Antifungal Potential of silver nanoparticles from Acacia nilotica Pod against Dermatophytes

Yunusa Saheed 

Microbiology Department, Yobe State University. Damaturu, Nigeria

Article Info:

_________________________________________

 Article History:

Received 14 August 2021      

Reviewed 27 September 2021

Accepted 03 October 2021  

Published 15 October 2021  

_________________________________________

Cite this article as: 

Saheed Y, Antifungal Potential of silver nanoparticles from Acacia nilotica Pod against Dermatophytes, Journal of Drug Delivery and Therapeutics. 2021; 11(5-S):85-95

DOI: http://dx.doi.org/10.22270/jddt.v11i5-S.5024       

_________________________________________

*Address for Correspondence:  

Yunusa Saheed, Microbiology Department, Yobe State University. Damaturu, Nigeria. E-mail Address: soy4kd@gmail.com

ORCID ID: https://orcid.org/0000-0003-4570-4936

Abstract

______________________________________________________________________________________________________

Background: Superficial fungal infections can lead to systemic infection in immune-compromised individuals. Acacia nilotica pod have been used ethnomedical to treat dermatophytes infection for ages. In this study, the anti-dermatophytes potential of silver nanoparticles biogenically synthesized using extracts from the pod of A. nilotica against dermatophytes isolated from secondary care hospital in Damaturu, North-East Nigeria. Experimental:Phytochemical screening and GC-MS analysis were conducted to screen the phytoconstituents of the plant material. The synthesized AgNPs were characterized by Uv-vis, FT-IR, and SEM. 133 samples (skin scraping) were screened for dermatophytes and antifungal susceptibility testing were conducted against the isolates using the aqueous, methanolic extracts, and AgNPs. Results: Phytochemical screening revealed the presence of alkaloids, flavonoids, glycosides, oxalate, quinones, phenols, saponins, terpenoids, GS-MS revealed the presence Polyphenolics including Hexadecenoic, Octadecanoic and Undecanoic acids, Catechol, pyrogallol, 3-methylpyridine, and methyl mannose. Uv-vis of synthesized AgNPs exhibited double sharp absorbance at 308nm and 311nm, FT-IR showed functional groups, thus, OH, C – H, C≡N, and C=O stretches of phenolics, alkenes, nitrile, and ketones respectively, and SEM showed various characteristic shapes and sizes. out of 133 samples collected, 54% were male and the age group with the highest clinical presentation were51 – 60, followed by 1 – 10years. according to clinical presentation, Tinea pedis (36%) and T. capitis (29%) were the commonest which may be due to constant contact with water and limited hair care. Aetiologic agents isolated include Trichophyton mentagrophyles (51%), T. rubrum (34%), and T. tonsurans (14%), although, there is no significant relationship between the clinical presentation and disease agent (p-value < 21.3 at 95% confidence level). AgNPs showed wider consistent zones of inhibitions against all the isolates. Discussion: A. nilotica is indeed very rich in polyphenolics. Foot and hair infections commonest in the study could be due to current weather conditions but sharing of footwear, caps, and brushes should be discouraged. Conclusion: This study opens up possibilities for exploration of this eco and economic approach of improving the medicinal value of plants, an opportunity the pharmaceutical industries can utilize. This study is the first to report prevalence of dermatophytes in the area and explored the AgNPs against it.

Keywords: Damaturu, susceptibility, catechol, ethnomedicinal, Acacia nilotica, AgNPs

 


 

1. INTRODUCTION

Dermatophytes are fungal infections on the surface and deep-rooted to the skin, hair, and nails1Dermatophytes and Candida spp constitute a serious problem especially in tropical and subtropical developing countries where hot and humid weather predisposes to fungal infections by increased sweating and maceration of the skin. Other predisposing factors include poor socio-economic conditions, overcrowding, poor hygiene, and lack of adequate water supply. Dermatophyte infections can also be acquired directly through fomites such as upholstery, hairbrushes, and combs. Dermatophytes in pupils cause morbidity and lower their quality of life leading to poor school attendance and irritation in adults2. They are believed to affect 20% to 25% of the world's population 3, and the incidence continues to increase4 due to immunosuppression procedures and diseases. They consist of about 40 fungal species from three genera of TrichophytonMicrosporum, and Epidermophyton leading to infections called dermatophytosis, ringworm, or Tinea (named according to the area of infection).

Due to the reduced efficacy of pharmaceutical products, possible side effects, resistance associated with some of the existing drugs, and increase pace of immunosuppressed individuals, management of dermatophytoses, though some considered it recurrent and seasonal, could be a public health concern shortly. Therefore, much of the attention has been paid to plant extracts to treat fungal infections, and treatment of these infections using nanoparticles is an alternative approach that can reduce treatment period and resistance 5. 

Natural medicinal plants promote self-healing, good health, and durability in ayurvedic medicine practices and have acknowledged that A. nilotica can provide the nutrients and therapeutic ingredients to prevent, mitigate or treat any diseases or conditions. Acacia nilotica pods are strongly constricted, white-grey, hairy, and thick 6.

A. nilotica is a pantropical and subtropical genus with species abundant throughout Asia, Australia, Africa, and America 7. It contains Polyphenol which can be of prime use to humans, the pods have potential antioxidants and are found effective in protecting plasmid DNA and human serum albumin protein oxidation induced by hydroxyl radicals though, the uses of Polyphenols in the plant is still vague8, other parts of the plant have been used for various purposes, the pod has been used ethnomedical for ages in the treatment of skin discomfort.

Silver Nanoparticle is environmentally friendly, non-toxic, chemically stable, low cost-effective when prepared green compared to other methods and, thus making it a cheaper alternative when available with the high potentiality to serve as coating agents to the phytocompounds in the plant extract for effective drug delivery and treatments.

The green synthesis does not utilize any potentially harmful chemicals that can add to global warming. If the enormous challenge is to develop an antifungal agent which has fungicidal properties and has the highest rate of mycological cure with the shortest duration of usage9 then, any drug developed using AgNPs synthesized from the pod of Acacia nilotica may be effective for the treatment of fungal diseases in plant and animal with the novelty of showing the possibilities of treatment success in the era of treatment failure.

This work is expected to scientifically back the use of the pod as anti-dermatophytoses, add to the existing literature about the advantages of Green AgNPs.

2. METHODOLOGY

2. 1. Collection of plant material and preparation of A. nilotica pod extracts

Fresh, ripe green pods of A. nilotica were collected from Nguru, Identified and authenticated in Yobe State University Biological Garden. The air-dried pods were crushed with the aid of pestle and mortar, sieved, and extracted at 25g/250ml distilled water for 72hrs at 200rpm in a shaking incubator. The extracts were filtered using Whatman no. 1 paper, concentrated, and evaporated using a rotary evaporator, and lyophilized for 30hours in a freeze dryer. The methanolic extract was extracted exhaustively using a soxhlet extractor.

2.2 Phytochemical screening 

Both aqueous and Methanolic extracts were tested for their chemical compositions. The tests carried out include:

2.2.1 Test for the presence of Alkaloids

About 0.5g of each extract was taken into a test tube and stirred with 3ml 1% aqueous hydrochloric acid on a steam bath. 1ml of the filtrate was treated with few drops of Mayer’s reagent and picric acid solution. Precipitation with either reagent was taken as preliminary evidence for the presence of alkaloids10.

2.2.2 Test for the presence of Anthraquinones

Bontrager's test was used for the detection of anthraquinones.0.5g of each extract was taken into a dry test tube and 5ml of chloroform was added and shaken for 5minutes. The extract was filtered and the filtrate was shaken with an equal volume of 100% ammonia solution. Pink, violet, or red colour in the lower layer indicate the presence of three anthraquinones11.

2.2.3 Test for the presence of steroid

About 100mg of each extract was taken into a dry test tube and dissolve in 2ml of chloroform. Concentrated sulphuric acid was carefully added down the side of the test tube to form a lower layer. The formation of reddish-brown colour at the interphase is indicative of the presence of steroidal ring11.

2.2.4 Test for the presence of flavonoids

Plant extract weighing 2.0g was taken into the test tube and detained with acetone on the water bath. The mixture was filtered while hot. The filtrate was cooled and used for sodium hydroxide test:

Sodium hydroxide test for flavonoids.5ml of 20% sodium hydroxide was added to an equal volume of the detained plant extract. A yellow solution indicates the presence of flavonoids.

2.2.5 Test for the presence of glycosides

100mg (0.1g) of each plant extracts were dissolved in 1ml of glacial acetic acid containing one drop of Ferric chloride solution in a test tube. Then 1ml of concentrated sulphuric acid was added. A brown ring obtained indicate the presence of deoxy-sugar characteristics of cardenolides 12.

2.2.6 Test for the presence of saponins

0.5g of each extract was taken into a dry test tube and dissolved in distilled water in a test tube and was shaken vigorously for two minutes and observed for frothing which tube and was shaken vigorously for two minutes and observed for frothing which persisted on warming. This was taken as evidence for the presence of saponins.

2.2.7 Test for the presence of Tannins

About 0.5g of each extract was taken into a dry test tube and stirred with 1ml of distill water, it was filtered and Ferric chloride solution was added to the filtrate. A blue, a black, or green, or blue-green precipitate formed indicate the presence of Tannins12.

2.2.8 Test for the presence of terpenoids

0.1g of each extract was taken into a test tube and dissolved in 1ml of chloroform and 1ml of acetic anhydride was added. Two drops of concentrated sulphuric acid were then added. A pink colour that changes to bluish green indicates the presence of terpenes.

2.3 GC-MS Analysis of A. nilotica Pod

GC-MS analysis of A. nilotica pod will be performed on Agilent GC-MSD (7890B-5977A) at the Research Laboratory in the Chemistry Department, Yobe State University.

2.4 Biosynthesis and Characterization studies on A. nilotica pod AgNPs.

2.4.1 Preparation0.0169g of AgNO3 was dissolved in 100ml distilled water in a dark place. 

Different ratios of 2:8 (2ml P.E +8ml AgNO3 solution), 1:9, 7:3, 3:7, and 5:5. 10ml aqueous extract (10g in 100ml D. H2O) of A. nilotica pod were added slowly to 90ml 1mM silver nitrate in 250ml conical flask,5. This solution was incubated in the dark at 370c until use. A control solution (without extract) was also incubated under the same condition13.

2.4.2 Uv-Vis: Optical Density (OD) using UV-2401, India, performed between 200-800nm with a resolution of 1nm and Scanning speed of 300nm/min. The reduction of Ag+ ion was monitored by measuring the UV-vis spectrum of 1ml aliquots sample and 2ml deionized water in a quartz cell. Silver Nitrate (1mM) was used to adjust the baseline as a blank14.

2.4.3 FTIR: Surface topology on the powder sample of AgNPs and the crude extract was carried out using IRAffinity-1S Spectrometer (Buck Scientific – 530). The AgNP solution was centrifuged at 10,000 rpm for20 min. The solid residue obtained was then dried at room temperature, and the powder obtained was used for FTIR measurement using KBr.

2.4.4 SEM: Scanning Electron Microscopy (SEM) was taken using. The morphological properties of the as-synthesized Ag nanoparticles were studied using Phenom Pro-X 800-07334 operated at 25 kV. After the preparation of the nanoparticles, the suspension of nanoparticles in distilled water was used for SEM analysis by fabricating a drop of suspension onto a clean carbon-coated corer grid and allowing water to completely evaporate. The dried sample was then mounted with the aid of a sample holder15,16. An Enlarged SEM image was observed5.

2.5 Isolation and identification of dermatophytes fungi

2.5.1 Sample collection: Skin Scraping will be collected, using a sterile surgical blade, placed in a clean labeled envelope, as described by17, and was transported to Yobe State University, Microbiology Laboratory.

2.5.2 Media preparation: The media (Dextrose Agar (PDA)) will be prepared according to the manufacturer’s specifications. The media will be poured into sterile Petri dishes and allowed to solidify.

2.5.3 Inoculations: Specimens (scales) collected from each patient enrolled in the study will be inoculated onto the culture plates with PDA and incubated for 96 to 168 hours at room temperature (25°C to 35°C). The culture will be examined for the growth of dermatophytes. The colonies will be Sub-cultured to obtain typical growth of dermatophytes18.

2.5.4 Macroscopy: Positive cultures were examined microscopically for colour, the texture of colonies, their topography, and the pigmentation produced as described18.

2.5.5 MicroscopySuspected cultures were also viewed microscopically the following staining with lactophenol cotton blue (LPCB) at 10x and 40x magnification for conidia as described by18

2.5.6 Tease Mount PreparationA clean glass slide will be placed on the workbench and a small drop of LPCB solution in the middle of the slide, a fragment of a fungal colony was removed (approximately 1-2 mm from the periphery) with a wooden stick and place on the LPCB solution. The fragment will be teased gently with two wooden sticks until it has been separated. It was covered with a coverslip. The slide was carefully examined under low (x10) and high power (x40) objectives of the microscope for the characteristic shape and arrangement of the spores, hyphae, etc. as described by18.

2.6 In- vitro Antifungal Assay

The anti-dermatophytic activity of methanolic, aqueous extracts, and AgNPs of A. nilotica pod were determined using the Agar well diffusion method as described by 5and 19. Cork borer was used to bore holes on solidified agar, with the extracts dissolved in 5%DMSO with sterile saline differently at different concentrations of 400, 200, 100, 50, and 25 g/ml. Using Micro pipete, extracts were poured into the agar well of about 25ml of molten Potato Dextrose ager (HKM – HCM 050, Guanadong Huankai) plate containing suspension of 0.5 McFarland turbidity in 0.85% saline with a few colonies from a 24 -48hours old culture of isolates and the plates incubated at 350c.  Zones of inhibition (ZOI) that appear as clear areas surrounding the well from which the substances with antimicrobial activity diffused and the diameter of the ZOI measured in millimeters with a ruler.

3. RESULTS

3.1 Collection of Plant Materials, phytochemical screening, and GC-MS analysis of Acacia nilotica pod.

The Acacia nilotica od greenish and brownish when dried, it is brown glittering crystals were observed after lyophilization. The methanolic extract is a little darker when compared to the aqueous extract. The yield is presented in table 1.

Table 1: The yield

The plant parts

Solvent

Yield in percentage (%)

Fruit pod of A. nilotica

Aqueous

6.60

 

Methanol

48

The percentage of yield is relatively close to the one earlier reported 20.


 

 

The aqueous extracts of Acacia nilotica Pod yielded

imageimage

Figure 1: (a.) A nilotica pod (b.) A. nilotica  pod extract


 

3.2 Phytochemical screening

Table 2: Phytochemical Screening of Acacia nilotica Pod

 

Phytochemicals

Acacia nilotica pod extract and Test Inference

        Aqueous                            Methanol

Alkaloid 

+

-

Flavonoids

+

+

Glycosides

+

+

Oxalate 

-

-

Phenols

+

+

Quinones

+

-

Saponins

+

+

Steroids

+

-

Tannins

+

+

Terpenoids

+

+

 

Table 2: shows the presence of Alkaloids, Flavonoids, Cardiac glycoside, phenols, Quinones, Saponings, steroids, Tannins, and Terpenoids in Aqueous extract while all except alkaloids quinones and steroids were detected in methanolic extract of A. nilotica pod.

The results in this regard agree with the work of 21 for aqueous extracts. 20 also identified Glycosides, Flavonoid, Terenoid, Saponins, and Tannins in both H2O and Methanolic extracts.

Flavonoids are found in food, responsible for taste, colour, and phenolic compounds therapeutic potential has led to an upsurge in plants research22. They have also been found to contain antifungal activity, a wide range of antibiotics, they are a good coating agent, chelate free radicals and reactive oxygen species, increases the formation of capillaries and fibroblast, form complexes with proteins of cell wells20. Phenols stimulate PGE formation based on their activities as a co-substrate for peroxidase reaction21Polyphenols have excellent anti-fungal capabilities as well as a very good candidate for AgNPs. Ethanol extracts also showed all including sterol which was not observed in the methanolic extract in this study, this may be due to variation in the polarity of the extracting medium or reaction conditions23,24.


 

 

3.3 GC – MS Analysis of A. nilotica pod

 

Figure 2: GC-MS spectrogram of A. nilotica pod

Table 3: Active fractions of Acacia nilotica pod from GC – MS.

Derivatives

RT

MW(g/mol)

Peak Area

MF

2,2'-Azoxybis[3-methylpyridine]

1.184

228

1015254.62

C12H12N4O

1,2,3-Benzenetriol (pyrogallol)

6.62

126

79112701.84

C6H6O3

3-Methylmannoside

8.973

194

11856552.66

C7H14O6

Hexadecanoic acid, methyl ester

10.436

270

1904458.56

C17H34O2

Undecanoic acid

11.014

186

1434748.76

C11H22O2

9,12-Octadecadienoic acid, methyl ester

12.542

294

4622270.58

C19H34O2

6-Octadecenoic acid, methyl ester, (Z)-

12.599

296

4827313.37

C19H36O2

Methyl stearate

12.863

298

1023216.63

C19H38O2

9,12-Octadecadienoic acid (Z,Z)-

13.172

280

8493950.49

C18H32O2

Catechol

13.395

110

1079240.11

C6H6O2

 


 

The GC-MS spectrogram (Fig.2) led to the identification of some compounds (Table 3). A total of 10 peaks of different but closely related phytocompounds with their retention time and area presented. They are mostly polyunsaturated fatty acids. Including Azoxysis, 1,2,3 benzenetriol (pyrogallol), methylmanoneside, hexadecanoic, undecanoic, octadecanoic, methyl stearate, pyrocatechol, and Linoleic acid.

 The result in this study has been reported by previous scholars,25,26. 27also, obtain similar phytocompounds from Methanol extract claiming their antibiotic potentials. Methyl stearate is an antifungal28. Undecanoic acid - inhibit biofilm formation for some of the capric acids and also antibiotics. Capric and capric acids are chain fatty acids rarely found in food, it is also called pyrocatechol or 1, 2, -dihydroxy benzene. Benzenediol isomers occurring along with polyphenols oxidase or catecholase. 

According to29, polyphenol oxidase was identified from propolis when purified from black poplar leaves. Catechol (O-diphenol) occurs naturally in fruits, vegetables, and plants PPO causes enzymes localized on the thylakoids of chloroplast with two distant possible catalyzes (1.) Hydroxylation of monophenols to O-diphenol and (2.) Oxidation of p and O quinones. 

The same claim was supported by30, after isolating catechol from poplar bud and argued that the origin of polyphenols oxidase is from poplar bud as they were not detected in other parts of bees. The vast majority of phenolic compounds in higher lant vacuole could be involved in the production of O-quinones or catechol is oxidized into a quinone.

24detected galloylated catechin and gallocatechin derivatives in Acacia extract using MSfragmentation pattern. Benzene, hexadecenoic, and heptacosane extracts of Acacia nilotica have demonstrated Anti-microbial and mosquitocidal activities31,25. Similar compounds and derivatives were also classified as asteroids – Octadecanoic acid, Tannins- ethyl gallate, pyrocatechol, and Terpenoids – Tetradecanoic acid, Hexadecanoic 

32demonstrated the catalytic role of Acacia nilotica pod using the phytoconstituent – gallic acid, ellagic acid, epicatechin, and rutin- suggesting them as well reducing agents for the synthesis of AgNPs and capping candidates. polyphenol and tannins from A. nilotica pod have potent antibacterial and significant anti-biofilm activities against G+ve and G-ve bacteria as well as promoting wound healing efficiently within 15 days33.

Flavonoids having catechol groups are fairly stable and various pyrcathechins have been suggested for therapeutic applications Octadecanoic serves as an anti-inflammatory34.

Acacia extracts Secondary metabolites – are a complex mixture of active reducing biomolecules like antioxidant polyphenols, flavonoids, and phenolic acids, responsible for the reduction of metal ions and stabilization of Nano articles35,36.

The polyphenols and phenolic acids which are the major constituents of Acacia nilotica pod have no doubt demonstrated nucleation with AgNO3 solution indicating the reduction of Ag+ to Ag0.


 

 

3.4 Synthesis and Characterization of AgNPs


b

aaa

 image image



d

c

  imageimage



f

e

  imageimage


Figure 3: (a) AgNPs at different ratio, (b)1:9 AgNPs,(c) FTIR Spectrogram ,(d) Uv-vis spectrogram, (e)SEM image and (f) SEM image with ruler showing some enlarged particles.


 

3.4.1 Synthesized particles

The clear silver nitrate solutions showed colours change immediately after addition of the extracts as presented in Fig. 3. (a.) showed the colour of the particles at different ratios. 2:8, 1:9, 3:7, 7:3, 5:5, and the extract alone. 1:9 was considered for the rest of the study, fig.3(b). The colour change to light brown has been attributed to plasmons resonance formation on the surface of the molecules and oscillation of metal nanoparticles conduction electrons16.

3.4.2 Uv-Vis

The A. nilotica pod green nanoparticle analyzed using UV-2401, India. Uv-vis spectrophotometer confirmed the surface plasmon resonance bands with the sharp double peaks at 308 and 311nm (Fig. 3d). This indicates that the nanoparticle started to form immediately after the mixture and the double bands indicate that the Ag0 has been reduced to form a nanoparticle.

Lower double bands have been reported at 217 and 283nm37, which is in contrast with the more common 430 – 450 ranges reported in other studies38,39&16.

3.4.3 FTIR

The A. nilotica Pod extract was used as a reducing agent in synthesizing the nanoparticle. The functional groups indicating on the surfaces of AgNPs were confirmed using FTIR spectrogram (Fig. 3(c). The FTIR showed absorption peak thus, 3428.3313 cm-1, 2830.9657, 2391.0141, 1951.7333, 1716.9535, 1575.1434. This absorption indicates the possibility of bio-reduction and stability of the nanoparticles leading to the formation of surface plasmon resonance.

3428.3313 cm-1 – OH bond stretching of phenolics40. The double stretch between 2980 and 2830cm-1 is due to the C-H stretching of nitrile or alkanes or C-H bending, at 2371, cm-1 C≡N stretching of nitrile, C=O stretch of ketones or carbonyl group, and N-0 symmetric stretch of nitrile.

It can be deduced from the FTIR that some of the phytocompounds present in the aqueous extract such as polyphenols have been involved in the redox reaction of silver salt to Ag0. The compounds would have capped and stabilized the solution by preventing the accumulation of the silver nanoparticles. As a good capping agent.

Other scholars have also reported similar peaks for A. Senegal and A. tottilis41 and A. nilotica 37.

3.4.4 SEM

Figure 3 (e and f) Shows the morphological properties of the nanoparticle as can be observed on the micrograph which confirmed that the nanoparticle development appeared to have close shapes and large surface diameters revealing the aggregation although only zoomed 500X. Other scholars have also reported diverse shapes and sizes in A. nilotica nanoparticles5


 

 

3.5 Sample collection, isolation, and identification of Fungal Isolates

Table 4: Distribution of Dermatophytes according to Dermographic Characteristics

Demographic Characteristics 

Number of Sample

Percentage Distribution

1   – 10

21

15.8

11 – 20

18

13.5

21 – 30

14

10.5

31 – 40

10

07.5

41 – 50

21

15.8

51 – 60 

33

24.8

61 – 70 

09

06.8

71 – 80

04

03.0

81 – 90

03

02.3

Total 

133

100

Sex

 

 

Male

73

54.9

Female

60

45.1

Total 

133

100

 


 

3.5.1 Distribution of Dermatophytes according to demographic Characteristics.

Out of 133 individuals enrolled in the study, 73(54%) were male while female counterparts took 60(45%) and the age group with the highest clinical presentation were 51 – 60 (24%) years followed by 1 -10 and 41 – 50 (15.8%) the lowest represented age groups were > 70 years with 7(5.3%) as shown in Table 4.

This agrees with the findings of 2, establishing that males were more affected by dermatophytes than the female counterpart in their work sampling children from Bauchi state and disagree with the age group with the highest prevalence is 9 – 12years only because, they have enrolled children alone in their study. In Maiduguri,41 also reported that children 7 – 11 years (8.1%) are more infected followed by 4 – 6years (6.9%) and 12 – 16years (3.6%). The isolates recovered according to children also followed the same order at 64%, 25.4%, and 10.5%.


 

Table 5: Number of Samples Collected According to Clinical Presentation 

Clinical Diseases

Number of Cases

Percentage Distribution (%)

Tinea barbae 

01

0.8

Tinea capitis

39

29. 3

Tinea corporis

13

9.8

Tinea cruris

04

3.0

Tinea mannum

11

8.3

Tinea pedis

49

36.8

Tinea unguium

16

12.0

Total

133

100

 

 


 

3.5.2 Number of Samples according to Clinical presentation

The data as presented in table 5, shows that the commonest clinical lesions were Tinea. Pedis, (36.8%), T. capitis (29.3%), T. unguium (12%), and the lowest representative were T. barbae (0.8%) and T. cruris (3%). Table 5. This result agrees with42

3.5.3 Distribution and frequency of dermatophytic fungi Isolated

49 isolates of Trichophyton mentagrophytes 25(51%), T. rubrum 17(34.7%), and Trichophyton tonsurans 7(14.3%) were recovered according to infected sites as presented in table 6. 

This corresponds to previous reports from the neighboring states of Bauchi, Borno, and Kano.42, 2020 described Trichophyton species (16%) as the leading cause of tineasis in Kano and the only contrary result is that they reported Tinea capitis as the leading clinical disease while this report presented Tinea corpori and T. unguium ahead of T. capitis. T. mentagrophyles (16.7%) and T. tonsurans (10.5%) were among the isolates identified in Maiduguri 41.

This study disagrees with the reports that non-dermatophyte molds (51.7%) were mostly isolated from dermatophytosis cases in Bauchi2.


 

 

Table 6. Distribution and Frequency dermatophytic fungi Isolated According to the Infected Sites

Diseases/ Isolates 

T. mentagrophyles

T. rubrum

T. tonsurans

Total 

Tinea barbae

-

-

-

-

Tinea capitis

02

05

01

08

Tinea corporis

10

04

03

17

Tinea cruris

-

-

-

-

Tinea mannum

03

01

01

05

Tinea pedis

02

05

-

07

Tinea unguium

08

02

02

12

Frequency 

25

17

07

49

Percentage 

51

34.7

14.3

100

 

 

 

 

 

 

 

 

 

 

 

Table 7: Antifungal activities

                                                                           Number of positive and sensitive Isolates

ZOI(MM)

Acacia Pod Aqueous Extract(mg/ml)

Acacia Pod Methanolic Extract(mg/ml

Acacia pod AgNPs

 

400

200

100

50

25

400

200

100

50

25

1:9

Trichophyton Mentagrophyles n=25

<15

00

00

00

02

10

00

00

00

00

09

00

15 – 20 

00

02

02

02

08

02

00

00

00

02

00

21 – 25 

17

16

18

18

07

14

15

13

17

14

00

26 – 30 

08

07

05

03

00

03

06

08

06

00

21

>30

00

00

00

00

00

06

04

01

00

00

04

Trichophyton rubrum n=17

<15

00

00

00

00

00

00

00

00

00

00

00

15 – 20 

00

00

00

00

03

00

00

00

00

1

00

21 – 25 

06

07

05

03

07

06

08

04

03

07

03

26 – 30 

06

08

12

14

07

05

05

13

14

06

13

>30

05

02

00

00

00

07

04

00

00

00

01

Trichophyton tonsurans n=7

<15

00

00

00

00

01

00

00

00

00

00

00

15 – 20 

00

00

00

02

02

00

00

00

00

00

00

21 – 25 

02

03

04

04

04

03

03

04

04

04

03

26 – 30 

02

03

03

00

00

01

04

03

03

03

04

>30

03

01

00

00

00

03

00

00

00

00

00

 


 

3.6 Antifungal Activities of A. nilotica pod Aqueous, Methanolic and AgNPs

Antifungal activities were expressed as an average diameter of the zones of inhibition calculated as the difference in diameter of the observed zones. The diameter of the wells is 6mm, only zones of inhibition greater than 6mm are regarded as a measure of antifungal activity 43, zones of inhibition less than 6mm are neglected 44.

All extracts (Aqueous, Methanolic and AgNps) were tested against 49 isolates of (Trichophyton mentagrophytes, Trichophyton rubrum and Trichophyton tonsurans) at different dilutions (400mg/ml, 200mg/ml, 100mg/ml, 50mg/ml, and 25mg/ml) as presented in table 7.

For Aqueous extracts – Antifungal activities against aqueous extract shows remarkable zones of inhibition – the highest zones observed (8) fall between 26-30mm against T. Mentagrophytes and maintenance activities between 21 -25mm for all extract dilutions. T. rubrum shows zones of inhibition observed above 30mm at both 400mg/ml and 200mg/ml aqueous extracts. All extracts dilution showed activity with the lowest falling between 15 and 20mm at 25mg/mlIn T. tonsurans, the lowest dilution showed only one (1) zones of inhibition below 15mm. all dilution showed activity against Tt with 3 zones of inhibition observed at 400mg/ml and one at 200mg/ml. T. tonsurans is more susceptible to Aqueous extract compared to other extracts.

For Methanolic extract – methanolic extract showed zones of inhibition against T. mentagrophytes at all concentrations. Above 30mm zones of inhibition were observed for 40mg/ml, 200mg/ml, and 100 mg/ml, and the zones of inhibition between 21 -25 were observed at all tested dilutions. T. rubrum, compared to T. mentagrophytes exhibited more susceptibility to the methanolic extract. T. tonsurans, only 3 zones of inhibition were above 30mm and most significant zones of inhibition were observed at 21-25 and 26-30 range for all extract dilutions.

For AgNPs – AgNPs didn’t show antifungal activities below 26mm for both T. mentagrophytes and T. rubrum, except in the case of T. tonsurans were zones of inhibition between 21 – 25mm (03) and 26 30 (04) were exhibited and observed and no zones of inhibition above 30mm. AgNPs have shown broader and more consistent activities against all extracts.

Similar results have been reported 36,37,45, 24, 46, 23,7, 5 and 47. 

36 using A. nilotica stem bark against Shigella sonnei and Bacillus subtilis observed antimicrobial activities with zones of inhibition ranging between 8.5mm and 18mm at 30mg/ml and 5mm and 16.5 at 25mg/ml.

37 using FeNps synthesized from A. nilotica pod observed zones of inhibition between 11mm and 25mm at 3 concentrations (10, 40, and 60) against E. coli, Salmonella, Staphylococcus aureus, and Candida albicans as found by 7. In other work, 5 revealed that there is a difference of 3mm between AgNps synthesized from A. nilotica leaves against Aspergillus niger.

45 used a methanolic extract from A. nilotica pod to obtain zones of inhibitions between 16mm and 19.5mm at 400mg/ml, 15mm and 19.5 mm at 200mg/ml against S. aureus, Bacillus cereus, Escherichia coli, and Acinetobacter baumannii.  At 100mg/ml, 50mg/ml, and 25mg/ml, 24, reported Zones of inhibition of 22mm against clinical E. coli isolate, 21.7mm against E. coli isolated from fish, 21mm against clinical isolate of Salmonella spp and 18mm against salmonella isolate from poultry meat. At 50mg/ml 18mm (clinical E. coli), 14.7mm (fish E. coli), 17.33mm (clinical Salmonella) and 18mm (Meat salmonella). At 25mg/ml 17.7mm (clinical E. coli), 11.33mm (Fish E. coli), 13.7mm (clinical Salmonella) and 12.7mm (meat Salmonella).

47 revealed the antifungal efficacy of ethanolic A. nilotica pod extract as it causes weak effective growth inhibition by between 4.05 and 37.48% as the concentrations increased from 100 to 1000 ppm while [46], showed how hexane stem bark extract of the same plant inhibits mycelial growth by 46% (Fusarium oxysporum), 58% (Rhizoctonia solani) and 52% (Alternaria brassicae).   

23explained how A. nilotica pod induces cythopathogenicity, antiplatelet aggregation activity, antioxidant, decreases the arterial blood pressure, antituberculosis, antibacterial and antifungal activities against C. albicans, and A. niger

It is said that silver interacts with thiol groups of protein on the cell membrane, which results in blocking respiration and producing ultimate death 48. It has also been suggested that the interaction of silver nanoparticles with the cell wall increases the membrane permeability by forming pores or pits and thereby causing the death of microorganisms 49 of the organisms 50,51.


 

 

imageimage

Figure 4: Sample Lates

 

 


 

4. CONCLUSION

Acacia nilotica pod extracted containing various secondary metabolites like polyphenols were used in the synthesis of AgNPs tested against dermatophytic fungi collected and isolated from clinical samples. 

This investigation established the prevalence of dermatophyte infections in Damaturu with males more affected than females and the highest percentage found among 61-70 years, age group. Fungal infection of the foot is commonest, followed by a fungal infection of the hair commonly caused by fungal of Trichophyton species. The phytochemical screening suggests the presence of the phytochemicals supported by the GC-MS analysis of the same plant material. silver nanoparticles synthesized showed double absorption 311 and 308nm with FT-IR spectrogram showing stretches of alkane, alkenes, nitriles, ketones. The SEM shows the morphology of the AgNPs. Fungal species were susceptible to all the extracts. The green synthesized AgNPs showed improved and consistent anti-fungal efficacy against T. mentagrophytes and T. tonsurans than T. rubrum. This study has supported ethnomedicinal use of Acacia nilotica pod for treatment of dermatophytes, opened up the possibility of exploring more about the use of the eco and economic approach of improving medicinal values of the plants and the possibility of adding GC-MS analysis to the methods used the pre-synthesis analysis of AgNPs. Further exploration of these plants using the green synthesis approach, could open up more understanding and add value to medicinal plant research.

 

 

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