دراسة الفعالية التثبيطية لمستخلص أوراق نبات الميريمية Salvia officinalis L. والمركب النانوي له ضد بعض أنواع المبيضات لدى الأطفال في محافظة كربلاء

Rp_ Study of the inhibitory activity of leave extract of Salvia officinalis L. and its nanoparticle against Candidiasis of children in Kerbala. pdf

Summary
The current study included isolating and identifying the Candida species that cause oral candidiasis, preparing a nano plant extract using sage leaves, separating and purifying the active ingredients in the leaves of the plant, and testing their inhibitory effects against the isolated Candida species. The results showed the following :
From 22/1/2021 to 5/2/2021, 83 samples were collected from children who were clinically diagnosed with oral candidiasis, and the results of isolation and diagnosis revealed the presence of growth in 63 samples (76%), of which 58 samples (92%), and it turned out that the majority of them belong to the genus Candida, with a percentage of 93.1% (54 samples), while other non-candida yeasts recorded a percentage of 6.9% (4 samples), and the Candida albicans Predominance Isolated with a rate of 53.7%, Followed by C.dubliniesis With a rate of 18.5%, then C.lusitaniae with a rate of 12.9%, followed by C.tropicalis with a rate of 7.4%, C.parapsilosis with a rate of 5.6% and C.kefyr with a rate of 1.9%. The Diagnosis of the species was confirmed using Vitek device with the Vitek 2 YST.
The isolated species were also molecularly characterized using polymerase chain reaction in the presence of the primers NS1 and NS8, which succeeded in doubling the SrRNA 18 region, with a duplication product whose sizes ranged between bp 1624 and 1656 and were deposited in the National Center for Biotechnology Information’s Genbank database, and each isolate was assigned a sequence code: MZ574137.1, MZ574410.1, MZ376949.1, MZ376951.1, MZ572966.1,MZ574180.1 .
The sensitivity of the isolated species towards six types of antifungals, namely Fluconazole, Flucytosine, Caspufungin, Amphotericin B, Micafungin, and Voriconazole, was examined by the Vitec device, and the minimum inhibitory concentration value for each type was determined. Two isolates from C.albicans and one from C.lusitaniae showed resistance to amphotericin, while one isolate from C.dubliniensis showed resistance to flucytocin.
A crude aqueous and alcoholic extract of the leaves of the sagebrush plant was prepared for the purpose of identifying the antagonistic activity against the isolated species. It was found that both contain saponins, tannins, carbohydrates, glycosides, resins, and flavonoids, while the alcoholic extract contains more alkaloids and phenols than the aqueous extract. The study proved that the alcoholic extract in all tested concentrations of 10, 25, 50, 75, and 100 mg/ml showed efficacy in inhibiting all isolates sensitive to and resistant to amphotericin, compared to the aqueous extract ,which showed efficacy against all species at a concentration of 100 mg/ml .When comparing the effect of the alcoholic extract with the effect of the antifungal amphotericin at the same concentration, it was found that the effectiveness of the crude extract was less than that of the antifungal amphotericin, and that the sensitive species C. lusitaniae, C. tropicalis, C.parapsilosis and C.kefyr are the most affected by the extract and the antifungal. The study also showed that the lowest inhibitory concentration (MIC) of the alcoholic extract against the sensitive species in the above was 12.5 µg/ml for each, while the minimum lethal concentration value was 25 µg/ml, and for amphotericin-resistant species, the value of the minimum inhibitory concentration was 200 and 50 µg/ml for each. of C.albicans and C.lucitaniae, respectively, while the lethal concentration values were 400 µg/ml and 100 µg/ml, respectively, for the aforementioned species.
The alcoholic extract was subjected to a fractionation process using the rarefied liquid chromatography (VLC) technique, and it was found that there were the largest number of compounds in the chloroform part: ethyl acetate, and this part showed high effectiveness in inhibiting sensitive and amphotericin-resistant species compared to the rest of the parts, as the rates of inhibition diameters reached sensitive species 14, 16, 19, 21, 20, and 21 mm for C.albicans, C.dubliniesis, C.lusitaniae, C.tropicalis, C.parapsilosis, and C.kefyr, respectively, and 14, 19 mm for C.albicans C. lusitaniae, which are resistant to amphotericin.
18 active compounds were isolated and purified using preparative high-performance liquid chromatography (Prep.HPLC) at a wavelength of 273 nm, and using analytical high-performance liquid chromatography, three active compounds were diagnosed: caffeic acid, ferulic acid, and kaempferol, and all these compounds showed inhibitory activity against Candida species. sensitive to amphotericin.
Also, during the study, a hybrid nano-extract of alcoholic extract of sagebrush was prepared after loading it on zinc oxide. This was confirmed by the results of the infrared spectroscopy (FTIR) examination, which showed a shift in the frequencies of some chemical groups towards high and low frequencies, as revealed by X-ray spectroscopy (XRD). The emergence of new levels in the spectrum of the hybrid nanocomposite compared to the spectrum of the carrier zinc oxide, which indicates that the compound under study is nano. It was clear from the results of the examination by atomic force microscope (AFM) that the average dimensions of the diameters of the particles of the hybrid nanocomposite amounted to 62.32 nanometers. As for the examination using a scanning electron microscope (SEM) SEM) revealed the appearance of regular geometric shapes and the formation of structures with high porosity.
The results of the analysis by EDS spectroscopy showed the presence of zinc, oxygen and carbon elements, which indicates the success of inserting the plant extract compounds between the layers of zinc oxide and then forming a new hybrid nanocomposite. This hybrid nanocomposite showed higher inhibitory activity than the crude extract and close to the effectiveness of the antibiotic amphotericin, and the sensitive species were more sensitive to the nanoextract, as the average diameters of inhibition by etching method were 18.33, 21.67, 21, 20, 22, 16 mm and the lowest inhibitory concentration. The sensitive species had about 6.25, 6.25, 3.125, 3.125, 3.125, and 6.25 µg/ml each, respectively, while the LCC values were 12.5, 12.5, 6.25, 6.25, 6.25. 12.5 µg/ml compared to the resistant species.