1. Quantitative phytochemical screening
The powders of examined plants were processed according to the specific protocols to assess the phytochemical composition belongs to each plant, all the data obtained were tabulated in Table 2 which revealed the concentration of each component.
Table 2
Phytochemical composition of the six examined plants.
Item | Item concentration for each plant |
Cichorium intybus | Cinnamomum camphora | Commiphora Myrrha | Foeniculum Vulgare | Nerium oleander | Spartium junceum |
Phenolics (mg eq./g gallic acid) | 237 | 223 | 247 | 261 | 213 | 215 |
Flavonoids (mg eq./g rutin) | 143 | 138 | 159 | 131 | 124 | 118 |
Conc. % |
Tannins | 4.31 | 4.13 | 4.05 | 3.87 | 3.74 | 3.27 |
Saponins | 2.47 | 2.09 | 2.11 | 2.69 | 2.55 | 2.28 |
Alkaloids | 3.92 | 3.85 | 3.49 | 3.85 | 2.99 | 2.87 |
Carbohydrates | 2.15 | 3.48 | 3.07 | 2.21 | 2.86 | 2.94 |
Proteins | 15.94 | 11.39 | 12.54 | 13.94 | 10.29 | 11.05 |
Total nitrogen | 26.76 | 21.53 | 21.96 | 23.41 | 21.47 | 20.98 |
Oils | 0.62 | 0.58 | 0.47 | 0.81 | 0.69 | 0.41 |
Regarding to total phenolic compounds; F. vulgare has the highest value 261 mg followed by C. myrrha 247 mg, while the lowest value 213 mg belonged to N. oleander. In case of flavonoid concentration; C. myrrha contains the highest value 159 mg followed by C. intybus 143 mg, while the lowest concentration belonged to S. junceum.
Regarding to tannins; the uppermost percentage went to C. intybus (4.31%) followed by C. camphora (4.13%) while, S. junceum contains the lowest concentration of tannins (2.87%). Referring to total saponin values; F. vulgare contains the highest percentage (2.69%) followed by N. oleander (2.55%) while, the lowest percentage (2.09%) belonged to C. camphora.
Concerning to alkaloids concentrations, the highest value belonged to C. intybus (3.92%) followed by both C. camphora and F. vulgare (3.85%) while, the lowest percentage belonged to S. junceum (2.87%).
In case of total soluble carbohydrates; C. camphora possessed the highest value (3.48%) followed by C. myrrha (3.07%) while, the lowest carbohydrates content belonged to C. intybus (2.11%). Regarding soluble proteins and total nitrogen content, the highest value belonged to C. intybus (15.94% and 26.76% respectively) and followed by F. vulgare (13.94% and 23.41% respectively) while, the lowest percentage of proteins belonged to N. oleander and the lowest nitrogen content belonged to S. junceum.
Finally, in case of oil percentage; the highest concentration was detected in F. vulgare (0.81%) followed by N. oleander (0.69%) while, the minimum value was detected in S. junceum (0.41%).
Regarding phytochemical screening, all examined plant are rich in phenolics, flavonoids, alkaloids, saponins, tannins and this is the reason of their pharmacological properties as good medicinal plants especially, fennel, chicory and myrrh, this result is in harmony with anthocyanin that had been from red chicory Cichorium intybus in aqueous solution at pH 2.5 [40].
Also, the current results of myrrh are compatible with those who recorded the presence of terpenoids, steroids, tannins, volatile oils and resins [41].
All data from Table 2 except total phenolics and total flavonoids (only phytochemical constituents that had been expressed in %) were configured in a stacked column chart (Fig. 2). By the first sight to Fig. 2, it could be noticed that C. intybus had the higher content followed by F. vulgare, C. myrrha, C. camphora, N. oleander and the lowest plant content is S. junceum.
2. Supercritical Fluid Extracting System
By applying selected parameters, the extraction capacity ranged from 0.4 to 0.6 ml which represents 4–6 wt.% approximately. The advantage of using this technique for extraction is the high precision, time saving, and with no solvent traces. This result expressed success of SFE equipment in extraction process, this is friendly with the results reported that the use of SFE in last years had been approved to be alternative for extraction of natural compounds like triterpenes which extracted by both SFE in corresponding to traditional extraction method; Soxhlet, and the result of SFE was more satisfying [42]. As well, SFE is a green extraction method providing a concentrated end product with no undesirable residues [43]. The effect of SFE parameters changing to obtain better extractability of phenolic compounds from lavender flowers, different ranges of SFE parameters were checked such as 200 bar, 40 °C and 15 min and the yield was 4.3 wt.% while 250 bar, 60 °C for 45 min gave 9.2 wt.% (more than double) [44].
3. Antimicrobial Activity Determination
Antimicrobial activity of these six different plants were estimated and the results had been expressed in mm as diameters of inhibition zones. All the data resulted were tabulated in table (3) which revealed the following observations:
All test organisms exhibited variation in their responses against the examined crude extracts, Candida albicans exhibited more resistance than C. libolytica among the examined yeast strains; only three extracts had moderate effects against C. albicans while all six extracts affected C. libolytica.
In case of Gram-positive bacteria; the most resistant strain was S. mutans followed by Micrococcus sp. and MRSA (had been affected only by three extracts) while E. faecalis was the most susceptible one that had been inhibited by all six investigated extracts.
Referring to Gram-negative bacteria; the most resistant strain was K. pneumonia followed by E. cloaca and P. vulgaris (only two extracts weakly inhibit its growth) while S. typhimurium was the most susceptible one that had been inhibited by four extracts.
On the other side, the investigated crude extracts might be categorized as potent, moderate and weak antimicrobial agents according to the applied concentrations, also, some of them could be classified as wide spectrum while the other are narrow spectrum. For example; crude extract of F. vulgare showed the highest potency against C. albicans, E. faecalis and S. typhimurirm, it also showed wide spectrum against 7 out of 10. This result is in line with those who had investigated phytochemistry, antimicrobial activity and GC-MS of Portuguese fennel fruits that gave very compatible results with the current study where the crude extract has antimicrobial activity slightly lower than those belong to positive standard used; tetracycline as antibacterial and nystatin as antifungal [45]. Also, our finding showed moderate effect of fennel extract against methicillin resistant Staphylococcus aureus (MRSA) clinical isolate and this result is relative to those findings which concluded that the combination between fennel essential oil and mupirocin has a significant eradicating effect against S. aureus and his finding will be useful antistaphylococcal agent [46].
Extract of Cichorium intybus showed a moderate activity especially against C. libolytica and MRSA with moderate range against 4 out of 10, while the extract of Commiphora myrrha gave a moderate to weak activity with wide spectrum against 6 out of 10. This result in a harmony with hexane and ethyl acetate extracts of chicory roots that showed pronounced antibacterial activity against both Gram-positive and Gram-negative bacteria [47].
SFE extract of Commiphora myrrha has a moderate antimicrobial activity according to the experimental circumstances, it is only active against 5 out of 10. This result slightly related to the conclusion related to myrrh extract which has a noticeable antibacterial activity against Enterococcus faecalis and Fusobacterium nucleatum involved in root canal infections especially when it combined with sodium chlorite [48].
Finally, Spartium junceum and Nerium oleander could be classified as weak and narrow spectrum antimicrobial agents; they only active against 3 and 2 out of 10 respectively. This finding is consistent with the previous published data that are very limited about antimicrobial activity of Spanish broom. Oleander leaves have antimicrobial activity against Bacillus pumilus, B. subtilis, S. aureus, E. coli and Aspergillus niger [49].
Table 3
Antimicrobial activity of SFE plant extract against some variable pathogenic microorganisms
Test organisms | Cichorium intybus | Cinnamomum camphora | Commiphora myrrha | Foeniculum vulgare | Nerium oleander | Spartium junceum | Positive Control |
Pathogenic yeasts Ketoconazol 100 µg (positive control) |
Candida albicans RCMB 005003 (1) ATCC 10231 | - | 11 | - | 14 | - | 12 | 20 |
Candida lipolytica | 18 | 16 | 17 | 15 | 10 | 8 | 18 |
Gram-positive bacteria Gentamycin 4 µg (positive control) |
MRSA clinical isolate | 11 | - | 7 | 8 | - | - | 30 |
Enterococcus faecalis ATCC 29212 | 18 | 12 | 13 | 22 | 10 | 13 | 26 |
Streptococcus mutans RCMB 017 (1) ATCC 25175 | - | - | - | - | - | - | 20 |
Micrococcus sp. RCMB 028 (1) | - | - | - | - | - | - | 22 |
Gram-negative bacteria Gentamycin 4 µg (positive control) |
Enterobacter cloaca RCMB 001 (1) ATCC 23355 | - | - | - | - | - | - | 27 |
Klebsiella pneumonia RCMB 003 (1) ATCC 13883 | - | - | - | - | - | - | - |
Proteus vulgaris RCMB 004 (1) ATCC 13315 | - | - | 8 | 7 | - | - | 25 |
Salmonella typhimurium RCMB 006 (1) ATCC 14028 | 14 | 16 | 11 | 20 | - | - | 17 |
Data expressed as diameters of inhibition zones in mm |
4. Gas Chromatographic Analyses
4.1. Cichorium intybus
Table 4
Analysis of GC-MS chromatograph which exhibits the predicted subcomponents of SFE Cichorium intybus extract.
Compound predicted | RT | M. wt. | M. formula |
Hydroquinone derivative | 15.95 | 206 | C13H18O2 |
Tetradecanol | 17.76 | 214 | C14H30O |
Tetradecanoic acid methyl ester | 20.68 | 256 | C16H32O2 |
Retinal | 22.82 | 284 | C20H28O |
Hexadecenoic acid methyl ester | 24.76 | 270 | C17H34O2 |
Heptadecanoic acid ethyl ester | 26.05 | 298 | C19H38O2 |
Octadecenoic acid methyl ester | 28.03 | 296 | C19H36O2 |
Linoleic acid ethyl ester | 29.11 | 308 | C20H36O2 |
Ethyl oleate | 29.22 | 310 | C20H38O2 |
Erucic acid | 29.67 | 338 | C22H42O2 |
Eicosenoic acid derivative | 33.96 | 310 | C20H38O2 |
Benzene dicarboxylic acid | 38.95 | 390 | C24H38O4 |
Spirostenone | 44.29 | 428 | C27H40O4 |
There 13 variable compounds were detected in Cichorium intybus by GC-MS analysis (Table 4 and Fig. 4); most of them belong to fatty acid whether saturated or unsaturated as well as fatty acid precursors. Also, some phenolic and terpenoids were detected. Retinal is also known as retinaldehyde, is a form of vitamin A produced by oxidation of retinol which functions as the active component of the visual cycle, this compound is unique for chicory.
4.2. Cinnamomum camphora
Table 5
Analysis of GC-MS chromatograph which exhibits the predicted subcomponents of SFE Cinnamomum camphora extract.
Compound predicted | RT | M. wt. | M. formula |
Hydroquinone derivative | 15.95 | 206 | C13H18O2 |
Spathulenol | 17.51 | 220 | C15H24O |
Nonadecene | 22.08 | 266 | C19H38 |
Hexadecenoic acid methyl ester | 24.76 | 270 | C17H34O2 |
9-Eicosene | 26.04 | 280 | C20H40 |
Octadecenoic acid methyl ester | 28.02 | 296 | C19H36O2 |
Methyl stearate | 28.50 | 298 | C19H38O2 |
Ethyl oleate | 29.21 | 310 | C20H38O2 |
Docosene | 29.66 | 308 | C22H44 |
Behenyl alcohol or Doconasol | 33.96 | 326 | C22H46O |
Benzene dicarboxylic acid | 38.94 | 390 | C24H38O4 |
Spirostenone | 44.29 | 428 | C27H40O4 |
Table 5 and Fig. 5 refer to prediction of the subcomponents of SFE Cinnamomum camphora extract that point to existence of 12 components belong to fatty acids and their precursors in addition to phenolics and terpenoids. Spathulenol is sesquiterpene alcohol; only detected in Cinnamomum camphora as unique compound for Cinnamomum camphora during this study. On the other hand, Spirostenone belongs to terpenoids (isoprenoids), this compound found only in both Cichorium intybus and Cinnamomum camphora.
4.3. Commiphora myrrha
Table 6
Analysis of GC-MS chromatograph which exhibits the predicted subcomponents of SFE Commiphora myrrha extract.
Compound predicted | RT | M. wt. | M. formula |
Hydroquinone derivative | 15.95 | 206 | C13H18O2 |
Tetradecanol | 17.76 | 214 | C14H30O |
Tetradecanoic acid methyl ester | 20.69 | 256 | C16H32O2 |
Hexadecanol | 22.09 | 244 | C16H34O |
Hexadecenoic acid methyl ester | 24.76 | 270 | C17H34O2 |
9-Eicosene | 26.04 | 280 | C20H40 |
Octadecenoic acid methyl ester | 28.02 | 296 | C19H36O2 |
Methyl stearate | 28.50 | 298 | C19H38O2 |
Eicosenoic acid | 29.21 | 310 | C20H38O2 |
Docosene | 29.66 | 308 | C22H44 |
Erucic acid | 31.74 | 338 | C22H42O2 |
Benzene dicarboxylic acid | 38.94 | 390 | C24H38O4 |
Flavone dioglucoside | 44.30 | 594 | C27H30O15 |
7. GC-MS chromatograph of SFE Foeniculum vulgare extract. |
Table 6 and Fig. 6 expressed the prediction of subcomponents of SFE Commiphora myrrha extract, analysis of GC-MS report displayed that 13 compounds had been detected; all these compounds belong to fatty acids and their precursors as well as phenolic and flavonoids.
4.4. Foeniculum vulgare
Table 7
Analysis of GC-MS chromatograph which exhibits the predicted subcomponents of SFE Foeniculum vulgare extract.
Compound predicted | RT | M. wt. | M. formula |
Trans isoeugenol | 12.28 | 164 | C10H12O2 |
Bezodoxepin derivative | 15.95 | 206 | C13H18O2 |
Eugenol | 16.31 | 164 | C10H12O2 |
Hexadecanol | 17.76 | 242 | C16H34O |
Tetradecanoic acid methyl ester | 20.68 | 256 | C16H32O2 |
Hexadecenoic acid methyl ester | 24.76 | 270 | C17H34O2 |
1-Eicosanol | 26.05 | 298 | C20H42O |
Octadecenoic acid methyl ester | 28.03 | 296 | C19H36O2 |
Methyl stearate | 28.50 | 298 | C19H38O2 |
Ethyl oleate | 29.22 | 310 | C20H38O2 |
Docosene | 29.67 | 308 | C22H44 |
Erucic acid | 31.74 | 338 | C22H42O2 |
Benzene dicarboxylic acid | 38.94 | 390 | C24H38O4 |
Docosanoic acid trihydroxy methyl ester | 40.26 | 402 | C23H46O5 |
Squalene | 44.30 | 410 | C30H50 |
Foeniculum vulgare SFE extract was analyzed by GC-MS (Fig. 7) to predict its subcomponents which were tabulated in Table 7 which revealed detection of 15 subcomponents including fatty acids and their precursors. Eugenol and trans isoeugenol are unique for only Foeniculum vulgare which are classified as phenolic compounds, also squalene is alkene belong to isoprenoid compounds, this compound is also unique for Foeniculum vulgare.
4.5. Nerium oleander
Table 8
Analysis of GC-MS chromatograph which exhibits the predicted subcomponents of SFE Nerium oleander extract.
Compound predicted | RT | M. wt. | M. formula |
Cyclohexane derivative | 13.13 | 202 | C15H24 |
Dihydro butyl bezodoxepin | 15.94 | 206 | C13H18O2 |
Isofuranodionone | 18.11 | 230 | C15H18O |
Tridecanoic acid methyl ester | 20.68 | 256 | C15H30O2 |
Hexadecanol | 22.08 | 242 | C16H34O |
Hexadecenoic acid methyl ester | 24.76 | 270 | C17H34O2 |
Nonadecene | 26.04 | 266 | C19H38 |
Nonadecanoic acid | 27.71 | 296 | C19H36O2 |
Octadecenoic acid methyl ester | 28.02 | 296 | C19H36O2 |
Octadecanoic acid methyl ester | 28.50 | 298 | C19H38O2 |
Eicosenoic acid | 29.66 | 310 | C20H38O2 |
Erucic acid | 33.97 | 338 | C22H42O2 |
Benzene dicarboxylic acid | 38.95 | 390 | C24H38O4 |
Nerium oleander SFE extract was examined by GC-MS (Fig. 8) to forecast its ingredients which were presented in Table 8 that showing presence of 13 kinds of compounds including fatty acids and their precursors. Isofuranodionone is a unique for only Nerium oleander which are classified as heterocyclic organic compound.
4.6. Spartium junceum
Table 9
Analysis of GC-MS chromatograph which exhibits the predicted subcomponents of SFE Spartium junceum extract.
Compound predicted | RT | M. wt. | M. formula |
Dihydro butyl bezodoxepin | 15.94 | 206 | C13H18O2 |
Hexadecanol | 22.08 | 242 | C16H34O |
Hexadecenoic acid methyl ester | 24.76 | 270 | C17H34O2 |
Nonadecene | 26.04 | 266 | C19H38 |
Octadecenoic acid methyl ester | 28.02 | 296 | C19H36O2 |
Octadecanoic acid methyl ester | 28.50 | 298 | C19H38O2 |
Erucic acid | 33.96 | 338 | C22H42O2 |
Benzene dicarboxylic acid | 38.95 | 390 | C24H38O4 |
Only 8 compounds were detected in Spartium junceum SFE extract and this is the least content diversity among the examined plants, data was obtained by Fig. 9 that represents GC-MS chromatograph of Spartium junceum SFE extract, subsequently this figure was analyzed to expect the ingredients which were arranged into Table 9.
Table 9 showed limited number and limited diversity of subcomponents which belong to fatty acid (erucic acid) and precursors of fatty acids.
GC-MS analyses for all examined plants showed presence of fatty acids and fatty acids precursors in all investigated SFE extracts, although some compounds are unique for a specific type of examined plant among this study, on the other hands there are some compounds being common between two or more types of examine plants, for example; eicosonoic acid is monounsaturated (omega 9) fatty acid and it was detected in Cichorium intybus, Commiphora myrrha and Nerium oleander. Erucic acid is also monounsaturated (omega 9) fatty acid and it was detected in Cichorium intybus, Commiphora myrrha, Foeniculum vulgare, Nerium oleander and Spartium junceum. Doxepin derivative is antidepressant molecule and it was detected in Foeniculum vulgare, Nerium oleander and Spartium junceum. Spirostenone belongs to terpenoids (isoprenoids), this compound found only in both Cichorium intybus and Cinnamomum camphora. GC-MS report indicated presence of residues of solvents involved in extraction process plus the components constituting ethanolic clove extract [50] but fortunately, in this study GC-MS report indicated absence of the residues of solvents as an evidence to the high purity degree of the plant extract yielded by SFE.