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HONORARY EDITOR

PROFESSOR GERALD BLUNDEN The School of Pharmacy & Biomedical Sciences,

University of Portsmouth, Portsmouth, PO1 2DT U.K.

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Composition at Different Development Stages of the Essential Oil of Four Achillea Species Grown in Iran Majid Azizia, Remigius Chizzolab*, Askar Ghania and Fatemeh Oroojalianc

aDepartment of Horticulture, Faculty of Agriculture, Ferdowsi University of Mashhad, Iran bInstitute for Applied Botany and Pharmacognosy, University of Veterinary Medicine Vienna, A – 1210 Vienna, Veterinaerplatz 1, Austria cMicrobiology Division, Biology Department, Faculty of Science, Isfahan University, Isfahan, Iran remigius.chizzola@vetmeduni.ac.at Received: October 14th, 2009; Accepted: November 25th, 2009 Four Achillea species, A. millefolium, A. nobilis, A. eriophora and A. biebersteinii, were grown in small field plots in Iran and harvested at four developmental stages: vegetative, at the appearance of the first flower heads, at full flowering, and at late flowering. The composition of the main volatile compounds in dichloromethane extracts and the essential oil obtained by microdistillation was established by GC/MS and GC. 1,8-Cineole (27-41%) was the main compound in the oils from A. millefolium and A. biebersteinii. These two species reached the highest amount of volatile compounds at the full blooming stage. α-Thujone was the main compound in A. nobilis oil (25-64%). Fully blooming plants of this species also had a high proportion of artemisia ketone (up to 40%) in the oil. The main oil compounds of A. eriophora were camphor (about 35%) and 1,8-cineol (about 30%). This species produces only a small number of flower heads and the composition of the essential oil did not change during development. Keywords: Achillea; essential oil composition; 1,8-cineol; α-thujone; camphor; development. The genus Achillea L. (Asteraceae), with about 115 species, is widely distributed in Europe, Asia and northern Africa and is naturalised in other parts of the world. The plants are perennial herbs and sub-shrubs with alternate, ordinary dentate to pinnatisect leaves and flower heads in dense corymbs [1]. A. millefolium L. agg. represents a complex of closely related, morphological similar species, present in different levels of ploidy that are widely distributed in the northern hemisphere. Used since ancient times to cure numerous ailments, the large number of medicinal properties may be due to the occurrence of different chemotypes. The plants may or may not contain proazulenes, which are pharmaceutically interesting compounds. They give, upon distillation, a dark blue essential oil [2]. The Pharmacopoeia Europea prescribes at least 2 mL/kg essential oil with at least 0.02% proazulenes in Millefolii herba (Pharm. Eur. 5.0/1382). An evaluation and selection of plant material to optimize the production of Millefolii herba has been carried out in various countries [3, 4, 5]. The use of A. millefolium as an additive to animal feed has recently been reviewed [6].

A. nobilis L. occurs in southern Europe and western Asia, mainly in open dry meadows. Ethanolic extracts of A. nobilis subsp. sipylea showed an antispasmodic effect on the rat duodenum [7]. A. biebersteinii Afan. is distributed in south western Asia, mainly in coniferous forests, steppes, dry meadows and rocky slopes, and is used as a folk remedy in Turkey [8]. The essential oil displayed antimicrobial activity [8]. The distilled oils and n-hexane extracts were also active against some phytopathogenic fungi and inhibited the germination and growth of selected common weeds [9]. A. eriophora DC is endemic in Iran, occurring mainly in the south, at altitudes between 700 and 3000 m [10]. The essential oil of this species showed some in vitro antimicrobial activity [11]. The present study investigated, in a field experiment, the essential oil composition of the four mentioned Achillea species during a vegetation period. The oil composition from the leaves and flower heads of the four species, as obtained by microdistillation, is

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284 Natural Product Communications Vol. 5 (2) 2010 Azizi et al.

displayed in Tables 1 and 3. As the available amount of plant material and the used microdistillation unit did not allow exact oil quantities to be measured by hydrodistillation, a quantification of the main oil components, as extracted with dichloromethane, has been attempted. These results are presented in Tables 2, and 4. Achillea millefolium: As can be seen in Table 1, the A. millefolium examined contained an oil without chamazulene. The main component of the distilled oil was 1,8-cineol, ranging from 27 to 41% with a maximum in the leaves of the fully blooming plants (stage 3). In the flowers, 1,8-cineol was in the same range (36%) and its proportion did not vary during further growth. α-Eudesmol, p-cymene, sabinene, β-pinene, cis-sabinene hydrate, α-terpineol and α-pinene were the predominant minor compounds in this species, occurring both in leaves and flower heads. In the flower heads β-bisabolene, δ-terpineol, bornyl acetate and γ-cadinene were also amongst the minor compounds. Evaluation of the volatiles in the dichloromethane extracts showed also the predominance of 1,8-cineole with the highest volatile levels in the leaves from the pre-blooming plants (stage 2) and the fully developed flowers (stage 3). Altogether, flowers accumulated more volatiles than leaves (Table 2). The content of the essential oil of A. millefolium is reported to be very variable. The highest contents (0.8-1.2% v/w) have been recorded shortly before flowering and then declining [2]. In A. millefolium, the relative chamazulene content of the flower heads decreased during development [12], whereas camphor increased. Under Iranian climatic conditions the best harvesting time for azulene-free field grown A. millefolium, rich in sabinene was in July [13]. Also, the composition may show great differences in accordance with the provenance of the plant material. Forty A. millefolium samples that were collected in the wild in Lithuania could be classified into four groups according to a cluster analysis evaluating the essential oil composition [14]. Another study from the same country that considered 19 A. millefolium samples defined 6 chemotypes according to the main essential oil components. In these samples, 1,8-cineol ranged from 2.3 to 21.6% [15]. From Iran, Afsharypuor et al. [16] described an essential oil of A. millefolium subsp. millefolium rich in α-bisabolol (23%), spathulenol (12%) and cis-nerolidol (6%). The main constituents of A. millefolium essential oil from the Balkans were β-pinene (33%), β-caryophyllene (17%), sabinene (11%), and chamazulene (6%) [17]. In A. millefolium subsp. millefolium from Turkey, 1,8-cineol (25%), camphor (17%) and α-terpineol (10%) were the main

essential oil components [18]. Two different diploid ecotypes of A. millefolium from the Indian Himalaya had β-caryophyllene (16%), 1,8-cineol (15%) and β-pinene (11% or 14%), β-caryophyllene (13%) and borneol (12%) as major compounds in the essential oil from the flowering parts. These plants were low in proazulenes [19]. Achillea biebersteinii: The essential oil of A. biebersteinii displayed, like that from A. millefolium, 1,8-cineol as its main compound, ranging from 46-60% in the leaves and 26-36% in the flower heads (Table 1). Besides p-cymene and camphor, piperitone, ascaridol and isoascaridol were further major components of this species. The last two compounds were highest in the late flowers with 25% and 7%, respectively (stage 4). The amount of volatiles in the dichloromethane extracts (Table 2) was highest in the leaves of the pre-blooming plants (stage 2) and the fully developed flowers (Stage 3). The leaves of the other stages had a comparable composition of volatiles and afforded only half as much volatiles. According to available literature reports, the essential oil of A. biebersteinii may vary greatly with the respective origin. Plants having 1,8-cineol as their main oil compound were described from Erzurum (Turkey) with 1,8-cineol (46%), camphor (18%) and α-terpineol (8%) [20], 1,8-cineol (38%) and camphor (24%) [9], and 1,8-cineol (30%) and camphor (17%) as main oil compounds [21]. Other plants from the same region had piperitone (31%), camphor (13%) and 1,8-cineol (11%) as the main essential oil components in flowering parts [22]. Also, samples from the Ankara region of Turkey displayed piperitone (50%) as the main oil compound, along with 1,8-cineol (11%) and camphor (9%) [21]. These three compounds were also reported as main oil constituents in Turkish plants by Sökmen et al. [8]. All these samples from Turkey either lack or were very low in ascaridol and isoascaridol. Similarly, 1,8-cineol (8%), camphor (7%) and α-fenchene (6%) were reported as main compounds from flowering A. biebersteinii plants in Iran [23]. Besides the main compounds, camphor and 1,8-cineol, further plants from Iran displayed appreciable proportions of germacrene D and spathulenol in their oils [24]. In contrast, other A. biebertsteinii accessions from Iran had ascaridol (37%) as their main oil compound, and piperitone (17%), camphor (12%), p-cymene (8%) and piperitone oxide (6%) as further major compounds, whereas 1,8-cineol reached merely 4% [25]. Furthermore, the same species collected in Azerbeijan had camphor (34-38%) as its main oil compound, along with borneol (7-23%) and 1,8-cineol (10-22%) [26]. In these samples leaves had less 1,8-cineol but more borneol than the flowers.

Essential oil of four Achillea species Natural Product Communications Vol. 5 (2) 2010 285

Table 1: Composition of the essential oil (%) from Achillea millefolium and A. biebersteinii during plant development.

Achillea millefolium Achillea biebersteinii Stage 1 Stage 2 Stage 3 Stage 4 Stage 3 Stage 4 Stage 1 Stage 2 Stage 3 Stage 4 Stage 3 Stage 4 Compound RI Leaves Inflorescences Leaves Inflorescences α-Thujene 929 0.5 1.1 1.4 0.4 0.4 0.5 0.3 0.5 0.6 0.5 0.4 0.2 α-Pinene 936 6.0 2.3 2.3 2.0 2.3 1.3 2.8 2.8 2.9 2.8 2.0 1.1 Camphene 950 0.3 0.5 0.5 0.7 1.1 0.6 1.5 1.3 1.8 2.2 1.2 1.2 Benzaldehyde 958 0.1 0.2 Sabinene 974 4.0 11.0 3.9 2.8 4.6 5.1 0.3 1.1 1.3 0.8 2.0 0.5 β-Pinene 977 11.4 5.1 6.0 5.1 4.4 3.6 1.8 1.9 2.3 2.1 1.6 0.8 1-Octen-3-ol 990 0.5 0.5 0.2 0.0 0.4 0.5 0.0 0.0 0.0 0.0 0.0 0.0 α-Phellandrene 1003 0.2 0.1 0.4 0.2 0.4 0.0 α-Terpinene 1016 0.7 1.0 1.0 1.0 1.0 1.0 0.3 2.4 2.3 0.9 9.7 1.6 p-Cymene 1024 4.9 4.4 6.2 7.3 3.1 4.1 7.0 9.4 5.9 8.5 8.0 10.4 1,8-Cineol 1031 27.7 27.7 41.5 34.2 36.1 36.8 59.6 46.1 53.8 49.7 35.5 26.4 γ-Terpinene 1060 1.4 2.4 1.7 1.8 2.2 1.8 0.3 0.5 0.7 0.6 0.7 0.5 cis-Sabinene hydrate 1067 3.3 3.3 2.9 4.0 1.3 0.7 0.2 0.3 0.4 0.5 0.1 α-Terpinolene 1087 0.5 0.6 0.1 0.1 0.4 0.5 0.1 0.3 0.1 0.5 0.4 0.3 trans-Sabinene hydrate 1095 1.0 1.1 1.4 2.3 0.6 0.5 0.2 0.3 0.5 0.1 0.2 Linalool 1098 0.9 4.9 0.9 1.3 1.8 1.1 0.0 0.1 0.9 0.2 cis-p-Menth-2-en-1-ol 1120 0.3 0.4 2.1 1.1 2.7 2.0 2.5 3.8 α-Campholenal 1124 0.7 0.6 0.4 0.6 0.5 0.4 trans-p-Menth-2-en-1-ol 1139 1.3 0.1 1.5 1.0 0.9 1.0 Camphor 1141 0.3 1.0 1.1 0.7 2.2 1.1 3.9 3.5 4.8 6.1 3.3 4.1 Sabina ketone 1154 0.2 0.4 0.8 0.8 0.8 0.7 0.6 0.5 Pinocarvone 1160 0.4 0.4 Borneol 1165 1.5 1.1 1.3 3.1 δ-Terpineol 1166 2.3 1.6 Lavandulol 1167 0.9 1.0 0.9 0.9 0.9 Terpinen-4-ol 1176 1.9 2.8 2.8 4.1 3.0 3.3 0.8 1.0 1.2 1.1 1.2 1.0 α-Terpineol 1189 2.7 2.8 2.2 2.0 2.4 1.6 1.5 2.9 2.1 1.2 2.1 0.8 Myrtenal 1191 0.5 0.6 cis-Piperitol 1193 0.4 0.2 0.7 0.4 0.4 trans-Piperitol 1205 0.5 0.1 0.7 0.4 0.4 0.3 Ascaridol 1235 3.4 12.2 2.5 6.5 8.9 25.6 Piperitone 1250 2.8 0.3 4.6 2.5 5.6 5.6 cis-Piperiton epoxide 1254 1.0 1.5 0.7 1.9 1.1 2.4 Bornylacetate 1283 1.6 1.4 p-Cymen-7-ol 1285 0.1 0.2 0.1 0.3 0.5 Lavandulyl-acetate 1289 0.3 0.4 0.2 0.6 Carvacrol 1294 0.4 0.7 0.2 0.4 0.8 Isoascaridol 1299 2.0 3.7 1.5 2.7 3.5 7.4 Eugenol 1354 0.6 0.6 0.1 0.2 0.3 0.2 Jasmone 1392 0.1 0.1 allo-Aromadendrene 1459 0.3 0.8 0.1 0.7 0.4 1.0 Lavandulyl-isovalerate 1506 0.3 γ-Cadinene 1512 0.5 0.9 δ-Cadinene 1522 0.3 0.1 Spathulenol 1577 1.5 1.1 1.7 1.9 0.4 0.7 Caryophyllene oxide 1582 1.3 0.9 1.1 1.2 0.4 0.6 γ-Eudesmol 1624 0.1 0.1 0.7 Sesquiterpenoid* 1643 2.8 3.6 1.3 3.1 1.4 3.0 tau-cadinol 1653 1.2 0.8 1.2 4.9 1.3 β-Eudesmol 1654 0.4 0.6 0.4 0.4 0.2 0.1 α-Eudesmol 1657 3.3 5.1 7.9 7.3 0.1 7.3 Cedren-13-ol-8 1686 1.0 1.8 1.0 0.9 0.7 1.6 Bisabolone 1747 5.5 1.5 Sum 81.5 88.1 90.8 89.7 88.4 90.5 97.9 97.8 98.7 97.8 97.7 97.9

* Sesquiterpenoid: 220 (M+):9, 159:100, 43:37, 97:36, 41:34, 202:33, 107:32, 109:29, 81:27, 105:27, 121:26 Achillea nobilis: In Achillea nobilis, α-thujone was the main volatile compound with 40-64 % in the leaf oil and 25 to 59% in that of the flower heads (Table 3). Further major compounds in this species were artemisia

ketone (up to 40%), 1,8-cineol (2-14%) and the sesquiterpene cadin-4-en-7-ol (4-10%). β-Thujone was 8-10 times lower than α-thujone.

286 Natural Product Communications Vol. 5 (2) 2010 Azizi et al.

Table 2: Content of selected volatile compounds (µg/g plant dry matter) in Achillea millefolium and A. biebersteinii during development. Calculated from dichloromethane extracts.

µg/g Achillea millefolium Achillea biebersteinii Stage 1 Stage 2 Stage 3 Stage 4 Stage 3 Stage 4 Stage 1 Stage 2 Stage 3 Stage 4 Stage3 Stage 4 Compound Leaves Inflorescences Leaves Inflorescences α-Thujene 11 34 15 7 3 21 α-Pinene 48 48 27 6 62 12 75 331 121 259 Camphene 5 8 4 24 27 Sabinene 70 247 46 23 176 157 280 109 398 β-Pinene 126 151 57 27 149 68 52 264 47 238 α-Terpinene 3 2 1 9 46 9 943 47 p-Cymene 51 109 61 69 58 75 380 1075 467 637 1502 973 1,8-Cineole 358 853 527 408 1194 665 3452 7318 3779 3631 6148 2230 γ-Terpinene 9 19 10 2 22 12 cis-Sabinene hydrate 72 197 92 92 224 109 α-Terpinolene 3 3 trans-Sabinene hydrate 7 19 22 54 7 24 Linalool 25 69 24 13 63 54 Camphor 9 10 3 50 50 293 447 488 527 650 443 Borneol 8 41 α-Terpineol 56 132 46 56 144 123 150 524 108 602 97 Ascaridol 201 595 116 1487 1380 Piperitone 195 543 395 270 1359 600 Isoascaridol 379 1334 406 443 2381 2579

Sum 832 1890 954 802 2162 1386 5201 12757 6072 5508 15965 8349 The highest proportions of α-thujone were found in the plants at the vegetative (stage 1) and late flowering stages (stage 4). Artemisia ketone showed the greatest dynamic: the levels were highest in both leaves and flower heads of the fully blooming plants (stage 3), where it became the major compound. Then it nearly disappeared in the late flowering plants (stage 4). 1,8-Cineol was highest in the pre-blooming (2) and late flowering (4) stages. The amount of the selected volatile compounds in the dichloromethane extracts from the leaves (Table 4) changed scarcely during ontogenesis, whereas the flowers, which were higher in volatiles than the leaves, came to a maximum content in the late flowers (stage 4). The essential oil composition of A. nobilis varied greatly with the origin of the plants. α-Thujone (34%) and 1,8-cineol (14%) were the main oil compounds in wild growing Iranian A. nobilis plants [27]. Similar to the present study, plants from Serbia displayed α-thujone as the main essential oil component (26%), followed by artemisia ketone (15%), borneol (10%) and camphor (8%) [28]. In contrast, A. nobilis samples from the western Italian Alps were particularly rich in germacrene D (46%) and virtually devoid of α-thujone and artemisia ketone [29]. However, other oil compositions have also been described in which fragranol (19%), chrysanthenone, (17%), linalool (16%) and dihydro-eudesmol (13%) were the major compounds in the essential oil from the flower heads of A. nobilis subsp. sipylea and 1,8-cineol, α-bisabolol, piperitone, chrysanthenone and linalool in subsp. neilreichii [7].

Achillea eriophora: The volatile fraction of Achillea eriophora, as isolated by microdistillation, contained two main compounds: camphor (33-36%) and 1,8- cineol (25-30%) in comparable proportions (Table 3). Additionally α-pinene, sabinene, camphene, β-pinene, borneol, α-terpineol and terpinen-4-ol were minor compounds, accounting for 1.5-8 % of the volatile fraction, each. During development the relative proportions of these compounds changed little. Also, the selected compounds measured in the dichloromethane extracts gave a largely stable composition during the experiment (Table 4). Plants collected from south Iran (Shiraz) displayed 1,8-cineol (34%) as the main essential oil component and contained very little camphor [30]. Santolina alcohol, α-thujone and cis-chrysanthenol were found as minor compounds, which could not be detected in the plants from the present study. Recent reports pointed out camphor (30%) and 1,8-cineol (25%) [27], 1,8-cineol (55%), linalool (9%) and α-terpineol (7%) [11], and camphor (10%) and germacrene D (19%) [24] as main essential oil compounds in Iranian A. eriophora. Comparison of the four species: According to the composition of the essential oils, the greatest similarities were between A. millefolium and A. biebersteinii, where 1,8-cineol was the main compound. In A. eriophora, 1,8-cineol was a major compound, but in A. nobilis a minor one. Camphor, the second major compound in the oil from A. eriophora, was also present as a minor compound in the other three species.

Essential oil of four Achillea species Natural Product Communications Vol. 5 (2) 2010 287

Table 3: Composition of the essential oil (%) from Achillea nobilis and A. eriophora during plant development.

Achillea nobilis Achillea eriophora Stage 1 Stage 2 Stage 3 Stage 4 Stage 3 Stage 4 Stage 1 Stage 2 Stage 3 Compound RI Leaves Inflorescences Leaves Santolina triene 907 0.1 0.3 0.7 <0.05 0.3 <0.05 Tricyclene 924 0.5 0.5 0.3 α-Thujene 929 0.3 0.6 0.2 0.1 0.1 0.4 0.5 0.5 0.5 α-Pinene 936 0.6 1.1 0.4 0.2 0.1 0.5 4.3 4.7 4.7 Camphene 950 0.9 0.1 7.2 9.4 5.9 Sabinene 974 1.3 2.1 1.1 0.9 0.5 1.6 1.6 1.6 0.9 β-Pinene 977 0.3 0.9 0.2 0.1 0.1 0.4 3.7 3.5 4.4 Dehydrocineol 989 0.1 0.1 0.6 0.3 0.4 Yomogi alcohol 998 0.6 0.4 1.7 <0.05 1.6 <0.05 α-Terpinene 1016 0.1 0.2 0.2 0.1 0.1 0.5 0.6 0.3 0.4 p-Cymene 1024 0.7 0.7 0.5 0.8 0.1 0.7 0.5 0.7 0.8 1,8-Cineol 1031 2.8 14.1 5.2 12.8 2.2 9.2 24.8 29.8 29.3 γ-Terpinene 1060 0.3 0.2 0.2 3.6 0.1 1.2 1.0 0.6 0.7 Artemisiaketone 1062 11.2 9.5 27.8 <0.05 40.1 <0.05 cis-Sabinene hydrate 1067 0.3 0.4 0.2 <0.05 0.1 0.1 0.3 0.2 0.2 Artemisia alcohol 1084 0.5 0.2 1.3 <0.05 0.8 <0.05 α-Terpinolene 1087 <0.05 0.1 <0.05 <0.05 <0.05 0.1 0.3 0.2 0.3 trans-Sabinene hydrate 1095 0.6 0.6 0.2 Linalool 1098 0.2 0.2 0.2 α-Thujone 1106 64.1 40.4 42.5 54.0 24.7 58.7 β-Thujone 1115 6.4 3.7 5.0 5.9 3.1 6.0 cis-p-Menth-2en-1-ol 1120 0.2 0.2 0.2 α-Campholenal 1124 0.2 0.1 0.3 cis-Sabinol 1140 0.3 <0.05 0.3 0.1 0.1 0.8 Camphor 1141 <0.05 3.4 <0.05 1.2 35.9 32.9 34.0 Pinocarvone 1160 0.8 0.9 1.9 Borneol 1165 2.5 1.8 1.4 Lavandulol 1167 <0.05 0.7 0.6 1.1 2.8 3.0 Pinocamphone 1171 <0.05 0.1 0.3 Terpinen-4-ol 1176 0.7 0.9 0.6 0.9 0.3 1.6 1.7 1.0 1.5 α-Terpineol 1189 0.1 1.3 0.2 0.1 0.1 0.3 2.5 1.6 1.0 Myrtenal 1191 0.7 0.6 1.3 Myrtenol 1194 0.8 0.6 1.0 Carvone 1240 0.0 0.1 0.1 Bornylacetate 1283 <0.05 0.1 <0.05 <0.05 1.0 1.1 1.1 Lavandulyl acetate 1289 0.1 0.1 0.1 0.1 0.4 0.2 Eugenol 1354 0.4 0.5 0.3 Lavandulyl propionate 1379 <0.05 0.1 0.1 0.9 Jasmone 1392 0.5 0.5 0.3 Methyleugenol 1399 1.1 0.9 0.8 β-Caryophyllene 1417 <0.05 0.1 0.1 0.2 0.3 0.3 0.1 Lavandulyl isobutanoate 1421 <0.05 0.1 0.1 0.4 Germacrene D 1479 <0.05 0.2 0.1 <0.05 Indipone 1492 0.3 0.1 0.4 0.3 0.6 Lavandulyl-isovalerate 1506 <0.05 0.4 0.4 2.6 0.1 Longipinocarvone 1574 0.4 0.1 0.3 1.8 0.5 0.1 0.6 0.5 0.5 Spathulenol 1577 0.1 0.4 <0.05 <0.05 <0.05 <0.05 Caryophyllene oxide 1582 1.8 1.8 2.6 5.4 2.7 3.0 1.1 0.8 0.9 γ-Eudesmol 1624 0.3 0.5 0.1 Cadin-4-en-7-ol 1637 4.6 9.7 3.1 6.7 5.7 4.3 β-Eudesmol 1654 0.7 0.5 1.0 1.9 1.4 0.6 neo-Intermedol 1658 0.2 0.3 0.9 1.2 1.8 0.4

Sum 98.9 96.2 98.0 99.2 94.5 94.8 97.4 98.3 96.5

α-Thujone, which was predominant in A. nobilis, could not be found in the other species. Flower heads usually contained more volatile oils than the leaves [15]. The fully blooming A. millefolium plants had the highest oil contents [3]. The most suitable

phase for the harvest of the various accessions of the A. millefolium complex rich in active compounds was the early flowering stage [31]. This maximum essential oil accumulation in the pre-blooming stage could also be observed in other plant species, such as Santolina etrusca [32] and Satureja rechingeri [33]. In the present

288 Natural Product Communications Vol. 5 (2) 2010 Azizi et al.

Table 4: Content of selected volatile compounds (µg/g plant dry matter) in Achillea nobilis and A. eriophora during development. Calculated from dichloromethane extracts.

Achillea nobilis Achillea eriophora Stage 1 Stage 2 Stage 3 Stage 4 Stage 3 Stage 4 Stage 1 Stage 2 Stage 3 Compound Leaves Inflorescences Leaves α-Thujene 20 68 20 22 35 116 199 93 112 α-Pinene 15 62 10 10 42 80 678 576 782 Camphene 1099 1084 891 Sabinene 12 241 89 106 171 398 442 314 244 β-Pinene 83 50 10 12 23 31 598 439 761 p-Cymene 32 44 13 59 27 88 78 116 129 1,8,Cineol 170 1073 378 868 526 1206 5322 4629 5460 γ-Terpinene 118 68 110 Artemisiaketon 916 349 1949 302 4183 0 cis-Sabinene hydrate 35 85 50 38 67 182 129 112 139 α-Terpinolene 161 104 157 trans-Sabinene hydrate 22 60 21 33 35 115 59 70 63 α-Thujone 4654 4765 4252 5578 6689 14889 β-Thujone 540 425 497 556 719 1301 cis-Sabinol 7 227 5 49 19 8 Camphor 7291 7621 6290 Pinocarvone 157 198 Lavandulol 0 105 99 59 659 902 Borneol 58 94 506 Pinocamphone 329 503 249 Terpinen-4-ol 14 46 17 α-Terpineol 708 642 660 Myrtenal 306 196 460 Myrtenol 156 152 80 Bornylacetate 472 386 336 Cadin-4-en-7-ol 522 1342 464 459 1763 1868

Sum 7028 8897 7856 8150 14957 21183 18374 17446 17446

study A. millefolium and A. biebersteinii displayed the maximum volatile contents in the leaves of the pre-blooming plants and the fully developed flowers. In older plants of these two species the volatile content decreased. In A. nobilis, the maximum volatile level is reached in the older flower heads. Experimental

Plant material: The research was conducted during 2005-2007. The plants of A. nobilis and A. biebersteinii were collected from "Golmakan" in Khorasan Razawi province, those of A. eriophora from "Jahrom" in Fars province, and A. millefolium from " Karaj " in Tehran province, Iran. They were authenticated in Ferdowsi University of Mashhad (FUM) herbarium and voucher specimens [No. 36534(FUMH), 29400(FUMH), 17683(FUMH) and 34876(FUMH) respectively] were deposited there. Experimental plots: The seeds were cultivated in pots in the greenhouse at 25-30°C for seedling production. After 8 weeks, as the seedlings had developed 6 true leaves, they were transplanted to the research field at Ferdowsi University of Mashhad, Iran. The plots (1.5 × 1.5 m with 30 and 40 cm within and between the rows, respectively) were arranged according to the

randomized complete block design (RCBD) with four replications. Definition of the developmental stages and harvest: The above ground plant parts were harvested at 4 defined developmental stages: vegetative (1), appearance of the first flower heads (2), fully flowering (3) and late flowering (4). The plant material was dried in the ambient air. Plants in stages 3 and 4 were separated into leaves and flower heads for analysis. In the case of A. eriophora, only developmental stages 1 to 3 were harvested and the plants were analysed as a whole because this species produces only a small number of flower heads on each plant. Microdistillation: As only small sample amounts from single plants were available the distillation was carried out using the automatic microdistillation unit MicroDistiller from Eppendorf (Hamburg, Germany), which allows the simultaneous distillation of 6 samples. About 0.2 to 0.3 g finely crushed dried plant material (leaves or flower heads) and 10 mL distilled water were filled into the sample vial. The collecting vial, which contained 1 mL water, 0.5 g NaCl and 300 µL n-hexane was connected with a capillary to the sample vial. The

Essential oil of four Achillea species Natural Product Communications Vol. 5 (2) 2010 289

heating program applied to the sample vial was 15 min at 108°C and then 45 min at 112°C. The collecting vial was kept at -2°C, where the volatiles were trapped in 0.3 mL n-hexane. CH2Cl2 extracts: About 0.15 to 0.25 g dried crushed plant material was extracted with 1 mL CH2Cl2 containing hexadecane as internal standard (0.15 mg/mL) for 30 min in an ultrasonic bath at room temperature. The filtered extracts were directly analysed by GC/MS and GC. To construct calibration curves for the main oil compounds, 20 to 1000 µg/mL decane, 1,8-cineol, α-thujone and camphor were prepared in n-hexane containing hexadecane as internal standard. Each of these calibration standards were analysed by GC in triplicate. The concentrations of 1,8-cineole, α-thujone and camphor were then calculated using the respective calibration curves. The other components were calculated by assuming the same response as determined for decane. GC/MS: To assure the identification of the volatile components of the extracts and oils, a HP 6890 GC equipped with a 5972 quadrupole mass selective detector was used. The separation was undertaken on a 30 m x 0.25 mm column coated with 0.25µm HP5-MS. The analytical conditions were: carrier gas He 1.3 mL/min in the constant flow mode; injector temperature

250°C; injection volume 1 µL; split ratio 15:1; temperature program: 2 min at 40°C, with 3°C/min up to 180°C, 10°C/min up to 280°C; transfer line to MSD 280°C; MSD 170°C. (m/z 40 to 350). A mixture of n-alkanes (C9 - C30) was analyzed under the same conditions to calculate the retention indices. The compounds were identified according to their mass spectra and their retention indices [34,35]. GC: An Agilent Technologies 6890N GC with FID was used. Separation was achieved on a DB-5 narrow bore column 10 m x 0.10 mm with 0.17 µm film thickness. The analytical conditions were: carrier gas He initial flow 0.5 mL/min (42 cm/sec), constant pressure 42.78 psi; injector temperature 250°C, split ratio 100:1, temperature program: 1 min at 60°C, with 6°C/min up to 85°C, with 12°C/min to 180°C, then with 20°C/min to 280°C and held for 3 min at 280°C. The injector temperature was set at 250°C, the injection volume was 1 µL. The FID was operated at 260°C with an air flow of 350 mL/min and a hydrogen flow of 35 mL/min. The percentage of the oil composition was calculated from the FID response without corrections. Acknowledgments - The authors wish to thank Mr Patrick Zwickl for technical assistance during the analyzes of the volatile oils.

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Antioxidant Activity and Total Phenolic Content of 24 Lamiaceae Species Growing in Iran Omidreza Firuzi, Katayoun Javidnia, Maryam Gholami, Mohammad Soltani and Ramin Miri 261

Preparation and Characterization of 5′-Phosphodiesterase from Barley Malt Rootlets Jie Hua and Ke-long Huang 265

Volatiles of Callicarpa macrophylla: A Rich Source of Selinene Isomers Anil K. Singh, Chandan S. Chanotiya, Anju Yadav and Alok Kalra 269

Volatile Components of Aerial Parts of Centaurea nigrescens and C. stenolepis Growing Wild in the Balkans Carmen Formisano, Felice Senatore, Svetlana Bancheva, Maurizio Bruno, Antonella Maggio and Sergio Rosselli 273

Compositional Variability in Essential Oil from Different Parts of Alpinia speciosa from India Rajendra C. Padalia, Chandan S. Chanotiya and V. Sundaresan 279

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Characterization of Some Italian Ornamental Thyme by Their Aroma Alessandra Bertoli, Szilvia Sárosi, Jenő Bernáth and Luisa Pistelli 291

Characterization of Szovitsia callicarpa Volatile Constituents Obtained by Micro- and Hydrodistillation Betül Demirci, Nurgün Küçükboyacı, Nezaket Adıgüzel, K. Hüsnü Can Başer and Fatih Demirci 297

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Chemical Composition and Antibacterial Activity of the Essential Oil from Fruits of Bursera tomentosa José Moreno, Rosa Aparicio, Judith Velasco, Luis B Rojas, Alfredo Usubillaga and Marcó Lue-Merú 311

Composition and Antioxidant Activity of Inula crithmoides Essential Oil Grown in Central Italy (Marche Region) Laura Giamperi, Anahi Bucchini, Daniele Fraternale, Salvatore Genovese, Massimo Curini and Donata Ricci 315

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Natural Product Communications 2010

Volume 5, Number 2

Contents

Original Paper Page

Antimosquito and Antimicrobial Clerodanoids and a Chlorobenzenoid from Tessmannia species Charles Kihampa, Mayunga H.H. Nkunya, Cosam C. Joseph, Stephen M. Magesa, Ahmed Hassanali, Matthias Heydenreich and Erich Kleinpeter 175

Two New Terpenoids from Trichilia quadrijuga (Meliaceae) Virginia F. Rodrigues, Hadria M. Carmo, Raimundo Braz Filho, Leda Mathias and Ivo J. Curcino Vieira 179

Effect of Miconazole and Terbinafine on Artemisinin Content of Shooty Teratoma of Artemisia annua Rinki Jain and Vinod Kumar Dixit 185

A New Triterpenoid Saponin from the Stem Bark of Pometia pinnata Faryal Vali Mohammad, Viqar Uddin Ahmad, Mushtaq Noorwala and Nordin HJ.Lajis 191

27-Hydroxyoleanolic Acid Type Triterpenoid Saponins from Anemone raddeana rhizome Li Fan, Jin-Cai Lu, Jiao Xue, Song Gao, Bei-Bei Xu, Bai-Yi Cao and Jing-Jing Zhang 197

Steroids from the South China Sea Gorgonian Subergorgia suberosa Shu-Hua Qi, Cheng-Hai Gao, Pei-Yuan Qian and Si Zhang 201

Auroside, a Xylosyl-sterol, and Patusterol A and B, two Hydroxylated Sterols, from two Soft Corals Eleutherobia aurea and Lobophytum patulum Dina Yeffet, Amira Rudi, Sharon Ketzinel, Yehuda Benayahu and Yoel Kashman 205

Anti-tuberculosis Compounds from Mallotus philippinensis Qi Hong, David E. Minter, Scott G. Franzblau, Mohammad Arfan, Hazrat Amin and Manfred G. Reinecke 211

Phenolic Derivatives with an Irregular Sesquiterpenyl Side Chain from Macaranga pruinosa Yana M. Syah and Emilio L. Ghisalberti 219

Hexaoxygenated Flavonoids from Pteroxygonum giraldii Yanhong Gao, Yanfang Su, Shilun Yan, Zhenhai Wu, Xiao Zhang, Tianqi Wang and Xiumei Gao 223

Comparative Study of the Antioxidant Activities of Eleven Salvia Species Gábor Janicsák, István Zupkó, Imre Máthé and Judit Hohmann 227

Dibenzocyclooctadiene Lignans from Fructus Schisandrae Chinensis Improve Glucose Uptake in vitro Jing Zhang, Lei Ling Shi and Yi Nan Zheng 231

Honokiol and Magnolol Production by in vitro Micropropagated Plants of Magnolia dealbata, an Endangered Endemic Mexican Species Fabiola Domínguez, Marco Chávez, María Luisa Garduño-Ramírez, Víctor M. Chávez-Ávila, Martín Mata and Francisco Cruz-Sosa 235

Design, Synthesis and Biological Evaluation of Novel Spin-Labeled Derivatives of Podophyllotoxin Jia-qiang Zhang, Zhi-wei Zhang, Ling Hui and Xuan Tian 241

Secondary Metabolites of the Phytopathogen Peronophythora litchii Haihui Xie, Yaoguang Liang, Jinghua Xue, Qiaolin Xu, Yueming Jiang and Xiaoyi Wei 245

Bioassay-guided Isolation of Antibacterial and Cytotoxic Compounds from the Mesophilic Actinomycete M-33-5 Mustafa Urgen, Fatma Kocabaş, Ayşe Nalbantsoy, Esin Hameş Kocabas, Ataç Uzel and Erdal Bedir 249

Aristolactams, 1-(2-C-Methyl-β-D-ribofuranosyl)-uracil and Other Bioactive Constituents of Toussaintia orientalis Josiah O. Odalo, Cosam C. Joseph, Mayunga H.H. Nkunya, Isabel Sattler, Corinna Lange, Gollmick Friedrich, Hans-Martin Dahse and Ute Möllman 253

Continued inside backcover