Assessing the impact of quercetin isolated from Ammi majus seeds upon Candida spp. isolants isolated from different sources

Quercetin has been extracted from Ammi majus plant using ethanol via soxhlet system. Further, the substance was diagnosed by three binary and monolayered chromatographical devices beside chronographical quantitative separation. Total of 45 patients was in licted with mouth infections and urinary tract in both genders. Themomentary vaginal infections among different ages of people settled in different regions ofNinevehdistrict / Iraqwere considered for the study. The study patients were identi ied using microscopic testing, selective differential medium (CHROM candida agar) and Vitek system. The isolation results inferred that the mouth infection is caused by most yeasts such as Candida parapsilosis (the most frequent),. The urinary tract infection is concerned, and most of the infections were reportedly caused by C. parapsilosis, C. tropicalis and C. albicans. with regards to the vaginal infections cases, C. albicans fungus has been the most frequent. Bio chemical tests were conducted using Vitek for the isolants studied, in which there were differences in the study results. Quercetin was extracted from Candid spp. The increase in the inhibition of quercetin is noted, whenever its concentration is increased. With regards to the anti-fungals Nystantin, for the concentration of 100IU/ml.and Clotrimazole 30mg/ml had inhibited the yeast Candid spp.


INTRODUCTION
Plants contain abundant numbers of different chemical compounds, whose discovery increases, thanks to the progress made in the ield of multiple chemical analysis methods. This progress enables the detection of compounds and get acquainted to it so as to harness its bene its. These compounds are of great importance because of the physiological effect and medicinal properties on human, animal and plant organs (Abboud et al., 2017). One of these plants is Ammi majus L. which contains quercetin. Ammi majus L. falls under the family, umbelliferae. Being a common perennial herb, it grows in all the cultivated areas and is a native of Mediterranean Sea region, especially Africa . The interest towards Ammi majus L. was revived in the recent years after its value was established through many clinical investigations. It is a well-known fact that furocomairns exhibit photosensitizing activity and their derivatives have been found to possess antibacterial properties. In Northern Iraq, especially Mosul, Erbil and Zakho, these plants grow abundantly in different areas. The studies published so far proved that the fruits of Ammi majus L. plants contain glycoside and glycoside comarius while few studies reported the presence of lavonoids in L. plants (Ayoub et al., 1998). This speci ic species and few other species under this family are considered as the source of khellin, which is used as a diuretic (Ziment, 1998). Ammi majus L. is used for gruelling in teeth and palate diseases (Al-Janabi et al., 2017). There are many diseases caused by fungi like yeasts and semi-yeasts including Candida genus while the latter consists of more than 200 species in it and morphologically, it looks similar to semi-yeasts and oval in shape. These cells are asexual ones i.e., breeding through budding or ission. Candida species descends from Saccharomycetaceae family, Saccharomycetales order, Saccharomyces class and Ascomycota phylum (Vazques et al., 2003). This species causes infections in the mouth, urinary tract and vagina. The infection occurs as a causative one or accompanies the bacterial or viral infection or a side effect to weak immunity of the body or excessive use of antibiotics and change in the natural lora of infected area (Al-Hadithi et al., 2007). The ability of some candida species to develop the anti-medicine biomembranes is considered as the important factor that contributes to the virulence in human diseases (Rajendran et al., 2010;Vila et al., 2020). Candida is generally observed in mouth among patients with weak immunity. Especially, it exists in mouth up to 20 % to 40 % among healthy people (Habib et al., 2015).Besides, Candida exists in respiration channel, vagina and in other such tracts (Lewis et al., 2000).
The transformation of yeast from antiretroviral to an opportunist is due to its possession of aggressive factors like sticking to the surface of epithelial cells, production of digesting enzymes for fats and proteins and the development of production tube (Panagoda et al., 2001). These yeasts are also found in tooth decay and mouth infections (Habib et al., 2015;Al-Kaaby et al., 2016). In addition, bacteria exist with the host round the mouth since it is important to have the ability to cause opportunist diseases so that they naturally exist in skin, nose, intestinal tract epithelium, and sexual organs. They contain a lot of antigens super icially and enzymes that enable the pathogen to penetrate body tissues (Nicholls et al., 2011). Candida spp. are considered as the most common cause of urinary tract infections in which they affect the urinary tracts through urine stream followed by the bladder and then the bloodstream (Pfaller et al., 1996). These infections are the most dangerous health problems reported among millions of people; especially women, because they are likely to get affected than men. The rate of the spread of this disease differs according to different health and geographical conditions. The urinary tract infections may vary from case to case according to the patient, gender and their age than the symptoms of urinary tract infections (Al-Rubaie et al., 2010). More than 90% of the fungal cases are caused by Candida albican, C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei (Pfaller et al., 2007). Nowadays, the increase in the number of anti-medicine yeasts and antifungal species are identi ied all over the world. Therefore, it is best to use the laboratory practical tests which may help the physician to choose the suitable remedy (Ingham et al., 2012). In the present study, the technique of Vitek 2 Compact system was used (Aubertine et al., 2006;Habib et al., 2015;Badr and Abaas, 2017).

Plant material
The plant was cultivated in Iraq using the seeds of Ammi majus, procured from local market. The seeds of Ammi majus L. that were originally collected from Al-Jazeera region were irst cleaned and then ground.

Preparation of plant extract
The plant was weighed upto 100 gms and extracted for 6 to 8 hours using soxhlet system with 1 litre of pet. Ether at 60 • C to 80 • C for delayed processing. This batch was again extracted with 70% of the ethanol. So, the ethanol got concentrated on vacuum votary that evaporated at 50 • C to give 15 ml as a residue (Ayoub et al., 1998).
A total of three batches of ifteen random samples each (total 45 samples) were collected from persons with a mouth infection, swab samples of patients affected with urinary tract infections and swab samples of women affected with vaginal infections. The samples were collected from patients who visited the clinics in Nineveh district and the swabs were cultured in a dish containing Sabouraud Glucose Agar (SGA) medium. All the samples were incubated at 37 • C for 24 hours. The results were recorded, and the isolates were reserved in slanting mediums for further use (Habib et al., 2015).
The isolated fungi were identi ied through mere observation and through microscopic testing in which the fungal colony shape and its structure were noted. The colonies were observed microscopically according to the method described earlier (Sood, 1994;Morello et al., 2003). The fungi cultures were developed in differential medium chrome agar candida. This medium was used to identify different types of candida genus depending on the enzymes that exhibit colour (Baumgartner et al., 1996;Pfaller et al., 1996;Ellis et al., 2007). Quercetin, which was extracted from Ammi majus (Ayoub et al., 1998) in the concentrations of 2, 3 and 4 mg/ml of the solved substance (DMSO), was prepared. The in luence of concentrations of quercetin was tested by making a dig of 5 cm in diameter. In SGA medium, the isolated samples considered for testing were cultured with 0.1 ml concentration. Each dig was added the solution thrice and was incubated at 37ºC for 24 hours. The results were recorded by measuring the diameter of the inhibition state, excluding the dig diameter (Ginnis, 1980).

RESULTS AND DISCUSSION
The quercetin was extracted from Ammi majus as follows

Separation of quercetin from ethanol extract
Crude ethanolic extract was obtained from the second step (preparation of plant extract) using 100 gms of seeds. This extract measured 15 ml. while 10 gm. of this crude ethanolic residue was applied onto a column packed with silica gel in the pet-ether 60-80 • C.
The elution affected the petroleum ether followed by chloroform and chloroform: methanol. The fractions obtained were examined using Thin-Layer Chromatography on silica gel. The elution with 10% methanol in chloroform attained a yellow ppt, which was crystallized from ethanol to give 120 mg. Yellow needles. The material was made to undergo acid hydrolysis in 2NHCI for 30 minutes. This was followed by the usual work for the given residue that is identical to quercetin (m.p = 314-315) C • (lit 313-314) C • (Ayoub et al., 1998) as shown in Table 1.   The Table ?? shows the data of isolates for 45 cases in both genders at different ages infected with the mouth, urinary tract infections while the samples also show the women infected with vaginal infections in Mosul district and its suburbs. The isolates were identi ied through microscopic testing and it has been observed that most of the pathogens belonged to candida genus as shown in Figure 1.
The species were identi ied through the medium (chrom candida agar) and the following isolants were found in the medium: C. albicans in green colour, C. parapsilosis in purple colour, and C. tropicalis in blue colour as shown in Figure 2 (Baumgartner et al., 1996;Pfaller et al., 1996;Ellis et al., 2007).
Then, the isolates were identi ied using Vitek system and their frequency for all the cases was observed as   Table ?? containing age, gender, residents and the diagnosis of the patients with mouth infections. The regions lying between AL Harmat and Garage Al Shimal in Mosul district showed a difference in the frequency of infection. It has also been observed that the patients' ages varied from 18 years to 60 years and the most frequent ages were 26, 28, and 35 years. The af liction among the males was found to be more than females. It is obvious that the most frequent yeasts observed were C. parapsilosis and C. tropicalis which caused mouth infections in the Mosul region. The following yeasts such as C. albicans, C. kefyr, and Rhodotorula glutinis took the next positions, respectively. These results support the study indings of (Al- Kaaby et al., 2016) in which the semi-yeast Candida spp. was isolated as %10.4 from teeth and gum surfaces of patients of both genders at different ages. Table ?? shows the urinary tract infections among the patients with information such as age, gender and residents. The patients identi ied with urinary tract infections reveal the fact that they were from different regions and the yeasts were isolated between Bahzani and Omar Kapchee regions. There was variations in the age range between 12 years and 60 years and the most frequent ones were 21, 25, 40 and 60 years. The table also reveals that the infection is high among females than the males. It is observed that the most common yeasts that cause urine tract infections in Mosul are C. tropicalis, C. parapsilosis and C. albicans (less frequent one). These results support the indings of (Pfaller et al.,    -+  +  ARG  25  -+  --AMYa  26  ----dCELa  27  ----dMELa  28  +  +  +  +  SACa  29  --+  -IARAa  30  +  +  +  +  ACEa  31  --+  +  dGNTa  32  ----ERYa  33  -+  +  +  dGALa  34  ----GGT  35  ----dMLZa  36  ----URE  37  ----dGATa  38  -+  -+  CITa  39  --+  +  GLYLa  40  ----GENa  41  +  -+  +  dMALa  42  ----ISBEa  43  +  -+  +  AGLU  44  ----ESC  45  _ -+ -GRTas 46 These results are in agreement with the study indings of (Habib et al., 2007) in which the authors noted that the vaginal infections were mostly caused by candida spp. Yeast. Henceforth, it is noticed that the yeasts have a great role in mouth, urinary tract and vaginal infections. They have an important relationship with natural lora that creates an environmental imbalance in the human body (Al-Hadithi et al., 2007). In other words, the environment plays a vital role in infection and the progression of disease in the isolant. Yeasts are the main causative agents of infection that differ in their capability of causing the disease from one type to another. The capability of the island in developing diseases also differs from one type to another.
Further, the position of af liction also has an important role in disease progression. It has been observed that most of the infections are dependent on the region and age (van den Braak et al., 2001;Badr and Abaas, 2017). This observation is supported by the study indings of (Habib et al., 2015) in which the author inferred that candida infection is not con ined to a speci ic geographical location. As per the Table ??, biochemical tests of both isolated and identi ied fungi were accomplished by Vitek system as follows, The tests results for different species such as C.albicans, C.parapsilosis, C.kefyr and gultinis Rhorlutoula, were arrived at, and each fungus has been found among the materials used in the above test.
But IMLTa, dMNEa and SACa were observed to be positive in the isolated and diagnosed yeasts such as C.albicans, C.parapsilosis, C.ke iy and Rodutorula gultinis.
With regards to the assessment of effect of quercetin isolated from Ammi majus, (Ayoub et al., 1998) concentrations (2,3,4 mg./ml), it can be observed that the inhibition (A) got increased with increase in the concentration of the inhibited material. It inhibited 2.1 cm at a concentration of 4 mg /ml in the fungus C.albicans. Further, the inhibition was measured at 2 cm when the concentration was 4 mg/ml for the fungus C. parapsilosis and 1.3 cm for C. tropicalis fungus of the similar concentration. There was no signi icant effect of stander DMSO on the previous yeasts whereas the antifungal Nystatin exhibited high inhibition rate (2.8cm, 2.3 cm. and 1.8 cm.) for the concentration (100U/ml) in both C. parapsilosis and C.albicans respectively. Meanwhile, the antifungal Clotrimazole, at a concentration of 30.mg/ml inhibited the yeasts and measured at 1.9 cm for C. albicans fungus, 1 cm for C.parapsilosis fungus and 1.5 cm for C. tropical as shown in the Table ?? and Figure 3.
Henceforth, it can be inferred that quercetin has inhibitory effect on yeasts. This inference is supported by the indings of (Al_Sana i, 2013) in which the quercetin plant consists of second oils with 18% and coumarin 0.5-0.2%. It also consists of quercetin in which these substances are considered effective in healing psoriasis. It is germicidal in nature and act against fungi, particularly, Tinia versicolors. They are also effective in treating skin cancer (Al-Janabi et al., 2017;Kasper et al., 2015) mentioned that Clotrimazole has a fatal effect on the cells of C.albicans fungus in its growth and formation stages.
The antifungal substance, Nystatin exhibits strong inhibition upon Candida spp. especially C.albicans (Yahya and Altae, 2013) Nystatin consists of polyene which helps in the prevention of membranes that form Candida species. It further inhibits its growth and impact it by replacing the ergisetrol in the membrane of yeasts. As a result, it stops the growth of yeast membrane, change the osmosis, free transportation, ionic exchange and consequently death of the yeast cell (Carrillo-Munoz et al., 2004;Zotchev, 2003;Carrillo-Muñoz et al., 2001).

CONCLUSIONS
The signi icance in this study is that, 1. The quercetin has an inhibitive effect on these yeasts. Thus, it is necessary to explore the substance in detail so as to use it as an antifungal medicine in the diseases caused by Candida sp. Quercetin can be used as a drug or as mouth wash too.
2. It has been noticed through research studies that the inhibition by Nystatin is superior to inhibition by Clotrimazole in yeasts.

Source of funding
Self Funding.

Con lict of Interest
The authors declare that they have no con lict of interest for this study.