Abstract
Volatile oils from the leaves, flowers and rhizomes of Dietes bicolor (Steud.) Sweet ex Klatt (Iridaceae) were analyzed using GLC/FID and GLC/MS. A total of 84 compounds were identified accounting for 94.65, 95.63 and 87.09% in the hydrodistilled oils from flowers, leaves and rhizomes, respectively. Spathulenol (48.44%) represented the major component in the leaf oil, followed by dihydro-edulan I (6.25%), cubenol (6.00%) and τ-cadinol (5.90%). For the flower and rhizome oils, fatty acids, their esters, aliphatic hydrocarbons and their derivatives predominate. The antimicrobial activity of both leaf and flower oils was investigated against four bacteria in addition to four fungi using the micro-broth dilution method. The leaf oil showed a more potent antimicrobial activity as compared to the flower oil against most of the assessed bacteria and fungi, with higher activities against Gram- positive organisms showing MIC values of 115 and 460 μg/ml for Bacillus subtilis and Streptococcus pneumonia, respectively. Gram-negative bacteria were generally less susceptible (MIC > 2 mg/ml for both oils against Escherichia coli) and being completely ineffective against Pseudomonas aeruginosa. A relevant antifungal potency of the leaf oil against Geotrichum candidum and Syncephalastrum racemosum was also observed with MIC values of 115 and 920 μg/ml, respectively.
1 Introduction
Essential oils represent a fascinating mixture of plant secondary metabolites that have gained interest in both industrialized as well as developing countries owing to their remarkable therapeutic and commercial benefits. Essential oils, also known as volatile or ethereal oils, consist of a complex mixture of volatile components biosynthesized by living organisms [1]. They play a crucial role for plants such as for cooling, attraction or repellence of arthropods, in addition to protection against microbial infections [2]. Besides, they possess various biological and pharmacological properties such as antimicrobial, anti-inflammatory, antioxidant as well as cytotoxic activities [3–10], which are the main reason for being the core of their widely use in aromatherapy. Moreover, the emergence of antibiotic resistance among microorganisms constitutes a leading driving force to search for new and safe natural antimicrobials exemplified by the essential oils that can not only reduce the appearance but also the dissemination of the resistant pathogens [11].
The family Iridaceae represents one of the most important plant families for horticulture comprising about 72 genera and 2000 species [12, 13]. Members of the Iridaceae are perennial herbs or subshrubs with rhizomes, bulbs, or corms [14] that are widely grown as ornamentals owing to their beautiful, magnificent, and often fragrant flowers [15]. Traditionally, plants belonging to Iridaceae have been used for the alleviation of cold, flu, toothache and bruises, in addition to being effective in the treatment of malaria [16].
The family produces a rich diversity of secondary metabolites that includes alkaloids, isoflavonoids, flavonoids, monoterpenes, triterpenoids, naphthoquinones, anthraquinones, naphthalene derivatives, xanthones and simple phenolics [17, 18] to which many pharmacological and biological activities embracing antibacterial, antiprotozoal, antiviral, antioxidant, antinociceptive, anti- inflammatory, cytotoxic, and immunomodulatory activities have been attributed [19, 20].
The analysis of essential oils obtained from a number of Iridaceae revealed the prevalence of oxygenated monoterpenes, aliphatic hydrocarbons and their derivatives, fatty acids and their esters, in addition to monoterpene hydrocarbons of the pinane skeleton. (+)-(Z)-γ-Irone and (+)-(Z)-α-irone were found to be predominating the essential oils of Iris rhizomes [15, 21, 22]. Irones, with a pleasant violet-like odor, are gradually formed in the ageing rhizomes through oxidative degradation of iridals contributing to the attractive fragrance of Iris oil, one of the most precious ingredients used in perfumery [23–25].
Dietes bicolor (Steud.) Sweet ex Klatt is an evergreen plant with rhizomes, native to South Africa [26]. It is commonly known as Yellow Wild Iris, Butterfly Iris or Peacock Flower, characterized by having sword-like leaves and white to creamy yellow Iris-like flowers with three dark spots, each surrounded by an orange outline [27–29]. The genus name “Dietes” is derived from the Greek word “dis” meaning twice and the Latin one “etum” meaning an association for the close relationship to both Moraea and Iris. The species name “bicolor” means two-colored [26]. In our ongoing research regarding the phytochemistry and pharmacology of D. bicolor, a widely unexplored South African plant, we have recently reported antimicrobial activity of the crude alcoholic extracts of leaves, flowers and rhizomes from D. bicolor, which lack measurable cytotoxicity to mammalian cells [30]. Although current literature was thoroughly screened, we did not find information on the chemical composition and the biological activity of D. bicolor and to the best of our knowledge; no reports have addressed the antimicrobial activity and the chemical composition of the essential oils of flowers, leaves, and rhizomes from D. bicolor.
In this study, a comparative analysis of the volatile oil composition of different parts of D. bicolor was carried out and the antimicrobial activity of the leaf and flower essential oils was assessed against various Gram-positive and Gram-negative bacterial strains in addition to four fungi aiming to explore their antibacterial and antifungal activities.
2 Materials and methods
2.1 Plant material
Fresh leaves, flowers and rhizomes of D. bicolor (Steud.) Sweet ex Klatt were collected in April 2011 from a private ornamental garden, Cairo, Egypt. The plant was kindly identified and authenticated by Mrs Therese Labib, Consultant of Plant Taxonomy at the Ministry of Agriculture and El-Orman Botanical Garden, Giza, Egypt and Prof. Dr. Mohamed El-Gebaly, Department of Botany, National Research Centre (NRC), Giza, Egypt. A voucher specimen was deposited in the herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University (voucher number: IA-10411).
2.2 Chemicals
All chemicals utilized in the chemical analysis were purchased from AppliChem (Darmstadt, Germany), Fluka (Buchs, Switzerland) and Sigma (Sigma Aldrich, Sternheim, Germany).
2.3 Essential oil isolation
The essential oils of the fresh leaves, flowers and rhizomes of D. bicolor were obtained individually by hydrodistillation (200 g each) for 6 h using a Clevenger-type apparatus. The oils were collected over n-hexane and then dried over anhydrous sodium sulfate and kept in separated sealed vials at –30 °C for further analyses. The yield in % (w/w) was determined in triplicate and based on the initial plant dry weight.
2.4 GLC/FID analysis
The GLC-FID analysis was performed using a Varian 3400 apparatus (Varian, Darmstadt, Germany) equipped with DB-5 fused bonded column (30 m × 0.25 mm i.d. × 0.25 μm film thickness) (Ohio Valley, OH, USA) and FID detector. Helium was used as a carrier gas at a flow rate of 2 ml/min. The initial column temperature was kept at 45 °C for 2 min (isothermal) and programmed to 300 °C at a rate of 4 °C/min, and kept constant at 300 °C for 20 min (isothermal). Detector and injector temperatures were 300 and 250 °C, respectively. The split ratio was 1:20. PeakSimple® 2000 chromatography data system (SRI Instruments, CA, USA) was used for the recording and integration of the chromatograms. Average areas under the peaks of three independent chromatographic runs were used for calculating the % composition of each component.
2.5 GLC/MS analysis
Mass spectra were recorded using a Hewlett-Packard gas chromatograph (GC 5890 II; Hewlett-Packard, Bad Homburg, Germany) equipped with a split-splitless injector and an FID detector. A DB-5 column was used and the operating conditions were similar to those applied for the GLC/FID. The capillary column was directly coupled to a quadrupole mass spectrometer (SSQ 7000; Thermo-Finnigan, Bremen, Germany). The injector and detector temperatures were maintained at 250 and 300 °C, respectively. Helium carrier gas flow rate was 2 ml/min. All the mass spectra were recorded applying the following conditions: filament emission current, 100 mA; ionization voltage, 70 eV; ion source, 175 °C. Diluted samples (0.5% v/v) were injected with split mode (split ratio 1:15). Essential oil components were identified by comparing their retention indices and mass spectra with those listed in the commercial MS libraries: (NIST Mass Spectral Library (December 2005), Wiley Registry of Mass Spectral Data 8th edition), our own laboratory MS library in Heidelberg built from spectra corresponding to pure substances and components of known essential oils, the co-injection with an authentic sample, whenever possible and reported literature data [6, 8, 31].
2.6 Microbial strains
The antimicrobial activity of essential oils from leaves and flowers was evaluated against standard microbial species including the Gram-positive bacteria Streptococcus pneumoniae (RCMB 000101), Bacillus subtilis (RCMB 000102) and the Gram negative bacteria, Escherichia coli (RCMB 000107) and Pseudomonas aeruginosa (RCMB 000104). Moreover, the antimicrobial activity was assessed against the fungi Aspergillus fumigatus (RCMB 002007), Syncephalastrum racemosum (RCMB 005005), Candidaalbicans (RCMB 005008) and Geotrichum candidum (RCMB 052009). All microorganisms and culture media were supplied by the Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University, Nasr City, Cairo, Egypt.
2.7 Inocula preparation
The bacterial and fungal cultures were prepared as follows: the microbial suspension equivalent to the turbidity of 0.5 McFarland (108 CFU/ml) standard was taken from a fresh subculture of the tested bacteria in Mueller Hinton Broth (MHB) and the tested fungi in Sabouraud dextrose broth (SDB), then the suspensions were diluted to 106 CFU/ml using MHB for bacteria and SDB for fungi. The antimicrobial activity was determined using the broth microdilution assay following the methods described by the Clinical and Laboratory Standards Institute (CLSI) for bacteria [32] and yeasts [33].
2.8 Assessment of viability
The adjusted microbial inoculum (100 μl) was added to each well of a sterile 96-well flat-bottomed microtiter plate containing the serial dilutions of the tested samples (100 μl/well). Consequently, an inoculum concentration of 5 × 105 CFU/ml was obtained in each well. Three wells containing microbial suspensions with no sample using only DMSO employed for dissolving the tested samples (growth control) and two wells containing only media (background control) were included in this plate. Ampicillin, gentamicin and amphotericin B were used as positive controls for Gram-positive, Gram-negative bacteria and fungi, respectively. The tested samples as well as the used standards were used in the concentration range of 460–5000 μg/ml. Optical densities were measured at 600 nm after 24 h at 37 °C for bacteria and after 48 h at 28 °C for fungi using a multi-detection microplate reader (Sunrise-Tecan, Durham, North Carolina, USA). The percentage of growth at each sample concentration was calculated using the following equation: % growth = [(OD600 of wells containing the test sample/OD600 of the sample-free well) × 100] after subtraction of background ODs (ODs of microorganism-free wells). Data were measured in triplicates (n = 3) and expressed as means ± S.D.
2.9 Determination of minimum inhibitory concentration (MIC)
The MIC value was assessed applying the micro-broth dilution method according to Kaya et al. [34]. The adjusted bacterial inoculum (50 μl/well, 106 CFU/ml) was added to each well of a sterile U based microtiter plate containing the target concentrations of samples and standards (50 μl/well). Thus, an inoculum concentration of 5 × 105 CFU/ml was obtained in each well. Then, the plates were incubated at 37 °C for 24 h for bacteria and at 28 °C for 48 h for fungi. The lowest concentration inhibiting the visible microbial growth was determined as the MIC value of the isolate.
3 Results and discussion
GLC analyses of the volatile oils from D. bicolor flowers, leaves and rhizomes were carried out aiming to assess the differences in their chemical composition and their subsequent effect on the antimicrobial activity. Generally, all the oils had a yellow color displaying a characteristic odor. Nevertheless, their yields varied, to some extent, being 0.005 and 0.003% (w/w) in fresh leaves and flowers, respectively. However, only traces of volatile constituents were detected in the rhizomes. GLC/MS analysis revealed the presence of a total of 84 components, accounting for 94.65, 95.63 and 87.09% of total flower, leaf, and rhizome oils, respectively. A list of the identified components is documented in Table 1 together with their retention indices (RIs) on a DB-5 column.
Composition of the essential oils isolated from the flowers (DBF), leaves (DBL) and rhizomes (DBR) of Dietes bicolor.
| Component | RIexpa | RIlitb | Content (%)c | Identificationd | |||
|---|---|---|---|---|---|---|---|
| DBF | DBL | DBR | |||||
| 1. | Ethylcyclohexane | 826 | 827 | tr | – | – | MS, RI |
| 2. | 2-Hexen-1-al | 845 | 847 | tr | – | – | MS, RI |
| 3. | p-Xylene | 861 | 862 | tr | – | – | MS, RI |
| 4. | 3-Methyloctane | 864 | 867 | tr | – | – | MS, RI |
| 5. | 1-Methyl-(E)-4-ethylcyclohexane | 880 | 882 | tr | – | – | MS, RI |
| 6. | o-Xylene | 885 | 888 | tr | 0.55 | – | MS, RI |
| 7. | Heptanal | 894 | 895 | 2.78 | – | – | MS, RI |
| 8. | Methyl caproate | 920 | 922 | tr | – | – | MS, RI |
| 9. | n-Propylcyclohexane | 924 | 924 | tr | – | – | MS, RI |
| 10. | 3,5-Dimethyloctane | 929 | 924 | tr | – | – | MS, RI |
| 11. | 2,6-Dimethyloctane | 930 | 933 | tr | – | – | MS, RI |
| 12. | Benzaldehyde | 956 | 957 | tr | – | – | MS, RI |
| 13. | m-Ethyltoluene | 958 | 959 | tr | tr | – | MS, RI |
| 14. | o-Ethyltoluene | 973 | 974 | – | tr | MS, RI | |
| 15. | 6-Methyl-5-heptene-2-one | 987 | 988 | 6.92 | – | – | MS, RI |
| 16. | 1,2,4-Trimethylbenzene | 988 | 990 | – | tr | – | MS, RI |
| 17. | 2-Methylnonane | 963 | 962 | tr | – | – | MS, RI |
| 18. | 1,3,5-Trimethylbenzene | 993 | 994 | 0.42 | tr | – | MS, RI |
| 19. | Decane | 1001 | 1000 | 0.88 | – | – | MS, RI |
| 20. | (E,E)-2,4-Heptadienal | 1012 | 1011 | tr | – | – | MS, RI |
| 21. | 1,2,3-Trimethylbenzene | 1023 | 1023 | tr | tr | – | MS, RI |
| 22. | 3-Carene | 1023 | 1025 | – | tr | – | MS, RI |
| 23. | n-Butylcyclohexane | 1033 | 1033 | tr | – | – | MS, RI |
| 24. | 1-Methyl-3-propylbenzene | 1054 | 1058 | tr | – | – | MS, RI |
| 25. | (E)-Decahydronaphthalene | 1056 | 1056 | tr | – | – | MS, RI |
| 26. | (E)-2-Octenal | 1061 | 1060 | tr | – | – | MS, RI |
| 27. | Undecane | 1103 | 1100 | 0.48 | – | – | MS, RI |
| 28. | Nonanal | 1107 | 1107 | 1.61 | – | – | MS, RI |
| 29. | Methyl caprylate | 1127 | 1125 | 0.90 | – | – | MS, RI |
| 30. | (E)-2-Nonenal | 1161 | 1058 | tr | – | – | MS, RI |
| 31. | n-Caprylic acid | 1175 | 1177 | 0.95 | tr | 17.11 | MS, RI |
| 32. | Myrtenol | 1195 | 1195 | – | tr | – | MS, RI |
| 33. | Dodecane | 1200 | 1200 | tr | – | – | MS, RI |
| 34. | Decanal | 1206 | 1206 | tr | – | – | MS, RI |
| 35. | β-Cyclocitral | 1218 | 1219 | – | tr | – | MS, RI |
| 36. | β-Citral | 1244 | 1240 | tr | – | – | MS, RI |
| 37. | Citral | 1274 | 1273 | tr | – | – | MS, RI |
| 38. | Dihydro-edulan I | 1285 | 1286 | – | 6.25 | – | MS, RI |
| 39. | Thymol | 1295 | 1295 | tr | – | – | MS, RI |
| 40. | (E,E)-2,4-Decadienal | 1320 | 1320 | tr | – | – | MS, RI |
| 41. | Methyl caprate | 1327 | 1327 | 7.90 | – | – | MS, RI |
| 42. | α-Longipinene | 1347 | 1348 | – | tr | – | MS, RI |
| 43. | 1,1,6-Trimethyl-1,2-dihydronaphthalene | 1354 | 1352 | – | 0.50 | – | MS, RI |
| 44. | Capric acid | 1369 | 1369 | 21.12 | – | 46.14 | MS, RI |
| 45. | α-Copaene | 1377 | 1376 | – | 0.50 | – | MS, RI |
| 46. | Isolongifolene | 1388 | 1386 | – | tr | – | MS, RI |
| 47. | β-Cubebene | 1389 | 1388 | – | 2.06 | – | MS, RI |
| 48. | β-Elemen | 1389 | 1390 | – | 2.69 | – | MS, RI |
| 49. | β-Bourbonene | 1390 | 1390 | – | 4.06 | – | MS, RI |
| 50. | Ethyl caprate | 1396 | 1397 | tr | 3.72 | – | MS, RI |
| 51. | Tetradecane | 1400 | 1400 | tr | – | – | MS, RI |
| 52. | β-Caryophyllene | 1419 | 1419 | – | tr | – | MS, RI |
| 53. | α-Ionone | 1427 | 1430 | – | tr | – | MS, RI |
| 54. | β-Gurjunene | 1433 | 1433 | – | 2.06 | – | MS, RI |
| 55. | Aromadendrene | 1441 | 1441 | – | 0.30 | – | MS, RI |
| 56. | β-(Z) Guaiene | 1451 | 1451 | – | 0.36 | – | MS, RI |
| 57. | Geranyl acetone | 1455 | 1455 | tr | – | – | MS, RI |
| 58. | Allo-Aromadendrene | 1460 | 1461 | – | tr | – | MS, RI |
| 59. | τ-Muurolene | 1465 | 1466 | – | 1.88 | – | MS, RI |
| 60. | α-Amorphene | 1485 | 1484 | – | tr | – | MS, RI |
| 61. | Germacrene D | 1485 | 1485 | – | 1.57 | – | MS, RI |
| 62. | β-Selinene | 1490 | 1490 | – | tr | – | MS, RI |
| 63. | Valencene | 1496 | 1496 | – | tr | – | MS, RI |
| 64. | α-Selinene | 1498 | 1494 | – | tr | – | MS, RI |
| 65. | α-Muurolene | 1500 | 1500 | – | tr | – | MS, RI |
| 66. | τ-Cadinene | 1510 | 1510 | – | tr | – | MS, RI |
| 67. | δ-Cadinene | 1515 | 1514 | – | 0.52 | – | MS, RI |
| 68. | Calamenene | 1524 | 1524 | – | 0.53 | – | MS, RI |
| 69. | Methyl dodecanoate | 1527 | 1527 | 4.17 | – | – | MS, RI |
| 70. | Globulol | 1556 | 1556 | – | 3.48 | – | MS, RI |
| 71. | Dodecanoic acid | 1576 | 1578 | 22.84 | – | – | MS, RI |
| 72. | Spathulenol | 1578 | 1578 | – | 48.44 | – | MS, RI |
| 73. | Viridiflorol | 1591 | 1591 | – | tr | – | MS, RI |
| 74. | Cubenol | 1618 | 1616 | – | 6.00 | – | MS, RI |
| 75. | τ-Cadinol | 1635 | 1634 | – | 5.90 | – | MS, RI |
| 76. | β-Cadin-4-en-10-ol | 1645 | 1646 | – | tr | – | MS, RI |
| 77. | Methyl myristate | 1727 | 1727 | 2.26 | – | – | MS, RI |
| 78. | Myristic acid | 1768 | 1768 | 14.32 | – | – | MS, RI |
| 79. | Methyl palmitate | 1928 | 1927 | 2.12 | – | – | MS, RI |
| 80. | Chloropyrifos | 1957 | 1967 | – | 4.26 | – | MS, RI |
| 81. | n-Hexadecanoic acid | 1961 | 1961 | 4.33 | – | 23.84 | MS, RI |
| 82. | Methyl linoleate | 2101 | 2099 | 1.05 | – | – | MS, RI |
| 83. | Oleic Acid | 2114 | 2115 | 0.55 | – | – | MS, RI |
| 84. | Ethyl linoleate | 2151 | 2155 | – | tr | – | MS, RI |
| Monoterpene hydrocarbons | – | – | – | ||||
| Oxygenated monoterpenes | tr | tr | – | ||||
| Sesquiterpene hydrocarbons | – | 16.53 | – | ||||
| Oxygenated sesquiterpenes | – | 70.07 | – | ||||
| Others | 94.65 | 9.03 | 87.09 | ||||
| Total identified components | 94.65 | 95.63 | 87.09 | ||||
aRIexp, Retention index determined experimentally on a DB-5 column; bRIlit, published Kovats retention indices [6, 8, 31]; cContent (%), an average of three analyses; tr, trace; –, not detected; dIdentification, was based on comparison of the compounds’ mass spectral data (MS) and retention indices (RI) with those of NIST Mass Spectral Library (December 2005), Wiley Registry of Mass Spectral Data 8th edition and literature [6, 8, 31].
Analysis of the flower volatile constituents as shown in Figure 1(A) resulted in the identification of 47 components, in which fatty acids and their esters predominated with dodecanoic acid (22.84%), capric acid (21.12%), myristic acid (14.32%), and methyl caprate (7.90%) representing the most abundant components. The analysis of the leaf volatiles led to the identification of 42 compounds, in which spathulenol constituted the major compound accounting for 48.44% of the total oil composition. Moreover, dihydro-edulan I, cubenol and τ-cadinol, were found to exist in considerable quantities in the leaf oil representing 6.25, 6.00 and 5.90%, respectively as illustrated in Figure 1(B). The rhizome oil showed only three fatty acids, namely capric acid (46.14%), n-hexadecanoic acid (23.84%) and n-caprylic acid (17.11%) representing 87.09% of the total oil content as shown in Figure 1(C).
Gas chromatogram on a DB-5 column of the volatile oils from D. bicolor A) flowers, B) leaves and C) rhizomes.
The oxygenated sesquiterpene fraction of the leaf oil accounted for 70.07% and was mainly attributed to its high content of spathulenol. Furthermore, sesquiterpene hydrocarbons represented 16.53% of the total leaf oil with β-bourbonene being the most predominant compound. However, only traces of oxygenated monoterpenes were detected in the leaf oil, and monoterpene hydrocarbons were completely absent. Regarding the volatile constituents of flowers and rhizomes, fatty acids and their esters together with other aliphatic hydrocarbon compounds showed high prevalence representing nearly the total oil composition. However, monoterpenes, oxygenated monoterpenes, sesquiterpenes as well as oxygenated sesquiterpenes in these oils were hardly present or even absent. The remarkable chemical variations observed among the diverse components and their relative amounts in the volatile oil of the leaves, flowers and rhizomes can be used as a reliable marker for discriminating and ensuring the authenticity of the supplied oils [35].
The prevalence of fatty acids and their esters, as well as aliphatic hydrocarbons and their derivatives in the volatile constituents of flowers and rhizomes seems to be a common feature among members of Iridaceae [15, 22, 36]. Myristic acid was found to be the major component in the volatile constituents of the rhizomes of Syrian wild irises including, I.germanica, I. barnumae, I. bostrensis and I. aurantiaca [36]. In addition, lauric acid, capric acid, palmitic acid and its methyl ester, octadecanoic acid methyl ester and elaidic acid methyl ester were obtained from Syrian wild Iris species [36]. Moreover, the main components characterized in I. pseudacorus flower oil include hexadecanoic acid, heptacosane and 6-methyl-5-hepten-2-one whereas I. pseudacorus rhizome oil contained non-acosane, triacontane, octacosane and pentacosane as major components [22].
The antimicrobial activity of essential oils from D. bicolor leaves and flowers was evaluated against four bacterial strains and four fungi. The results are documented in Table 2. The essential oils of the leaves and flowers exhibited notable antimicrobial activity against most of the tested organisms showing minimum inhibitory concentrations (MICs) ranging from 115 to 1860 μg/ml. The leaf oil showed higher activity against the tested Gram-positive bacteria exceeding that of the flower oil with MIC values of 115 and 460 μg/ml for B. subtilis and S. pneumonia, respectively. Gram-negative bacteria were generally less susceptible, where both oils produced similar effects on E. coli (MIC > 2000 μg/ml) and being completely ineffective against P. aeruginosa.
The Half maximal inhibitory concentration (IC50) and minimum inhibitory concentrations (MICs) of essential oils from leaves and flowers of D. bicolor against different microorganisms using the broth micro-dilution method.
| Microorganisms | DBLa | DBFb | Ampicillin | Gentamicin | Amphotericin B | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| IC50c | MICd | IC50 | MIC | IC50 | MIC | IC50 | MIC | IC50 | MIC | |
| Gram-positive bacteria | ||||||||||
| Streptococcus pneumoniae (RCMB 000101) | 3.55±0.37 | 460 | >5 | 1860 | 0.72±0.08 | 0.25 | NTe | NT | NT | NT |
| Bacillus subtilis (RCMB 000102) | 0.88±0.11 | 115 | >5 | 920 | 0.72±0.07 | 0.25 | NT | NT | NT | NT |
| Gram-negative bacteria | ||||||||||
| Escherichia coli (RCMB 000107) | >5 | >2000 | >5 | >2000 | NT | NT | 8.85±0.9 | 1.95 | NT | NT |
| Pseudomonas aeruginosa (RCMB 000104) | NIf | NI | NI | NI | NT | NT | 115±10.4 | 15.63 | NT | NT |
| Fungi | ||||||||||
| Aspergillus fumigatus (RCMB 002007) | >5 | 1860 | >5 | >2000 | NT | NT | NT | NT | 0.61±0.05 | 0.25 |
| Syncephalastrum racemosum (RCMB 005005) | 4.87±0.67 | 920 | >5 | >2000 | NT | NT | NT | NT | 11.72±1.05 | 1.95 |
| Candidaalbicans(RCMB 005008) | NI | NI | NI | NI | NT | NT | NT | NT | 0.74±0.08 | 0.25 |
| Geotrichum candidum (RCMB 052009) | 0.94±0.09 | 115 | >5 | 1860 | NT | NT | NT | NT | 0.61±0.07 | 0.25 |
aDietes bicolor leaf volatile oil; bD. bicolor flower volatile constituents; cIC50 expressed in mg/ml for DBL and DBF but in μg/ml for antibiotics, data are measured in triplicates (n = 3) and presented as means ± SD; dMIC expressed in μg/ml; enot tested (NT); fno inhibition (NI).
Concerning their antifungal activity, the leaf oil exerted a more pronounced effect than the flower oil on the tested fungi namely G. candidum, S. racemosum and A. fumigatus showing MIC values of 115, 920 and 1860 μg/ml, respectively except for C.albicans that is unsusceptible to the effect of both oils as shown in Table 2.
The use of multiple assays is generally recommended to evaluate the antimicrobial activity of herbal extracts and natural products with subsequent careful comparison of their results aiming to reach a solid conclusion [37]. In our study, a strong correlation was observed between the two screening methods employed, where lower IC50 values were correlated to lower MIC values, and this observation was consistent throughout the two experiments. Moreover, the results obtained in this study appear to be consistent with previous reports on the antimicrobial activity of essential oils of different species of Iridaceae including Irisbulleyana [38], Iris pallida [39], as well as Gynandriris sisyrinchium [40].
The antimicrobial activity exerted mainly by the leaf oil could be attributed to its high content of sesquiterpenes represented by spathulenol being its major constituent or as a result of the synergy between the major and minor constituents within the oil. Although spathulenol has been previously described in the literature as a potent antimicrobial agent [41, 42], its exact mechanism of action is still unknown [43]. Nevertheless, the leaf oil and its isolated components are lipophilic in nature. They can easily permeate and dissolve in the biomembrane of microbes resulting in a disturbance of permeability, leading to cell disruption, leakage of cell content and ultimately cell death [44, 45].
However, Gram-negative bacteria were not susceptible as compared to the Gram-positive bacteria, which is probably due to their different cell membrane structures among these two bacterial groups. Generally, Gram-negative organisms have an outer membrane enclosing the cell wall with a lipopolysaccharide coat that is crucial for the restriction of hydrophobic compounds. Its absence in the Gram-positive bacteria makes them more accessible to volatile oil components [42].
4 Conclusions
The current investigation revealed qualitative and quantitative variations in the essential oil components of leaves, flowers and rhizomes from D. bicolor. Furthermore, it shed a light on the potential use of the leaf oil as an antimicrobial agent owing to its high content of oxygenated sesquiterpenes, represented by spathulenol. These findings should be supported by further in vivo studies to confirm the claimed activity.
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