In Vitro Evaluation of Antimicrobial potential of Some Medicinal Plants Extracted by Supercritical Fluid Extractor; Their Phytochemistry and GC-MS Analyses

Along with swift economy upgrowing and continuous amelioration of lifestyle, people at present are paying more attention to health issues. Synthetic drugs will be compensated with other natural ones which belong to natural origin. Plants have always been considered as sources of several compounds that are used in many fields, especially human and animal health, starting from boosting the immunity to treatment of infectious diseases caused by some pathogenic microbes such as bacteria, fungi as well as viruses. This study aimed to incorporate some types of plants within antimicrobial portfolio through examination of different six plants which were Cichorium intybus, Cinnamomum camphora, Commiphora myrrha, Foeniculum vulgare, Nerium oleander and Spartium junceum . As well, attempting to identify the active constituents of their extracts using GC-MS.

crude extract of F. vulgare showed the highest potency against C. albicans, E. faecalis and S. typhimurirm, it contains some unique compounds such as squalene, eugenol and isoeugenol while, Extract of Cichorium intybus showed a moderate activity especially against C. libolytica and MRSA and it includes Vitamin A like compound which indicates antioxidant property.

Conclusion
Conclusively, fennel gave a promising result as a good wide spectrum antimicrobial agent because it contains some compounds act as antimicrobial agents such as eugenol which was used as food preservative in addition to squalene which acts as antioxidant and antimycotic agent so, it will be useful especially while it was used in highly purified form excluding all undesirable subcomponents.

Background
The majority of microorganisms are free-living and perform useful activities however, some microorganisms caused diseases are called pathogens. They include bacteria, viruses, fungi, and protozoa. Infection occurs when a pathogen invades a body that resulted in clinical infection, such an infection is referred to as subclinical asymptomatic leaf extracts (methanol, distilled water, chloroform, petroleum ether, and acetone) of Cichorium intybus were investigated for antibacterial activity against Gram-negative pathogenic bacteria, the extracts showed a wide spectrum of inhibition against the test pathogens. Methanolic extract of root and leaf proved to have the strongest antibacterial activity [14].
Cinnamomum camphora Cinnamomum spp. belong to Lauraceae and had been examined extensively for their essential oil constituents. This genus includes about 250 species in the tropical and subtropical districts, mostly in Asia and some in South and Central America, and Australia [15].
Cinnamomum camphora was known for its medicinal traits in folk medicine. Phytochemical screening showed a presence of alkaloids, Tannins, and carbohydrates, as well as its methanolic extract, presented the maximum antimicrobial activity when compared with the other extracts; chloroform and petroleum ether [16].

Commiphora myrrha
Commiphora myrrha belongs to Bruseraceae and it was commonly known as "Myrrh" which is one of the most important medicinal plants. Traditionally, its resin was used in the tackling of wounds, gastrointestinal tract GIT disorders, diarrhea, coughing, thoracic pain [17], gingivitis [18] also, it is very effective in the treatment of urinary tract infection UTI [19].

Foeniculum vulgare
Foeniculum vulgare Mill is called fennel and belongs to Apiciaceae, it features with its aromatic fruits. It was commonly used for the treatment of different disorders as well as it acts as a digestive, carminative and diuretic agent [20]. Preliminary phytochemical screening confirmed the occurrence of flavonoids, tannins, saponins, steroids, glycosides, terpenoids besides its antimicrobial activity due to its potential essential oil constituents [21] as well as several pharmacological advantages through its bioactive constituents that is very important for human health [22].

Nerium oleander
Nerium oleander Linn; belongs to Apocynaceae family. It is commonly known as kaner, an evergreen flowering shrub, and extensively cultivated for its aroma. Its extracts have many pharmacological properties such as diuretic, expectorant and sternutatory agent. It is also a highly toxic plant due to presence of a cardiac toxin hence, it was used topically [23].
Methanolic and water extract of different parts of the plant gave a reasonable activity against some pathogenic microorganisms [24].

Spartium junceum
Spartium junceum is one of the medicinal plants that belong to the Fabacea family that was cultivated as ornamental plant [25], its flowers were used for the treatment of gastric ulcer in Turkey [26] it has miscellaneous functions as antioxidant, antifertility, simulator for uterine and GI contraction thus causing vomiting [27][28][29].
The current study aimed to screen the phytochemical composition of all mentioned plants in the review and extraction by more sophisticated technique known as supercritical fluid extracting equipment SFE under certain conditions and subsequently investigation of the ability of these SFE extracts to inhibit the growth of some pathogenic microorganisms including Gram-positive and Gram-negative bacteria in addition to some pathogenic yeasts and finally, these SFE extracts would be analyzed by gas chromatography to predict their ingredients as an attempt to identify the bioactive components. Materials And Methods

Plants collected
Six different unusual plants were collected and had been finely ground by aggressive blender until turning to a fine powder for easy extraction and further investigations, the following table (Table 1) includes their scientific, common and Arabic name as well as the parts used for investigations and analyses.

Quantitative Assessment For Phytochemical Constituents
Ten grams of the air-dried plant powder were extracted independently with 80% methanol three successive times. The extracts were concentrated, and the dried matter was then dissolved in 50 ml methanol. The alcoholic extracts were then completed to the volume of 100 ml by adding distilled water and used for the following determinations.

Total Phenolics
One ml of the prepared extract of each specimen was completed to the volume of 10 ml by adding distilled water, then 1 ml of Folin-Ciocaltu reagent was added. The mixture was then shaken vigorously for 5 min. then 10 ml of 70% Na 2 CO 3 was added and diluted immediately up to 25 ml by adding distilled water. The latter mixture was incubated for 90 min. at room temperature. The absorbance was measured at 750 nm against the reagent used as a blank. A standard calibration plot was generated at 750 nm using known concentrations of gallic acid. The concentrations of phenols in the tested samples were calculated from the calibration plot and expressed as mg gallic acid equivalent of phenol/g of sample [30].

Total Flavonoids
One ml of the prepared extract of each specimen was completed to the volume of 5 ml by adding distilled water. Immediately 0.3 ml 5% NaNO 2 was added and the mixture was then left for 5 min. Respectively 0.3 ml 10% AlCl 3 and 2 ml 1 M NaOH were added. The mixture was then diluted up to a volume of 10 ml with distilled water and the formed pink color was measured at 550 nm against the reagent used as a blank.

Total Tannins
The total tannin content in the plant extract was estimated according to Folin-Deins reagent method [32], the absorbance was measured at 755 nm.

Total Saponins
One gm of each plant powder was dispersed in 10 ml of 20% ethanol. Heating of the over a hot water bath for 4 h with persistent tilting at about 55 °C. The mixture was filtered, and the residue re-extracted with another 10 ml of 20% ethanol. The combined extracts were reduced to 2 ml over a water bath at about 90 °C. The concentrate was transferred into a 250 ml separator funnel and 5 ml of diethyl ether was added and shaken vigorously.
The aqueous layer was retrieved whilst the ether layer was thrown away. The purification was done once again then 15 ml of n-butanol was added. The merged n-butanol extracts were washed twice with 10 ml of 5% aqueous sodium chloride, then the mixture was heated in a water bath. After evaporation, the samples were dried in the oven at 65 °C to a constant weight. The total saponin content was expressed as a percentage [33]. According to the described method by Chaplin and Kennedy [35], the plant powder samples were dried at 65 °C till a constant dry weight and grounded again to a very fine powder by a clean mortar. One gram of the powder was put in a 100 ml conical flask, to which 5 ml of 2% phenol/water and 10 ml of 30% trichloroacetic acid were added. The mixture was shaken and kept overnight before being filtered; the filtrate was made up to 50 ml.

Estimation:
Contents of total soluble carbohydrates were determined using anthrone technique [36], the developed color was measured using an electric colorimeter at 620 nm. A blank mixture containing distilled water and reagent was used to set up the apparatus at zero optical density.

Extraction
In this regard, one gram of the oven-dried powder at 60 °C and then transferred to 250 ml conical flask, then 10 ml distilled water and 5 ml of 2% phenol solution were added. The contents of the flasks were shaken well and kept overnight before being filtered, and then they were used for the estimation of soluble proteins.

Determination
The optical density of the resulted color was then read at the wavelength of 750 nm. The concentration of soluble protein present in the sample was then calculated making use of the constructed standard curve of proteins [37].

Total Nitrogen
The total nitrogen content of each plant powder was determined according to Kjeldahl digestion [38].

Volatile Oil
Fifty grams of each plant powder were exposed to steam distillation to extract volatile oils according to Balbaa's method [39].

Extraction By Supercritical CO2 Fluid Extractor (SFE)
Ten grams of each plant powder were used to be extracted via SFE equipment at SFE lab, the regional center for mycology and biotechnology RCMB, Al-Azhar university. The supercritical fluid extraction equipment (Teledyne ISCO SFX 200) includes carbon dioxide injection pumps, extractors, separators, compressors, carbon dioxide tanks, chillers.
The operation of extraction depends upon pushing the CO 2 through pipelines to be mixed with the samples under programmed critical conditions which gives CO 2 the solvent property, its ability as a tunable solvent differs with changing the critical conditions, these parameters include pressure, temperature, flow rate. In this experiment; the conditions applied were 300 bar, 55 °C and CO 2 flow rate of 6 g/min for 50 min.

Antimicrobial Activity Of Plant SFE Extracts
Agar well diffusion method was applied to determine the antimicrobial activity of six extracts against 10 pathogenic microorganisms; the first four belonged to Gram-positive bacteria, the next four belonged to Gram-negative bacteria and the last two belonged to yeasts. This assay was done in corresponding to gentamycin as a positive control antibacterial and ketoconazole as a positive control antifungal. The medium used for bacterial growth was Mueller-Hinton agar medium (code; CM0337, Thermo Scientific,  Results And Discussion

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.  [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.

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].

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. 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].   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.     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. 4.6. Spartium junceum Table 9 Analysis of GC-MS chromatograph which exhibits the predicted subcomponents of SFE Spartium junceum extract. 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. as well as high purity of the crude extract with no organic residues. There is a very crucial need to antimicrobial agents to be incorporated more and more into the pharmaceutical market especially those belong to natural sources to overcome the problem of microbial resistance, and the current study presented a primitive inspiring trial to help in complementary and alternative medicine. Also, combination between those examples of natural antimicrobial agents and other established drugs may offer synergistic powerful effect in tackling the resistance problem. GC-MS reports displayed that some examined plants contain very useful unsaturated fatty acids (omega 9) and hence, those antimicrobial extract will be have additional advantage or dual action.

Declarations
The author declares the following: That ethics approval is not applicable.
That consent participate is not applicable.
That all data and materials are available.
That all this work was done by himself as a single author without any contribution from any other authors.
That all the experiments and costs are totally funded personally by himself as a single author; including all analyses, collecting and buying plant samples and there is no conflict of interest.
That there is no conflict of interest.        GC-MS chromatograph of SFE Spartium junceum extract.