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The present work was designed to provide an in-depth phytochemical and biological study carried out on the fruits (seeds) of Daucus carota var. boissieri (Apiaceae); with elucidation of the chemical composition of the volatile oil and petroleum ether extract, validation of some of the seed traditional uses and a study of the potential topical anti-inflammatory effect of the petroleum ether extract with elucidation of the molecular mechanism underlying this effect.
For the fulfillment of these goals, our study was divided into three parts:
Part I: Phytochemical investigation
The chemical compositions of the volatile oil, total petroleum ether extract as well as the saponifiable and unsaponifiable fractions of the petroleum ether extract were evaluated qualitatively and quantitatively by GC/MS and GC/FID techniques, respectively.
Compounds were identified by comparison of their spectral data and retention indices with Wiley Registry of Mass Spectral Data 8th edition, NIST Mass Spectral Library (December 2005), Mass Finder 2.3 and the literature. Also, mass spectral deconvolution using Automated Mass Spectral Deconvolution and Identification System (AMDIS, v. 2.71) was used for the interpretation of the overlapping gas chromatography/mass spectrometry peaks especially in the complicated fatty acids region. Quantitative estimation was based on peak area integration.
Thirty three compounds accounting for 98.7% of the essential oil (6 h distillation) were unambiguously identified. Unidentified components were mainly present as traces with relative abundances less than 0.1%. Sesquiterpenes were the main components of the oil accounting for approximately 92% of the total oil composition. The main oil component was β-bisabolene (sesq. Hydrocarbon, 57.7% of the total oil composition) followed by carotol (oxygenated-daucane sesquiterpene alcohol, 23.2%).
Eighty compounds accounting for 97% of the extract were identified. Similarly, sesquiterpenes were the major constituents of the petroleum ether extract (≈30% sesquiterpene hydrocarbons and 23% oxygenated sesquiterpenes), mainly represented by β-bisabolene and carotol (21.6% and 17.6% of the total extract composition, respectively). Another major class of compounds was fatty acids; accounting for ≈25% of the total extract composition, mainly C18 fatty acids. Sterols accounted for 0.65% of the total extract composition, represented mainly by stigmasterol (0.328%) and β-sitosterol (0.24%).
Twenty different fatty acid methyl esters accounting for 94.88% of the saponifiable fraction were unambiguously identified by direct comparison with published data as well as computer library search. The sap. fraction contained mainly unsaturated fatty acids (77.4%); while saturated fatty acid resembled 17.5% of the total composition. The main fatty acid was petroselinic acid (Z6-octadecenoic acid, 74.8% of the total composition), Followed by palmitic acid (16:0, 8.85%).
Fifty seven compounds accounting for 97.46% of the unsaponifiable fraction were identified. The main component was β-bisabolene (31.8%), followed by carotol (25.01%) and α-asarone (19.49%). Sterols represented 1.02% of the total composition, represented mainly by stigmasterol(0.55%) and β-sitosterol (0.31%). α-Tocopherol and the pentacyclic triterpenes, β-amyrin and α-amyrin, were also detected (0.50%, 0.59% and 0.36%, respectively).
Part II: Biological screening
To validate some of the folk medicinal uses of the plant and to compare the biological activity of both the volatile oil and petroleum ether extract of Daucus carota var. boissieri (Apiaceae), the following experiments were adopted:
1. Anti-oxidant activity (radical scavenging activity experiment)
Both, volatile oil and petroleum ether extract of Daucus carota seed (fruit) exerted a potential antioxidant activity, as indicated by their ability to reduce the purple-colored DPPH• radical to the yellow-colored diphenyl picryl hydrazine in a concentration dependent manner. Comparison revealed that the petroleum ether extract exhibited higher antioxidant activity (lower IC50 value).
Higher antioxidant activity of the petroleum ether extract may be attributed to the presence of pharmacologically active components which were not present in the volatile oil, such as: phytosterols, linoleic acid,α-tocopherol, methyl palmitate and the pentacyclic triterpenes, α- and β-amyrin.
2. Anti-inflammatory activity (prostaglandin E2 assay)
Both, volatile oil and petroleum ether extract exerted anti-inflammatory effects as indicated by their abilities to suppress the production of PGE2 in MIA PaCa-2 cells. The petroleum ether extract exerted higher inhibitory effect on PGE2 production; resulting in higher inhibition of cyclooxygenase enzyme.
The potential radical scavenging activity of the petroleum ether extract and its significant inhibition of cyclooxygenase enzyme validate its potential anti-inflammatory activity and clarify the possible mechanism underlying this effect.
3. Cytotoxicity and apoptosis assay
3.1. Cytotoxicity and cell proliferation MTT assay
Both, volatile oil and petroleum ether extract exerted cytotoxic effect, as indicated by their ability to reduce the viability of the two cancer cell lines, MCF-7 (human breast cancer),and HeLa(human cervical cancer), in a concentration dependent manner.
Comparison of these values showed that the petroleum ether extract had a significantly higher cytotoxic activity in both cell lines. HeLa (human cervical cancer) cell line was more susceptible to the cytotoxic effect of both samples especially the petroleum ether extract.
This cytotoxic effect could be attributed to some specific components. For example, β-Caryophyllene, β-Caryophyllene oxide, limonene, α-humulene, phytosterols, α-amyrin, α-asarone.
3.2. Apoptosis assay
Both volatile oil and petroleum ether extract showed the ability to initiate the apoptotic cascade as indicated by the significant increase in activation of caspase 3/7 enzyme in all tested samples and in a dose dependent manner.
Comparison revealed that the petroleum ether extract had higher ability to initiate the apoptotic cascade, as compared to the volatile oil, indicated by exerting more potent activation of caspase 3/7 enzyme.
These findings explain, in part, the cytotoxic effect observed for the volatile oil and the petroleum ether extract.
In conclusion, these findings can validate the traditional use of carrot in the management of carcinomas and various inflammatory disorders.
Part III: Evaluation of the topical anti-inflammatory activity of the petroleum ether extract using ointment formulation
1. In-vivo Assessment of the topical anti-inflammatory activity of the petroleum ether extract’s formulations
In the present study, in-vivo carrageenan-induced rat paw edema model was used to assess the anti-inflammatory effect of ointment formulations containing different concentrations of the petroleum ether extract of Daucus carota var. boissieri and the results were compared to that of the positive control (indomethacin, commercial Farcomethacin® gel). Histopathological examination of hind paw soft tissue from different tested groups was adopted to further confirm the obtained results.
Different extract formulations with different concentrations significantly decreased the edema induced by carrageenan injection (except for the 0.1% formulation) indicated by the lower edema rate percentage, as compared to the inflamed non-treated rats.
(0.25%) Formulation exerted the highest anti-inflammatory effect, which was supported by the histopathological improvement of hind paw soft tissue as compared to the inflamed non-treated rats. The anti-inflammatory effect exerted by (0.25%) formulation was even higher than that exerted by the standard indomethacin.
Thus, (0.25%) was the most effective concentration of the extract which was chosen to study the mechanism of the anti-inflammatory effect exerted topically by the extract.
2. Elucidation of the molecular mechanism underlying the anti-inflammatory activity of the petroleum ether extract (using the most effective concentration)
In the present study, the petroleum ether extract (0.25%) significantly reduced the expression of NF-κB and hence inhibited the downstream inflammatory cascade as evidenced by decreasing the expression of iNOS as well as the tissue levels of TNF-α. It also exerted a potential inhibitory effect on COX-II isoenzyme in human peripheral blood mononuclear cells (PBMC), which was more potent than the standard indomethacin. The higher anti-inflammatory effect of the extract, as compared to indomethacin may be explained by the higher inhibition of NF-κB and COX-II expressions.
In conclusion, the petroleum ether extract (0.25%) provides satisfactory topical anti-inflammatory effects, which suggests its clinical use as a promising anti-inflammatory agent in dermatological preparations, after the study of its safety profile.