hasnah Mohd. Sirat and Farediah Ahmad Chemical Composition and Synergistic Antioxidant Activities of Essential Oils from Atractylodes macrocephala and Astragalus membranaceus Jinkui Li, Feng Li, Yan Xu, Wenjian Yang, Lili Qu, Qian Xiang, Cong Liu and Dapeng Li Chemical Analysis and Antioxidant Activity of the Essential Oils of Three Piperaceae Species Growing in the Central Region of Cuba Elisa Jorge Rodríguez, Yanelis Saucedo-Hernández, Yvan Vander Heyden, Ernesto F. Simó-Alfonso, Guillermo Ramis-Ramos, María Jesús Lerma-García, Urbano Monteagudo, Luis Bravo, Mildred Medinilla, Yuriam de Armas and José Manuel Herrero-Martínez The Composition, Anti-mildew and Anti-wood-decay Fungal Activities of the Leaf and Fruit Oils of Juniperus formosana from Taiwan Yu-Chang Su, Kuan-Ping Hsu, Eugene I-Chen Wang and Chen-Lung Ho

1279 1285 1289

1291 1297

1301

1305

1309 1313 1317

1321

1325 1329

Meeting/Report Meeting Report: First National Meeting on Aloe, April 20-21, 2013, Isernia, Italy New Perspectives in Aloe Research: from Basic Science to Clinical Application Raffaele Capasso, Massimiliano Laudato and Francesca Borrelli

1333

Review/Account Alkaloids of the South African Amaryllidaceae: a Review Jerald J. Nair, Jaume Bastida, Carles Codina, Francesc Viladomat and Johannes van Staden

1335

Natural Product Communications 2013 Volume 8, Number 9 Contents Original Paper

Page

Alternate Biosynthesis of Valerenadiene and Related Sesquiterpenes Shashikumar K. Paknikar, Shahuraj H. Kadam, April L. Ehrlich and Robert B. Bates A Facile Synthesis of (±)-Heliannuol-D Tao Zhang, Liang-Zhu Huang, You-Qiang Li, Yimg-Meng Xu and Zhen-Ting Du A New Bioactive Diterpene Glycoside from Molinaea retusa from the Madagascar Dry Forest Alexander L. Eaton, Liva Harinantenaina, Peggy J. Brodie, Maria B. Cassera, Jessica D. Bowman, Martin W. Callmander, Richard Randrianaivo, Roland Rakotondrajaona, Etienne Rakotobe, Vincent E. Rasamison and David G. I. Kingston Nitric Oxide and Tumor Necrosis factor-alpha Inhibitory Substances from the Rhizomes of Kaempferia marginata Kanidta Kaewkroek, Chatchai Wattanapiromsakul, Palangpon Kongsaeree and Supinya Tewtrakul Biscembranoids from the Marine Sponge Petrosia nigricans Nguyen Xuan Nhiem, Ngo Van Quang, Chau Van Minh, Dan Thi Thuy Hang, Hoang Le Tuan Anh, Bui Huu Tai, Pham Hai Yen, Nguyen Thi Hoai, Do Cong Thung and Phan Van Kiem Isolation of Cycloeucalenol from Boophone disticha and Evaluation of its Cytotoxicity Emmanuel Adekanmi Adewusi, Paul Steenkamp, Gerda Fouche and Vanessa Steenkamp Chemical Constituents from an Endophytic Fungus Chaetomium globosum Z1 Chun-Yan Zhang, Xiao Ji, Xuan Gui and Bao-Kang Huang Determination of C-23 Configuration in (20R)-23-Hydroxycholestane Side Chain of Steroid Compounds by 1H and 13 C NMR Spectroscopy Alla A. Kicha, Anatoly I. Kalinovsky, Alexander S. Antonov, Oleg S. Radchenko, Natalia V. Ivanchina, Timofey V. Malyarenko, Alexander M. Savchenko and Valentin A. Stonik Oxasetin from Lophiostoma sp. of the Baltic Sea: Identification, in silico Binding Mode Prediction and Antibacterial Evaluation against Fish Pathogenic Bacteria Muftah Ali M. Shushni, Faizul Azam and Ulrike Lindequist Chemical Constituents from the Fruit Body of Chlorophyllum molybdites Zushang Su, Ping Wang, Wei Yuan, and Shiyou Li Pulchranins B and C, New Acyclic Guanidine Alkaloids from the Far-Eastern Marine Sponge Monanchora pulchra Tatyana N. Makarieva, Ekaterina K. Ogurtsova, Yuliya V. Korolkova, Yaroslav A. Andreev, Irina V. Mosharova, Ksenya M. Tabakmakher, Alla G. Guzii, Vladimir A. Denisenko, Pavel S. Dmitrenok, Hyi-Seung Lee, Eugene V. Grishin and Valentin A. Stonik Cloning and Characterization of a cDNA Encoding Calcium/Calmodulin-dependent Glutamate Decarboxylase from Scutellaria baicalensis Yeon Bok Kim, Md Romij Uddin, Do Yeon Kwon, Min-Ki Lee, Sun-Ju Kim, Chanhui Lee and Sang Un Park Biflavonoids, Main Constituents from Garcinia bakeriana Leaves Ahmed Al-Shagdari, Adonis Bello Alarcón, Osmany Cuesta-Rubio, Anna Lisa Piccinelli and Luca Rastrelli Analysis of Flavonoids and Iridoids in Vitex negundo by HPLC-PDA and Method Validation Somendu K. Roy, Khemraj Bairwa, Jagdeep Grover, Amit Srivastava and Sanjay M. Jachak Chemical Constituents of the Leaves of Triumfetta semitriloba Alejandra Barraza-Morales, Deisy Medrano-Nahuat, Sergio R. Peraza-Sánchez Phytochemical Evaluation of Lythrum salicaria Extracts and Their Effects on Guinea-pig Ileum Tímea Bencsik, Loránd Barthó, Viktor Sándor, Nóra Papp, Rita Benkó, Attila Felinger, Ferenc Kilár and Györgyi Horváth New Flavonol Glycosides from the Leaves of Triantha japonica and Tofieldia nuda Tsukasa Iwashina, Minoru N. Tamura, Yoshinori Murai and Junichi Kitajima Cytotoxic Activity of Dihydrochalcones Isolated from Corema album Leaves against HT-29 Colon Cancer Cells Antonio J. León-González, Miguel López-Lázaro, José L. Espartero and Carmen Martín-Cordero Immunomodulatory Activities of α-Mangostin on Peripheral Blood Mononuclear Cells Pimolkan Kasemwattanaroj, Primchanien Moongkarndi, Kovit Pattanapanyasat, Supachoke Mangmool, Ekkarat Rodpai, Jutima Samer, Julaporn Konlata and Kasama Sukapirom Antiplasmodial Quinones from the Rhizomes of Kniphofia foliosa Martha Induli, Meron Gebru, Negera Abdissa, Hosea Akala, Ingrid Wekesa, Robert Byamukama, Matthias Heydenreich, Sylvia Murunga, Ermias Dagne and Abiy Yenesew Biphenyl Derivatives from Garcinia schomburgkiana and the Cytotoxicity of the Isolated Compounds Chihiro Ito, Takuya Matsui, Eri Noda, Nijsiri Ruangrungsi and Masataka Itoigawa Anticarcinogenic Effect and Carcinogenic Potential of the Dietary Phenolic Acid: o-Coumaric Acid Alaattin Sen, Pelin Atmaca, Gulsum Terzioglu and Sevki Arslan Bioproduction and Optimization of Rosmarinic Acid Production in Solenostemon scutellarioides through Media Manipulation and Conservation of High Yielding Clone via Encapsulation Ranabir Sahu, Saikat Dewanjee and Moumita Gangopadhyay Continued inside backcover

1195 1197

1201 1205

1209 1213 1217

1219

1223 1227

1229

1233 1237 1241 1245 1247 1251 1255

1257

1261 1265 1269

1275