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Variation in essential oil composition of Iris nigricans Dinsm. (Iridaceae) endemic to Jordan at different flowering stages
⁎Tel.: +962 6 4790333/4790359x108; fax: +962 6 4790350. rhhjaber@yahoo.com (Hala I. Al-Jaber)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
Fresh aerial blooming parts of Iris nigricans Dinsm. (Iridaceae) were collected at different flowering stages. The essential oils obtained from the fresh aerial parts and from the rhizomes at the post flowering stage were investigated by gas chromatography–mass spectrometry (GC/MS) analysis. The essential oil at the pre-flowering stage was characterized by a high proportion of oxygenated monoterpenes (40.93%), while the oils obtained during the full flowering and post flowering stages were dominated by aliphatic hydrocarbons and their derivatives (81.28%, 79.52%, respectively). The oil of I. nigricans rhizomes was dominated by monoterpene hydrocarbons (35.47%) of the pinane skeleton (32.05%), represented by α- and β-pinenes (24.05% and 7.99%, respectively). In addition, rhizome's aqueous and ethanolic extracts were investigated for their antimicrobial activity and were found to be inactive against Escherichia coli, Klebsiella pneumonia, Salmonella typhi, Staphylococcus aureus, Bacillus subtilis and Micrococcus luteus.
Keywords
Iris nigricans
Iridaceae
Essential oil
Monoterpenoids
α-Pinene
β-Pinene
1 Introduction
Iridaceae, taking its name from the genus Iris, is an interesting plant family from the viewpoint of the diverse floral structures of its species, many of which are grown in parks and gardens as ornamental plants due to their beautiful flowers (Baytop, 1984). It is a large family of perennial herbaceous plants with rhizomes or corms comprising nearly 1750 species in 82 genera (Devoto and Medan, 2008). Plants belonging to this family are distributed world-wide in tropical and temperate regions of the world, with the highest diversity occurring in South Africa, followed by South America, Europe and temperate regions of Asia (Bahali, 2006). In Jordan, Iridaceae family is represented by four genera including Gladulus, Crocus, Gynandriris and Iris. There are 13 Iris species (Al-Eisawi, 1982) occurring wild in Jordan. Iris nigricans Dinsm. commonly known as “Black Iris” is endemic to Jordan.
Plants of the Iridaceae family have been used in the traditional medicines to treat cold, flu, malaria, toothache, bruises and burns (Lin et al., 2002). The use of several Iris plant rhizomes in traditional medicine dates back in time, especially in European communities. The dry rhizomes of several Iris species, particularly those of Iris germanica L., were collectively used as ingredients of the toothpowders. The drug Rhizoma iridis, which is composed of small selected rhizome species, enjoyed popularity due to its emetic, cathartic, diuretic, stimulant, expectorant and errhine properties in addition to its use in relieving pain as masticatory for teething children (Rigano et al., 2006; Wollenweber et al., 2003). Traditional European healers recommended the aqueous extracts of the I. germanica roots as an enema or topically rubbing the oil on arthritic limbs (Adams et al., 2009). In Iran, medicinal plants belonging to the genus Iris are known as “Irsa” and were known to have diuretic and expectorant properties at low doses and strong purgative and emetic properties at high doses. Also, “Irsa” was considered to be useful in the treatment of pulmonary, liver and uterus diseases as well as in the treatment of hemorrhoids (Ayatollahi et al., 2004).
I. nigricans is an elegant endemic perennial herb, 20–30 cm long with underground rhizomes. The leaves are 0.5–1.0 cm wide, 10–15 cm long forming a tuft, curved, basal leaves, flattened, canal-like arranged in two opposite rows. The plant has one green flower stalk rising from a rhizome and bearing a single flower. Usually the leaves are much shorter than the stalk of the flower. The flowers are 12–15 cm in diameter, black-dark lilac, glossy with transparent veins (Al-Eisawi, 1998). Recognized as the flower emblem of Jordan, this plant is known to grow wild in marginal land spots and mountains of Amman, Madaba and Karak. The flowering period of this plant is short, extending from April to May (Al-Eisawi, 1998).
Phytochemical investigations on this plant were very limited, most of them dealt with determining the secondary metabolites of the rhizomes. Two new isoflavones named nigricin and nigricanin were isolated from I. nigricans rhizomes (Al-Khalil et al., 1994). In addition, the ethanol extracts of I. nigricans rhizomes afforded seven xanthones (Al-Khalil et al., 1995). A careful literature survey revealed that the essential oil of I. nigricans has not been studied before. Thus, this current investigation was aimed to study the composition of the essential oil of the aerial blooming parts of I. nigricans at different flowering stages and the composition of the oil of the rhizomes at the post flowering stage. In addition, the antimicrobial activities of the ethanol and aqueous extracts obtained from the rhizomes were evaluated.
2 Experimental
2.1 Plant material
The aerial blooming parts of I. nigricans at the pre-flowering, flowering, and post flowering stages and the rhizomes at the post flowering stage were collected from Shafa-badran, Amman, during the period extending from March to April, 2011. A voucher specimen (BAU/011/II 3010) has been deposited in the Department of Applied Sciences, Faculty of Engineering Technology, Al-Balqa Applied University, Amman, Jordan.
2.2 Preparation of the volatile oils and extracts
Each 340 g of fresh blooming aerial parts at different flowering stages and the rhizomes during the post flowering stage, was coarsely powdered and then hydrodistilled separately using a Clevenger apparatus for 3 h. The extraction was repeated twice and the obtained oils were pooled separately, dried over anhydrous sodium sulfate (Na2SO4) and stored at 4 °C in amber glass vials until analysis.
For the preparation of the ethanol and aqueous extracts, each 10 g plant material was refluxed with 100 ml solvent (70% ethanol or water, respectively), kept overnight, filtered, solvent evaporated and dissolved in DMSO (1 g extract in 10 ml DMSO). The DMSO solutions were then used in biological tests at different concentrations.
2.3 GC–MS and GC–FID analysis
About 1 μl aliquot of each oil sample, diluted to 5 μl in GC grade n-hexane, was subjected to GC/MS analysis. The GC/MS analysis was performed using Varian Chrompack CP-3800 GC/MS/MS-200 (Saturn, Netherlands) equipped with DP-5 (5% diphenyl, 95% dimethyl polysiloxane) GC capillary column (30 m × 0.25 mm i.d., 0.25 μm film thicknesses), with helium as a carrier gas (flow rate 0.9 ml/min). The actual temperature in MS source was 180 °C and the ionization voltage was 70 eV. The column temperature was kept at 60 °C for 1 min (isothermal), and programmed to 246 °C at a rate of 3 °C/min, and kept constant at 246 °C for 3 min (isothermal). A hydrocarbon mixture of n-alkanes (C8–C20) was analyzed separately by GC/MS under the same chromatographic conditions using the same DP-5 column.
For the quantitative analysis (% area), a Hewlett–Packard HP-8590 gas chromatograph equipped with a split–splitless injector (split ratio 1:50) and an FID detector was used. The column was an optima-5 (5% diphenyl, 95% dimethyl polysiloxan) fused silica capillary column (30 m × 0.25 mm, 0.25 film thickness). The temperature of the oven was increased at a rate of 10 °C/min from 60 to 250 °C and then held constant at 250 °C for 5 min. The temperatures of the injector and detector were maintained at 250 and 300 °C, respectively. The relative peak areas of the oil components were measured and then used to calculate the concentration of the detected compounds. Each sample was analyzed twice.
2.4 Identification of the components
The components of the essential oils at different flowering stages were identified using the built in libraries (Nist Co. and Wiley Co., USA) and by comparing their calculated retention indices relative to (C8–C20) n-alkanes literature values measured with columns of identical polarity (Adams, 2001), or with authentic samples. The compounds, α- and β-pinenes, p-cymene, limonene, linalool (Fluka, Buchs, Switzerland) and sabinene hydrate (Sigma–Aldrich, Buchs, Switzerland) were used as reference substances in GC/MS analysis. GC-grade hexane and analytical reagent grade anhydrous Na2SO4 were purchased from Scharlau (Barcelona, Spain) and UCB (Bruxelles, Belgium).
2.5 Antibacterial activity
Overnight bacterial cultures of Gram-negative bacteria, Escherichia coli (ATCC 8739), Klebsiella pneumoniae (ATCC 10031), Salmonella typhi (ATCC 6539) and Gram-positive bacteria, Staphylococcus aureus (ATCC 6538P), Bacillus subtilis (ATCC 6633), Micrococcus luteus (ATCC 9341), were used to evaluate the antibacterial properties of the ethanol and aqueous extracts of the rhizomes. Bacteria were grown in nutrient broth medium (Oxoid, UK). Batches of medium (20 ml) were inoculated from fresh culture slopes and incubated overnight at 37 °C. Long-term maintenance was on nutrient agar plates at 4 °C.
The antimicrobial activity of the extracts was initially assessed using the agar diffusion method as recommended by the Clinical Laboratory Institute (CLSI). Impregnated discs were prepared by the addition of 20 μl of the extract (stock concentration of 1 mg/ml ethanol) to “susceptibility blank discs” (Oxoid, UK). These were subsequently applied to the inoculated agar plates and then incubated at 37 °C for 24 h.
3 Results and discussion
GC/MS analysis of the essential oils of I. nigricans – obtained from aerial fresh blooms at different flowering stages and rhizomes at the post flowering stage – led to the identification of a total of 97 compounds (Table 1). In the essential oil obtained from aerial blooming parts at the pre-flowering stage, 24 compounds amounting to 97.98% of the total oil content were identified. Terpenoids accounted for 52.35% of the total oil composition, 40.93% of which were attributed to oxygenated monoterpenes. Among the oxygenated monoterpenes detected, pipertenone oxide was the major contributor accounting for 32.42% of the total oil. Other components detected in this fraction included cis-sabinene hydrate (2.90%), linalool (1.45%) and terpinene-4-ol (1.22%). Aliphatic hydrocarbons and their oxygenated derivatives represented 45.62% of total oil content. Also n-tetradecanol (19.86%), n-nonanal (9.67%) and n-nonadecane (7.72%) were among the compounds detected in this fraction. Sesquiterpene hydrocarbons were detected at this stage (9.32% of the total oil content) with α-humulene (3.31%), trans-E-caryophyllene (2.86%) and germacrene D (1.41%) being the major contributors to the fraction. Oxygenated sesquiterpenes accounted only for 2.11% of the total oil content and were represented by germacrene D-4-ol and α-cadinol (1.15% and 0.96%, respectively).
| No. | RI (reported) | RI (exp) | Compound | % Pre-flower | % Flowering | % Post-flower | % Rhizomes |
|---|---|---|---|---|---|---|---|
| 1 | 836 | 832 | Isovaleric acid | – | – | 0.28 | – |
| 2 | 939 | 934 | α-Pinene | – | – | – | 24.05 |
| 3 | 954 | 952 | Camphene | – | – | – | 1.04 |
| 4 | 968 | 955 | Verbenene | – | – | – | 0.36 |
| 5 | 979 | 980 | β-Pinene | – | – | – | 7.99 |
| 6 | 979 | 980 | 1-Octen-3-ol | 0.45 | – | – | – |
| 7 | 986 | 985 | 6-Methyl-5-hepten-2-one | – | 0.26 | – | – |
| 8 | 1017 | 1018 | α-Terpinene | – | – | 3.37 | – |
| 9 | 1025 | 1026 | p-Cymene | – | – | 0.87 | 0.43 |
| 10 | 1029 | 1031 | Limonene | – | – | – | 2.02 |
| 11 | 1031 | 1035 | 1,8-Cineol | 0.59 | – | – | 0.25 |
| 12 | 1068 | 1072 | n-Octanol | – | 0.80 | – | – |
| 13 | 1070 | 1076 | cis-Sabinene hydrate | 2.90 | – | – | 4.43 |
| 14 | 1091 | 1092 | Dehydrolinalool | – | – | – | 0.88 |
| 15 | 1097 | 1098 | Linalool | 1.45 | – | – | – |
| 16 | 1100 | 1099 | n-Undecane | – | – | 1.12 | – |
| 17 | 1097 | 1101 | Camphenone | – | – | – | 0.41 |
| 18 | 1101 | 1107 | n-Nonanal | 9.67 | 6.87 | 0.80 | – |
| 29 | 1108 | 1112 | cis-Rose oxide | – | – | – | 0.32 |
| 20 | 1122 | 1123 | exo-Fenchol | – | – | – | 0.88 |
| 21 | 1127 | 1129 | Methyloctanoate | 1.66 | 0.45 | 0.84 | – |
| 22 | 1126 | 1130 | α-Campholenal | – | – | – | 0.47 |
| 23 | 1141 | 1146 | cis-Verbenol | – | – | 0.45 | 1.63 |
| 24 | 1145 | 1150 | trans-Verbenol | 1.15 | – | 3.08 | 3.62 |
| 25 | 1145 | 1155 | Geijerene | – | – | – | 0.37 |
| 26 | 1162 | 1163 | E-2-Nonenal | – | 0.66 | – | – |
| 27 | 1165 | 1167 | Pinocarvone | – | – | 0.31 | 0.33 |
| 28 | 1169 | 1177 | Borneol | 0.61 | – | 0.49 | – |
| 29 | 1169 | 1174 | n-Nonanol | – | 0.23 | – | – |
| 30 | 1170 | 1176 | p-Mentha-1,5-dien-8-ol | – | – | – | 1.94 |
| 31 | 1175 | 1181 | cis-Pinocamphone | – | – | – | 0.25 |
| 32 | 1177 | 1185 | Terpinen-4-ol | 1.22 | – | – | 0.64 |
| 33 | 1200 | 1199 | n-Dodecane | – | – | 8.95 | – |
| 34 | 1189 | 1200 | α-Terpineol | 0.60 | – | – | 6.56 |
| 35 | 1202 | 1202 | n-Decanal | – | 1.39 | – | 0.26 |
| 36 | 1215 | 1214 | iso-Dihydrocarveol | – | – | – | 0.49 |
| 37 | 1217 | 1224 | trans-Carveol | – | – | 0.36 | 0.25 |
| 38 | 1226 | 1230 | Citronellol | – | – | – | 0.98 |
| 39 | 1270 | 1267 | n-Decanol | – | – | 1.69 | – |
| 40 | 1288⁎ | 1290 | Dihydroedulan I | – | – | 0.33 | – |
| 41 | 1300 | 1299 | n-Tridecane | 0.86 | 1.30 | 33.92 | – |
| 42 | 1299 | 1307 | Carvacrol | – | 1.59 | – | – |
| 43 | 1329 | 1327 | Silphiperfol-5-ene | – | – | – | 0.45 |
| 44 | 1337 | 1335 | Persilphiperfol-5-ene | – | – | – | 0.40 |
| 45 | 1348 | 1347 | 7-Epi-silphiperfol-5-ene | – | – | – | 3.51 |
| 46 | 1361 | 1358 | Silphiperfol-4,7 (14)-diene | – | – | – | 0.19 |
| 47 | 1369 | 1366 | Piperitenone oxide | 32.42 | 4.00 | 0.96 | – |
| 48 | 1370 | 1370 | n-Undecanol | – | – | 15.01 | – |
| 49 | 1379 | 1376 | Silphiperfol-6-ene | – | – | – | 1.46 |
| 50 | 1384 | 1386 | Hexyl hexanoate | – | – | – | 1.15 |
| 51 | 1391 | 1391 | β-Elemene | 0.44 | 0.17 | – | – |
| 52 | 1389 | 1392 | 1-Tetradecene | – | – | 0.33 | – |
| 53 | 1400 | 1400 | n-Tetradecane | – | – | 5.86 | – |
| 54 | 1409 | 1412 | Lauric aldehyde | – | 0.27 | – | – |
| 55 | 1419 | 1422 | trans-E-caryophyllene | 2.86 | 0.34 | – | 0.08 |
| 56 | 1434 | 1425 | β-Gurjunene | – | – | – | 0.25 |
| 57 | 1455 | 1443 | Khausimine | – | – | 0.29 | – |
| 58 | 1455 | 1450 | Geranyl acetone | – | 1.08 | – | – |
| 59 | 1455 | 1459 | α-Humulene | 3.31 | – | – | – |
| 60 | 1455 | 1460 | trans-Carvyl propaonate | – | 4.39 | – | – |
| 61 | 1460 | 1462 | Alloaromandrene | – | – | 1.87 | – |
| 62 | 1466 | 1466 | 9-Epi-E-caryophyllene | – | – | 0.32 | – |
| 63 | 1454 | 1467 | α-Neoclevene | – | – | – | 0.43 |
| 64 | 1471 | 1478 | n-Dodecanol | – | 0.42 | – | – |
| 65 | 1485 | 1485 | Germacrene D | 1.41 | 0.46 | 0.90 | – |
| 66 | 1500 | 1503 | n-Pentadecane | 3.73 | 2.24 | 4.18 | 1.06 |
| 67 | 1510 | 1510 | Tridecanal | – | 0.16 | – | – |
| 68 | 1508 | 1514 | Silphiperfol-6 α -ol | – | – | – | 0.18 |
| 69 | 1512 | 1519 | Cameroonan-7 α -ol | – | – | – | 1.92 |
| 70 | 1522 | 1523 | 7-Epi-α-salinene | 0.85 | 0.50 | – | – |
| 71 | 1529 | 1524 | trans-Calamanene | – | – | – | 0.60 |
| 72 | 1523 | 1526 | β-Sesquiphellandrene | 0.45 | 0.21 | – | – |
| 73 | 1521 | 1527 | Silphiperfol-7 β -ol | – | – | – | 0.19 |
| 74 | 1522 | 1535 | α-Irone | – | – | – | 1.42 |
| 75 | 1549 | 1547 | 1,10-Decanediol | – | – | 1.06 | – |
| 76 | 1572 | 1571 | n-Tridecanol | – | 0.21 | 1.49 | – |
| 77 | 1576 | 1581 | Germacrene D-4-ol | 1.15 | 1.14 | – | – |
| 78 | 1583 | 1585 | Caryophyllene oxide | – | 0.40 | – | 2.84 |
| 79 | 1585 | 1600 | Globulol | – | – | – | 0.52 |
| 80 | 1600 | 1601 | n-Hexadecane | 1.66 | 1.92 | 0.38 | – |
| 81 | 1608 | 1613 | Humulene epoxide II | – | – | – | 1.25 |
| 82 | 1619 | 1620 | 1,10-Di-epi-cubenol | – | 0.22 | – | – |
| 83 | 1626 | 1622 | silphiperfol-6-en-5-one | – | – | – | 1.19 |
| 84 | 1631 | 1627 | γ-Eudesmol | – | – | – | 1.49 |
| 85 | 1640 | 1647 | τ-Cadinol | – | 0.23 | – | – |
| 86 | 1654 | 1661 | α-Cadinol | 0.96 | 1.00 | – | 0.54 |
| 87 | 1667 | 1668 | Intermedeol | – | – | – | 0.25 |
| 88 | 1673 | 1675 | n-Tetradecanol | 19.88 | 16.75 | 0.56 | – |
| 89 | 1675 | 1677 | Valeranone | – | – | – | 2.94 |
| 90 | 1700 | 1703 | n-Heptadecane | – | 18.91 | 3.05 | – |
| 91 | 1730 | 1742 | Iso-longifolol | – | – | – | 0.53 |
| 92 | 1774 | 1773 | n-Pentadecanol | – | 0.59 | – | – |
| 93 | 1800 | 1800 | n-Octadecane | – | 3.54 | – | – |
| 94 | 1830 | 1832 | Isopropyl tetradecanoate | – | – | – | 12.87 |
| 95 | 1870 | 1876 | n-Hexadecanol | – | 4.50 | – | – |
| 96 | 1900 | 1905 | n-Nonadecane | 7.72 | 17.10 | – | 0.52 |
| 97 | 2000 | 2000 | Eicosane | – | 2.73 | – | – |
| Monoterpene hydrocarbons | – | – | 3.37 | 35.47 | |||
| Oxygenated monoterpenes | 40.93 | 4.00 | 5.65 | 24.32 | |||
| Sesquiterpene hydrocarbons | 9.32 | 1.69 | 3.39 | 7.14 | |||
| Oxygenated sesquiterpenes | 2.11 | 4.07 | – | 15.25 | |||
| Aliphatic hydrocarbons and their derivatives | 45.62 | 81.28 | 79.52 | 15.86 | |||
| Aromatic monoterpenes | – | 5.98 | 0.87 | 0.43 | |||
| Total identified | 97.98 | 97.02 | 92.79 | 98.47 | |||
RI (reported): Retention Index on DB-5 column in reference to n-alkanes as reported in Adams (2001); RI (exp): experimentally obtained Retention Index.
Upon flowering, the chemical composition changed greatly. A total of 35 compounds were identified comprising 96.56% of the total oil constituents. Aliphatic hydrocarbons and their oxygenated derivatives had the highest contribution at this stage, accounting for 81.28% of the total oil content. The major contributors to this fraction were n-heptadecane (18.91%), n-nonadecane (17.10%), n-tetradecanol (16.75%) and n-nonanal (6.87%). As compared to the previous stage, the total terpenoid content dropped to 9.30% of the total oil content. Piperitenone oxide was the only oxygenated monoterpene detected in the essential oil of fresh flowering I. nigricans at a concentration of 4.00%. The concentration of sesquiterpene hydrocarbons decreased compared to the previous pre-flowering stage (1.23%, 9.32%, respectively), 7-epi-α-salinene was the major component among the five sesquiterpene hydrocarbons detected during fresh flowering stage (0.5%). The amount of oxygenated sesquiterpenoids increased slightly to 4.07% of the total oil content, with germacrene D-4-ol (1.14%) and α -cadinol (1.00%) being detected as major components of this fraction. Interestingly, during the pre- and fresh flowering stages of the plant's life, monoterpene hydrocarbons were completely absent. However, two aromatic monoterpenes were detected in the oil obtained at the fresh flowering stage of the plant life amounting to 5.98%. These two compounds were carvylpropanoate (4.39%) and carvacrol (1.59%).
Analysis of the volatile oil obtained from fresh aerial blooming parts at the post flowering stage led to the identification of 29 components accounting for 92.79% of the whole oil. Aliphatic hydrocarbons and their oxygenated derivatives (79.52%) predominated this oil with n-tridecane (33.92%), n-undecanol (15.01%) and n-dodecane (8.95%) being the main contributors to this fraction. Terpenoids accounted for 12.40% of the total oil content. Oxygenated monoterpenes accounted for 5.62% of the total oil content, trans-verbenol (3.08%) was the most abundant component among the five oxygenated monoterpenes detected at this stage. It is noteworthy that the concentration of piperitenone oxide, the major oxygenated monoterpene detected during the pre- and fresh flowering stages of I. nigricans, dropped to a minimum level (0.96%) at the post flowering stage. Sesquiterpene hydrocarbons represented 3.39% of the total oil content. The most abundant components were alloaromandrene (1.87%) and germacrene D (0.90%). Oxygenated sesquiterpenoids were completely absent in this stage while aromatic monoterpenoids were represented by p-cymene (0.87%). Investigation of the essential oil of I. nigricans at the post flowering stage revealed the presence of monoterpene hydrocarbons that were represented only by α-terpinene which amounted to 3.37% of the total oil content.
The findings of the current investigation clearly indicated that the chemical composition of I. nigricans varies significantly with the physiological stage of the plant. In addition, remarkable differences were found between the oils obtained from the blooming parts and rhizomes at the post-flowering stage. Interestingly, the essential oil obtained from rhizomes was found to be rich in terpenoids (82.18%), especially monoterpene hydrocarbons (35.47%). The oil obtained from aerial blooms at the post flowering stage was dominated by aliphatic hydrocarbons (75.92%) and only 12.40% of the total oil contents were attributed to different classes of terpenoids. Monoterpenoids detected in the rhizomes oil basically belonged to the pinane (α- and β-pinenes, 24.05% and 7.99%, respectively) skeleton. Other monoterpenoid classes were also detected in rhizome's oil including p-menthane (limonene, 2.02%), camphane (camphene 1.04%) and aromatic monoterpenes (p-cymene, 0.43%). Alpha terpinene (3.37%), belonging to p-menthane skeleton, was the only monoterpene hydrocarbon detected in the aerial blooms during the post flowering stage. Oxygenated monoterpenes represented the second major fraction in rhizome's oil, comprising about 24.32% of the total oil. This fraction was represented by α-terpineol (6.56%) and cis-sabinen hydrate (4.43%). It is worth noticing that the content of oxygenated monoterpenes in the blooming aerial parts at the post flowering stage was much lower than that in the rhizomes (5.65%, 24.32%, respectively) with trans-verbenol being the major component in this class. Sesquiterpenoids were detected in much lower concentrations as compared to monoterpenoids (22.39%, 59.79%, respectively). Sesquiterpene hydrocarbons were detected in lower concentrations compared to their oxygenated derivatives (7.14%, 15.25%, respectively). Sesquiterpene hydrocarbons contained a mixture of silphiperfol derivatives amounting to 6.01% of all sesquiterpene hydrocarbons, among them, 7-epi-silphiperfol-5-ene (3.51%) and silphiperfol-6-ene (1.46%) were detected as major components. Oxygenated sesquiterpenoids were dominated by valeranone (2.94%) and caryophyllene oxide (2.84%). Interestingly, the oxygenated sesquiterpenoid α-irone was detected in the rhizomes's volatile oil amounting to 1.42% of the total oil content. Generally, irones are known to be formed due to oxidative degradation of iridals during rhizomes aging (Roger et al., 2010). Aliphatic hydrocarbons were detected in much lower concentrations as compared to the aerial blooming parts at different flowering stages (15.86%). Fig. 1 illustrates the variation of the essential oil composition between the blooms at different flowering stages and the oil of the rhizomes at the post flowering stage.
Variation in the chemical composition of I. nigricans essential oil at different flowering stages.
The present investigation revealed that I. nigricans contained different types of terpenoids with a wide range of concentrations, most of which were accumulated in the rhizomes. It is well established that plants synthesize different classes of terpenoids (C5–C40) at different stages of their growth for various biological purposes (Baby et al., 2009). The biosynthesis of monoterpenes is known to require relatively low molecular weight precursors and enzymatic activity, making their biosynthesis more feasible as compared to that of sesquiterpenes. However, due to their high vapor pressure, monoterpenes are easily emitted to the surrounding space, which might help in providing a chemical defense to the plants (Baby et al., 2009). Sesquiterpenoids possibly have a preferential role as defense compounds against direct attacks on the plant (rhizomes) by pathogens, nematodes, degrading microbes and other biotic/abiotic factors (Baby et al., 2009). The role that these terpenoids play as defense agents may account for their accumulation in I. nigricans rhizomes.
The aqueous and ethanol extracts of I. nigricans rhizomes have been screened for their antimicrobial activities according to the cup method using agar (Panda et al., 2009) and the disc method (Ghalem and Mohamed, 2009) against gram negative and gram positive bacteria represented by E. coli (ATCC 8739), K. pneumoniae (ATCC 10031), S. typhi (ATCC 6539), S. aureus (ATCC 25923), B. subtilis (ATCC 6633), M. luteus (ATCC 9341), respectively. All extracts were found to be inactive at the stock solution concentration of 1 mg/1 ml.
Acknowledgment
The author wishes to thank Mr. Ismaeil Abaza, Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Jordan for his assistance during the course of this study.
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