INTRODUCTION:

Moringa oleifera Lam. is a Moringaceae tree that grows quickly and is drought tolerant. Moringa, drumstick tree (for the long and slender seedpods), horseradish tree (for the roots that taste like horseradish), ben oil tree (for being high in behenic acid), and miracle tree are some of the common names given to it (for the medicinal properties)^1,2,3. For many years, moringaolifera has been used for health benefits. According to the WHO, over 5.6 billion people, or 80 percent of the world's population, rely on medicinal plants as part of their primary health care needs^4,5. In locations where medicinal plants are important, there is still a lot of knowledge about how to use them for various conditions. Medicinal plants are used to treat a variety of acute and chronic illnesses⁶. In certain circumstances, it's difficult to tell the difference between a medical herb and a food supplement. In most situations, the medicinal and/or nutritional value of a plant is found in numerous compounds within the plant, the direct action of which may not yet be fully understood^7,8.

Candida albicans is an opportunistic fungal pathogen that lives as a harmless commensal in the gastrointestinal and genitourinary tracts of around 70% of individuals and approximately 75% of women at some point in their lives^9,10,11. For immune compromised patients, some immunologically weak individuals, and even healthy people, it becomes an opportunistic pathogen. Candida albicans infection is generally referred to as candidiasis. Candidiasis is divided into two categories based on the severity of the infection.

The first category includes mucosal infections, the most well-known of which is thrush, which is marked by white spots on diseased membranes. Infections of the gastrointestinal epithelial cells, vaginal, or or opharyngeal mucosa are the most common. Furthermore, Vulvo Vaginal Candidiasis (VVC) is quite frequent among women, and some of them suffer from recurrent Vulvo Vaginal Candidiasis (RVC) (RVVC). It, on the other hand, causes life-threatening, systemic infections in critically ill patients; with a high fatality rate is about 30%^12,13,14,15. HIV-positive patients, transplant recipients, chemotherapy patients, and low-birth-weight newborns are all susceptible to systemic Candida infections^16,17,18,19.

The current research aimed to extract the active materials from oleifera leaves, and investigate its bioactivity against certain microbes

MATERIALS AND METHODS:

Preparing of plant extracts:

Three extracts of moringa leaves are prepared as fallowing:

Cold Water Extract:

Anesini and Perez (1993)⁹ method is adopted in preparing aqueous by weighed 100gm of powder and leaves put it in a special beaker and added 500ml of distilled and sterile water to it. The beaker was left in the vibrating incubator for 24 hours at a temperature of 37 °C, then the aqueous extract is filtered first by using a Buchner funnel with a piece of gauze to remove the large plant parts and secondly using Whatman No.1 filter paper. The filtrate was exposed to centrifuge with a force of 2500rpm for 15 minutes and then the filtrate is taken and put in the incubator at a temperature of 37°C for 48-72hours to obtain the powder, where it was put in the refrigerator under 4°C until use.

Hot Water Extract:

Anesini and Perez (1993)⁹ method is adopted in preparing aqueous by weighed 100gm of Moringa leaves and add 500ml of sterile distilled water to it, then put it in a saxolith device for 24hours, then filter it by a piece of gauze and then filter it by the filter unit (whatman1), then itake the filter and collect it in suitable glass containers, It is placed in the incubator at a temperature of 40-45 degrees Celsius until it dries up until it is ready for use.

Cold Alcoholic Extract:

Method is adopted in preparing aqueous by weighed 100 gm of powder and leaves put it in a special beaker and added 600ml of ethanol alcohol to it. The beaker was left in the vibrating incubator for 24hours at a temperature of 37°C, then the alcoholic extracts is filtered first by using a Buchner funnel with a piece of gauze to remove the large plant parts and secondly using Whatman No.1 filter paper. The filtrate was exposed to centrifuge with a force of 2500rpm for 15 minutes and then the filtrate is taken and put in the incubator at a temperature of 40-45°C for 48-72hours to obtain the powder, where it was put in the refrigerator under 4 °C until use. (Ladd et al, 1978)¹⁰.

Hot alcohol Extract:

method is adopted in preparing aqueous by weighed 100 gm of moringa leaves and placed in a piece of gauze and then placed inside the saxolites device and 700ml of ethanol alcohol was added to it at a concentration 99% for 24hours. The process was repeated several times to obtain a sufficient amount of the active substance, then the sample was transferred to a glass beaker with a known weight after filtering it with a glass funnel and a piece of gauze. It was placed in the incubator at a temperature of 40-45 degrees Celsius until it dries up until it is ready for use. (Ladd et al, 1978)¹⁰.

Preparation of 70% alcoholic water extract:

Method is adopted in preparing by weighed 100gm of Moringa leaves were placed in 70percent, equivalent to 420ml of ethanol alcohol and 180ml of distilled water, and 400ml of the extract was obtained (Vongsak et al., 2013)¹¹.

Activation and Preparation of the Fungal Suspension:

The fungal suspension was prepared by taking a few colonies by loop and put them in tubes containing mullerhinton broth for activating the fungal, then incubated the tubes to 18 hours. For each 1ml of the suspension, where it is preferred that the fungal number be close to (1.5 x 108) cells/ml. The isolates in our study were cultivated by using the agar well diffusion method ¹⁹0.1ml of the fungal suspension with a number of 10^8 cells/ml were spread on the surface of the culture medium, then we make a well in 10mm. In the culture medium, then equal quantities of different concentrations of the plant extract were placed in these wells. Then left for 15 minutes until the extract was absorbed. Then incubated at 37°C for 24 hours, the inhibition zone was measured in mm by Vernia.

Qualitative analysis of some bioactive components of plant extracts:

Glycosides:

The detection is performed by adding 2ml of Benedict's reagent to 1ml of the plant extract in a test tube, shaken the solution well and put in a boiling water bath for 5 minutes, then left the tube to cool and a red sediment is observed and this is evidence of the presence of glycosides²⁰.

Tannins:

Several drops of a 1% FeCl3 solution are added to a test tube containing 0.5ml of the extract and the appearance of a bluish green color was evidence of the presence of tannins²¹.

Phenols:

It was detected by using a solution of ferric chloride (Ferric Chloride), which is prepared by dissolving ferric chloride salt in distilled water at a ratio of (1%). This reagent gives a green or blue color when added to the amount of extract in the watch glass containing the phenolic compounds.

Flavonoids:

The detection solution is prepared by adding 10ml of ethyl alcohol at a concentration of 50% to 10ml of a solution of potassium hydroxide (KOH) at a concentration of 50%, after which equal quantities of the solution and the plant extract are mixed, and the appearance of the yellow color was evidence of the presence of flavonoids ²².

Saponin:

The test is performed by following the two methods below:

A- An aqueous solution of plant powder is prepared in the study and separately. The solution was placed in a test tube and was shaken very strongly. The appearance of thick and persistent foam for a long time was evidence of the presence of soap ²³.

B- (1-3) ml of mercuric chloride solution (HgCl2) at a concentration of 1% is added to 5 ml of the plant extract, and a white precipitate appeared as evidence of positive detection ¹⁹.

Coumarins:

Coumarin was detected according to what was mentioned in ²⁴. A little plant extract was placed for each of the aforementioned extracts in test tubes. The tubes were covered with filter papers moistened with diluted sodium hydroxide (NaOH) solution, and the tubes were placed in a boiling water bath for a few minutes, then the filter papers were exposed to a source of ultraviolet light. The appearance of a bright greenish-yellow color indicates the presence of coumarin.

Resins:

I followed the method presented in Al-Zubaidi 2020 ²⁵ for the detection of resins, (10) ml of each extract was taken and (20) ml of distilled water acidified with hydrochloric acid (4% HCl) was added to it. The presence of resins was indicated by the appearance of turbidity.

Alkaloids:

There are two methods for detection:

To begin, take one mL of the extract and mix in a few drops of Marquis reagent. The tint changed to a gritty gray color, indicating that alkaloids were present. Second, Mayer's reagent was applied to one ml of the extract, and the formation of a white precipitate or cream indicated the presence of alkaloids ²³.

Examination of the synergistic effect of moringa extracts with anti-fungal Saginstc. albicans:

According to the method of the Veterinary Drugs Research and Production Center, The following antifungals have been used (nystatin - clotrimazole - miconazole nitrite)It worked like this:

A. 0.1 gm of dried samples of moringa extracts were dissolved in 10 ml of distilled water and then placed in the Vortex device in order for the dissolution process to take place well.

B. 0.01 g of the three anti-fungals (nystatin-clotremazole-miconazole nitrate )used in this research is dissolved in 10 ml of distilled water(pH 6) and supplement the dissolution with a Vortex device and a little heat if necessary in order for the dissolution to take place well, and then we make a number of dilutions, which are as follows:

1. First dilution, 50 ul of moringa extract and 50 ul of anti-fungals are taken and placed in culture media.

2. The second dilution is taken 75 ul of moringa extracts and 25 ul of anti-fungal and placed on culture media.

3. The third dilution is taken 95 ul of Moringa extracts and 5 ul of anti-fungal and applied to culture media, The dishes are placed in the incubator for 24 hours, after which we observe the results and measure the diameters.

RESULTS AND DISCUSSION:

The results as presented in Table 1; the phytochemical screening conducted on leaf extract of Moringaoleifera revealed the presence of some bioactive components such as alkaloids, tannins, phenol, flavonoids, glycosides, saponins, tannins and protein . The presence of some of these bioactive components confirms similar research conducted by ^{26, 27, 28}.

Table 1: The Chemical constituents of leaf extracts of Moringa Oleifera

	Detection type	Cold Alcoholic Extract	Cold Water Extract	Hot water Extract	Hot Alcohol Extract	70% Alcoholic water Extract
1	Tannins Test	+	+	+	+	+
2	Carbohydrate Test	_	_	_	_	_
3	Glycosides Test	+	+	+	+	+
4	Phenols Test	+	+	++	_	+
5	Resins Test	+	_	_	+	_
6	Flavonoids Test	+	+	+	+	+
7	Saponin Test	+	+	+	+	+
8	Alkaloid Test	+	+	+	+	+
9	Protein Test	+	+	+	+	+
10	Coumarins Test	+	+	+	+	+
11	Terpenes Test	_	_	_	_	_
12	Steroids Test	_	_	_	_	_

Table No. ( 2) and Fig (1) shows significant differences for the effect of Moringa extracts and nystatin on Candida albican, as it found that the concentration 50/50 hot alcoholic extract showed the highest inhibition Zone 25mm followed by the cold alcoholic extract 24.67 in compared with other extract hot water extract, 70% cold aqueous alcohol (20, 1.33, 16mm) respectively.

It was also found that the 25/75 dilution showed significant differences between the extracts, as it was found that the hot alcoholic extract had the highest inhibition zone on Candida albican 25.33mm in compared to other hot water extracts, 70% cold water alcoholic (19.33, 19, 15, 15mm), respectively. While at 95/5 dilution, it was found that the hot alcoholic extract showed the highest inhibitory activity against Candida albican mm 26 in compared to the other extracts: hot water, cold alcoholic, and 70% alcoholic water ( 17, 16, 14.33) ), respectively.

Table 2: Shows the results of the synergistic efficacy of Moringa leaf extracts with the antifungal Nystatin on Candida albicans

Moringa extracts	50:50:00	75:25:00	95:05:00	p value
Cold Alcoholic	24.67 ±0.58 A a	19 ±1 A b	16 ±1 A c	sig
Cold water	16 ±1 B a	15 ±2 B a	-	not sig
70%Alcoholic	18.33 ±2.08 C a	15 ±1 B a	14.33 ±2.52 B a	not sig
Hot Alcoholic	25 ±1 A	25.33 ±1.53 C	26 ±1 C	not sig
Hot Water	20 ±1 C	19.33 ±3.06 A	17 ±2 A	not sig
p value	Sig	sig	Sig

Figure 1: The inhibition zone of C. albicans against moringa extract and nystatin

Table No. ( 3) and Fig(2)shows significant differences for the effect of Moringa extracts and miconazole on Candida albican, as it found that the concentration 50/50hot alcoholic extract showed the highest inhibition Zone 26.33mm followed by the cold alcoholic extract 23 in compared with other extract Cold water, Hot water, 70% alcoholic l (17, 16, 14.67mm) respectively. It was also found that the 25/75 dilution showed significant differences between the extracts, as it was found that the hot alcoholic extract had the highest inhibition zone on Candida albican 24 mm in compared to other Cold alcoholic,hot water, 70% alcoholic, cold water (21.33, 17, 16.33, 13.67mm), respectively While at 95/5 dilution, it was found that the hot alcoholic and cold alcoholic extract s showed the highest inhibitory activity against Candida albican 25 mm in compared to the other extracts: hot water, 70% alcoholic, cold water (16, 14.33, 13), respectively.

Table 3: Shows the results of the synergistic efficacy of Moringa leaf extracts with the antifungal Miconazole on Candida albican

Moringa extract	50:50:00	75:25:00	95:05:00	Pvalue
Cold Alcoholic	23±1 A a	21.33±2.08 A a	25±1 A a	Not sig
Cold water	17±1 B a	13.67±0.58 B b	13±1 B b	sig
70%Alcoholic	14.67±1.15 B a	16.33±2.52 C a	14.33±1.53 B a	Not sig
Hot Alcoholic	26.33±0.58 C a	24±1 A a	25±1 A a	Not sig
Hot Water	16±1 B	17±1 C	16±1 B	Not sig
P value	Sig	Sig	sig

Figure 2: Inhibition zone of C. albicans against moringa extract and Miconazole nitrate

Table No. ( 4) and Fig(3)shows significant differences for the effect of Moringa extracts and clotremazol on Candida albican, as it found that the concentration 50/50hot alcoholic extract showed the highest inhibition Zone 23mm followed by the cold alcoholic 22.67 in compared with other extract Hot water, Cold water, 70% alcoholic l (22, 20, 17mm) respectively. It was also found that the 25/75 dilution showed significant differences between the extracts, as it was found that the hot alcoholic extract had the highest inhibition zone on Candida albican 27 mm in compared to other Cold alcoholic, Hot water, 70% alcoholic,and Cold water, (23.33, 19, 18, 17mm), respectively While at 95/5 dilution, it was found that the Cold alcoholic showed the highest inhibitory activity against Candida albican 24.33mm in compared to the other extracts: Hot alcoholic, hot water, cold water,70% alcoholic, (23.33, 18, 16.33, 16) ), respectively.

Table 4: Shows the results of the synergistic efficacy of Moringa leaf extracts with the antifungal Clotremazol on Candida albican

Moringa extract	50:50:00	75:25:00	95:05:00	P value
Cold Alcoholic	22.67±0.58 A a	23.33±1.53 A a	24.33±0.58 A a	Not sig
Cold water	17±1 B a	17±1 B a	16.33±1.53 B a	Not sig
70% Alcoholic	20±1 C a	19±1 B a	16±1 B b	Sig
Hot Alcoholic	23±1 A a	27.67±0.58 C b	23.33±2.52 A a	Sig
Hot Water	22±1 A a	18±1 B b	18±1 B b	Sig
P value	Sig	Sig	Sig	P value

Fig 3: The inhibition zone of C. albicans against moringa extract and clotremazole

CONCLUSION:

1. The leaf of Moringa Oleifera has shown the presence of some bioactive components which possesses activity against some microorganisms.

2. Candida albicans is most sensitive to moringa extracts and antifungal (Nystatin, Miconazole nitrate, Clotremazole).

3. Moringa extracts and the antifungal nystatin showed the highest efficacy at 95/5 on Candida albicans.

4. Moringa extracts and the antifungal miconazol showed the highest efficacy at 50/50 on Candida albicans.

5. Moringa extracts and the antifungal clotremazol showed the highest efficacy at 75/25 on Candida albicans.

RECOMMENDATION:

· Further researches should be carried out on the dosage and in vivo evaluation of the leaf extract.

· Candida albicans is most sensitive to moringa extracts and antifungal (Nystatin, Miconazole nitrate, Clotremazole), it was recommended to carry out more studies toward other cell lines microbes.

ACKNOWLEDGMENT:

The authors would like to thank Mustansiriyah University (www.uomustansiriyah.edu.iq) Baghdad-Iraq for its support in the present work.

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Received on 25.05.2022 Modified on 02.07.2022

Research J. Pharm. and Tech 2023; 16(3):1369-1374.

DOI: 10.52711/0974-360X.2023.00225