Effect of Alcoholic Extract of Carrichtera annua Plant on the Growth of Leishmania donovani Parasite, In vitro Study

The current study was conducted to find out the efficacy of the alcohol extract of Al-Khashin plant Carrichtera annua plant in treating the Leishmania donovani parasite In vitro, where the concentration of 1.5 mg/ml resulted in an inhibitory effect on the growth of the promastigote of parasites at a percentage of 50% (LCD50) during 96 an hour at a rate of 38.1 x 103 cells/cm3 compared to the control treatment 76.2 x 103 cells/cm3, the results showed the significant inhibitory effect of the alcohol extract of Al-Khashin plant on the growth of Leishmania parasites, which led to a decrease in the number of generations and an increase in their time, as the number of generations reached 108.7 generation compared to the control treatment reached 232.5 generations using the same concentration within 96 hours of treatment, an inverse relationship was observed between increased concentration and generation time. The conclusion of the present study is the high efficiency of C. annua alcoholic extract in treating the L. donovani parasites as it led to an apparent inhibition of the growth of promastigote that was treated with different concentrations of the alcoholic extract of the plant.


Introduction
The Leishmania parasite is one of the protozoa unicellular parasites, and it includes more than 30 species that parasitize the various types of vertebrates, especially mammals [1], as it infects about 20 species, including humans, causing many serious pathological effects [2].The host that transmits this parasite is the sand fly insect (Figure 1), which belongs to a Diptera order [3].The parasite causes a number of diseases that differ according to the type of parasite, including visceral leishmaniasis caused by Leishmania donovani, which is the most dangerous, as it infects approximately 200 -400 thousand people annually worldwide, also called black fever or Kala-azar [4].
The parasite migrates to the internal organs such as the liver, spleen and bone marrow, therefore it is called visceral and often leads to death if left untreated [5].It causes many symptoms such as fever, fatigue, anemia, weight loss, and enlarged liver and spleen, the disease is spread in many countries of the world such as East Africa, Latin America, Southeast Asia, and the Mediterranean [6].The Flagellated form is found inside the invertebrate host or in the culture medium, where it appears singular or in the form of clusters the parasite transforms in this stage from the oval to the elongated, fusiform-like shape equipped with a delicate anterior flagellum, and this is called the promastigote [7].
2 Due to the resistance of the Leishmania parasite to many chemical treatments and also because of the high toxicity of these treatments, which harm human health, therefore medicinal plants and herbs were used as an alternative to these treatments, which is characterized by being safe to use and highly effective in eliminating pathogens, including parasites, because it contains many active substances [8].Al-Khashin or Carrichtera annua plant belongs to the Cruciferae (Brassicaceae) and it is an annual herb with upward and densely branched stems, with a height ranging between 15-30 cm, it spreads in the slopes, valleys, hillsides, and roadsides, the plant grows in all types of soils [9], it blooms in mid-March to early May, the alcoholic extract of the plant has a superior ability to treat leishmaniasis, and its most important compounds are glycosides [10].This study aimed to investigate the effect of the alcoholic extract of the Carrichtera annua plant and its inhibitory efficacy on the growth of the parasite Leishmania donovani In vitro, to find safe alternatives to chemotherapy treatments that have many side effects.

Preparation of the alcoholic extract of Al-Khashin plant
Al-Khashin plant (Figure 2) was collected from a region 160 km west of the city of Ramadi, and it was diagnosed in the herbarium of Anbar University.After cleaning the plant parts, they were dried and the extract was prepared by the method of Taskin et al., [11], by adding 50 gm of the dried plant parts to 500 ml of ethyl alcohol at a concentration of 70% in a 1:10 ratio, the mixture was placed in the Magnetic stirrer device for homogeneous mixing at a medium speed at 40-45 °C and left for 72 hours.
The mixture was filtered in a Buchner Funnel, the filtrate was collected, and it was evaporated using a rotary evaporator at medium speed at a temperature of 45 °C to get rid of the solvent, and the solution was filtered by filter papers to remove the chlorophyll dye.Then the extract was returned to an evaporator to get rid of the water and to obtain the concentrated extract, and then the mixture was sterilized using the pasteurization process at 62 ° C for 10 minutes, and thus the standard solution of the alcoholic extract was obtained, and from it, the concentrations were prepared 0.25, 0.5, 1.5 and 2, 2.5, 3 mg/ml used in this study, and the number and time of generation were calculated according to Parker et al., method [12].

Development of Leishmania parasite
The stock culture of the promastigote stage of Leishmania donovani parasites MHOM/IQ/2005/ MRU15 was obtained from the Medical Research Center -College of Medicine -Nahrain University, which was enzymatically diagnosed by the Isoenzymes method.The prepared liquid culture medium [13] RPMI 1640 was used with 9 ml and 1 ml of fetal calf serum [14] added to it and 3 μl of the antibiotic Gentamycin, these components were filtered using 0.22 μl filter paper to purify the culture.The parasite development process was carried out in vitro by taking 0.1 ml from the original culture 2 x 10 3 cells / cm 3 and added to a sterile tube containing 0.9 ml of the culture medium and placed in the incubator at a temperature of 26 ° C for 3 days, and the growth of the parasite was confirmed and it was free from fungal and bacterial contamination, by examining a sample of it under a light microscope at a force of 10 X.

The effect of alcohol extract concentrations of the plant on the Leishmania parasite
To know the effect of the alcoholic extract of the plant on the parasite, 2.6 ml of the culture medium was placed in 21 sterile test tubes and divided into 7 groups, 0.3 ml of alcohol extract concentrations 0.25, 0.5, 1.5, 2, 2.5, 3 mg/ml were added to 6 groups and the first group was left as a control group 0.1 ml of the culture medium containing 5.6 x 10 3 cells/cm3 was added to the different groups, the tubes were placed in the incubator at 26 °C and the parasite growth was followed daily for four days.The number of the promastigote was calculated using the Hemocytometer, where 0.02 ml of culture was taken and 0.4 ml of physiological saline solution containing 1% formalin was added to kill the parasites and stabilize them during the counting process.And physiological saline was used to dilute in the case of dense growth, and the counting process was done using light microscopy at a force of 40X, thus the number of promastigotes was obtained per 1 cm 3 of the culture [15].[16] 2.4.1.Detection of Glycosides 5 ml of Benedict reagent was added to 1 ml of extract in a test tube and within a water bath heated to 100°C.After 5 minutes, the tube was cooled, and the presence of a red deposit indicated the existence of glycosides.

Detection of flavonoids
A 10 ml solution of 50% ethanol was added to 10 ml of 50% potassium hydroxide and mixed in an equal amount; the presence of a yellow color indicated the existence of flavonoids.

Detection of alkaloids
10 grams of plant extract was boiled with 50 ml of distilled water containing 4% hydrochloric acid (HCL).The solution was cooled and filtered.Following this, 0.5 ml of leachate was tested in a watch glass with 0.5 ml of Meyer reagent; the emergence of a white deposition confirmed the presence of alkaloids.

Detection of Terpenes
A mixture of the following chemical materials was prepared: 1 gram of plant extract was dissolved in 2 ml of chloroform, followed by a drop of anhydrous acetic acid and a drop of concentrated sulfuric acid.The presence of brown sediment indicated the existence of terpenes.

Detection of Saponins
3 ml of mercuric chloride solution (1%) was added to 5 ml of the extract; the appearance of a white sediment indicated the presence of the saponins.

Detection of Tannins
A few drops of lead acetate solution (1%) were added to 5 ml of the plant extract; the emergence of white gelatin deposits confirmed the presence of tannins.

Detection of Volatile Oils
A few drops of plant extract were added to the filter paper to reduce saturation and exposure to ultraviolet radiation, the appearance of gray color indicates the presence of volatile oils.

Statistical analysis
Statistical analysis was performed using Chi-square, and the test is less significant difference (L.S.D.) using the SAS statistical program [17].

Results and Discussion
The results showed the high efficiency of the alcoholic extract of C. annua plant in the treatment of the L. donovani parasite, as it led to an apparent inhibition of the growth of promastigote that was treated with different concentrations of the alcoholic extract compared to the control group (Figure 3) in which the promastigote numbers increased steadily during different exposure periods 24, 48, 72, and 96 hours, which amounted to (32.2, 48, 66.1, 80.2) x 103 cells/cm³, respectively, Whereas, the concentrations used for the alcoholic extract of 0.25, 0.5, 1.5, 2, 2.5, and 3 mg/ml led to an apparent inhibition of the growth of the Leishmania parasites, and a decrease in the number of parasites was observed with an increase in the concentration during the different growth periods, and significant differences were found between the growth rate of the treated and non-treated promastigote (control group), where the highest concentration of 3 mg/mL led to complete inhibition of parasite growth within 48 hours of treatment (Figure 4).
while the concentration of 2.5 mg/mL completely inhibited growth within 72 hours of treatment, while the concentration of 0.25 mg / ml led to a decrease in the average number of parasites to (28.6, 34, 52.3, 72.4)× 10 3 cells/cm 3 , respectively during the above exposure periods compared to the control treatment, while the inhibition increased at a concentration of 0.5 mg/ml to record the average number of parasites to (20.7, 27.1, 41, 55.4) × 10 3 cells/cm 3 , The concentration of 1.5 mg/ml had an inhibitory effect of 50% (LCD 50 ) during 96 hours of treatment compared to the control group, while the inhibition of parasite growth increased at a concentration of 2 mg/ml, leading to a clear decrease in the rate to (10, 12.5, 16.3, 18) × 10 3 cells/cm 3 compared to the control group in which no growth inhibition was obtained (Table 1).This is consistent with the study of Al Douri et al., [18] in their use of the alcoholic extract of the Viscum album in the treatment of the L. donovani parasite In vitro, and the study of Al-Doori [19] in its use of extract of Prunus avium on the growth of the cutaneous Leishmaniasis parasite in the culture medium.
The effectiveness of the alcoholic extract in inhibiting the growth of the L. donovani parasites is attributed to the fact that it contains many active substances such as alkaloids, flavonoids, glycosides, sterols, and volatile oils that make the cell membrane completely permeable, which leads to the entry of some substances that cause toxicity cytotoxic or the merging of the entering materials with other substances inside the cells, which leads to the disruption of their work or turning them into harmful substances causing the parasite to die or it may affect the parasite's cell membrane, causing it to necrosis [20].Alkaloids also inhibit the work of the topoisomerase enzyme, which participates in the various vital activities of the parasite, including the process of DNA replication and thus stopping the physiological activities and the parasite's destruction [21], [22].In addition to inhibiting the activity of Acetylcholinesterase enzyme (AchE), which prevents the passage of ions through the cell membrane and affects the vital activities of the parasite, thus causing cell breakdown and parasite death [23], [24].
(Table 2) shows the effect of the alcoholic extract of the plant C. annua plant on the number of generations of the promastigote, as the results showed the high inhibitory effect of this extract on the of generations, which led to a significant decrease in the number compared with the control group in which the number increased during different exposure periods 24, 48, 72 , 96 hours as the average number of generations was 76.5, 120.8, 147.3 and 232.2 generations, respectively, the highest inhibition was observed at the highest concentration of 3 mg/ml, which led to complete inhibition in the number of generations within 48 hours of treatment, while the concentration of 2.5 mg /ml led to a total inhibition of the number of generations during 72 hours of treatment.As for the rest of the concentrations, they caused an obviously increased decrease, as the lowest concentration of 0.25 mg/ml led to a slight decrease in the average number of generations to reach 68.9, 103.8, 125.6, 226.0 generations, respectively, during the above exposure periods.
While the concentration of 2 mg/ ml led to a significant decrease, as it reached 14.7, 43.1, 36.8, and 77.9 generations respectively, the number of generations decreased to 108.7 at a concentration of 1.5 mg /ml after 96 hours, which was considered (LCD50), compared with the control treatment, which reached 232.5 generations.This is in agreement with the Al-Fahadi study [25] which demonstrated the effect of high concentrations of Equisetum ravens and Urtica puluifera on the 6 promastigote number of Leishmania tropica in vitro.The reason for the number of generations is affected by the alcohol extract is due to the great inhibition of the growth of parasites, which is due to the suppression of the genetic material and the defect in the process of replication and the lack of its production for new generations or the multiplication process of the parasite affected by the presence of toxic substances and compounds that led to the lack of ideal conditions for the parasite to reproduce.The results showed the inhibitory effect of C. annua plant on the generation time, which led to an increase in the generation time for the production of generations due to inhibition of parasite growth compared with the control treatment (Table 3).Where the concentrations from 0.25 -3 mg/ml led to a change in the generation time by gradually increasing it, which affected the time needed to produce generations, as the L. donovani parasite needed 1.94, 1.31 hours at a concentration of 2.5 mg/ml and the highest time needed was 2.88 hours at a concentration of 3 mg /ml, as for the concentration of 1.5 mg/ml, it increased the time by 0.75, 0.64, 0.91, and 0.65 hours, respectively, compared with the control treatment, in which the parasite needed 0.35, 0.47, 0.59, and 0.39 hours, respectively, during different exposure periods.A direct correlation was observed between the concentration of the alcoholic extract of the plant and the generation time, so the greater the 4.0 0.0 0.0 0.0 concentration, the greater the generation time, and this was consistent with the results of the Al-Jabouri [26] using aqueous extracts of Melia azedarach fruits and Nerium oleander fruits and leaves on the growth and metabolism of Promastigote of the L. tropica parasite, which their study showed the inhibitory effect of these plants, which caused an increase in generation time during different exposure periods.* The number represents an average of three replications * Different letters indicate the presence of significant differences p ≥ 0.05.
(Table 4) shows the Chemical reagents used on the C. annua plant to find out its effective compounds, as the results showed that this plant contains many active compounds, which are alkaloids, glycosides, flavonoids, sterols, and volatile oils, which greatly affected the growth of the parasite and led to its death by 100%, as it had the primary role in inhibiting the growth of the parasite and eliminating it, and this is what El-Saadony et al., [27] indicated in the containment of many plants on substances and effective compounds and have the effectiveness of antibiotics in the effect on bacteria, fungi and parasites.The results demonstrate the efficiency of the alcoholic extract of Al-Khashin plant C. annua in treating the L. donovani parasite for the effectiveness of the chemical compounds it possesses, which greatly affected the parasite and led to its elimination.

Conclusion
The conclusion from the current study is the effectiveness of the alcoholic extract of the C. annua plant and its efficiency in eliminating the L. donovani parasite ex vivo through the significant inhibitory effect on promastigote growth in the culture medium.The concentration of 1.5 mg/ml led to an inhibition of promastigote growth by 50% at a rate of 38.1 x 10 cells/cm3, and a decrease in the number of generations and an increase in their time, as the number of generations reached 108.7 generations within 96 hours.An inverse relationship was observed between increasing the concentration and generation time, the effectiveness of the plant in eliminating the parasite is due to it containing many active substances that lead to the death of the parasite.For this reason, it is necessary to treat parasitic diseases using medicinal plants and herbs that are safe for the health of humans, animals, and the environment.

Table 1 .
Effect of alcoholic extract concentrations of C. annua plant on the number of promastigote of L. donovani parasite (x10 3 ) in different growth periods (number of Promastigote used in Culture 5.8x10 3 ) *The number represents an average of three replications * Different letters indicate the presence of significant differences p ≥ 0.05 Table 2: Effect of alcoholic extract concentrations of C. annua plant on the number of promastigote generations of L. donovani parasite in different growth periods (number of Promastigote used in Culture 5.8 x10 3 ) * The number represents an average of three replications * Different letters indicate the presence of significant differences p ≥ 0.05

Table 3 :
Effect of alcoholic extract concentrations of C. annua plant on the time of promastigote generations of L. donovani parasite in different growth periods (number of Promastigote used in Culture 5.8 x10 3 )

Table 4 .
Chemical reagents on active compounds in extract of the C. annua plant.