A.C. Ofochebe , S.O. Anyadoh-Nwadike 1 · sub-cultured and reconfirmed using standard microbiological procedures including both morphological and direct microscopic method as described

GSJ: Volume 7, Issue 7, July 2019, Online: ISSN 2320-9186 www.globalscientificjournal.com

PHYTOCHEMICAL AND ANTIFUNGAL PROPERTIES OF HEDRANTHERA

BARTERI ON PATHOGENIC FUNGI OF THE HUMAN SKIN A.C. Ofochebe

1, S.O. Anyadoh-Nwadike

2

1(Department of Biotechnology, School of Biological Sciences, Federal University of Technology, Owerri, Imo State, Nigeria)

2(Department of Biotechnology, School of Biological Sciences, Federal University of Technology, Owerri, Imo State, Nigeria)

ABSTRACT

The phytochemical and antifungal properties of crude extracts of Hedranthera barteri were tested against Microsporum canis,

Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton violaceum, Epidermophyton floccosum and Candida albicans

which was compared with the efficacy of a known commercial antifungal drug (fluconazole). Extraction of active ingredients from the

leaves and fruits of H. barteri was done using ethanol and hot distilled water as solvents. Determination of antifungal activities of

crude extracts from the leaves and fruits against the test isolates were assessed using the disc diffusion method to detect zones of

inhibition while macro - broth dilution technique to assay the Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal

Concentration (MFC). Phytochemical and food component composition of the leaf and fruit extracts was also determined. Results

obtained showed that the ethanolic leaf and fruit extracts were more potent inhibiting all the isolates with diameters of zone of

inhibition ranging between 1 and 15 mm and 1 and 10 mm respectively compared with the hot distilled water leaf and fruit extracts

which did not inhibit growth of the isolates. The ethanolic leaf and fruit extracts inhibited growth of the fungal isolates in a

concentration dependent manner with MICs ranging between 25 and 100 mg/ml and MFCs also ranging between 25 and 100 mg/ml.

The phytochemical and food components analysis showed presence of alkaloids, flavonoids, glycosides, saponins, oils, carbohydrates,

proteins, and absence of tannins.

KEYWORDS: Antifungal activities, ethanol, extracts, fungal isolates, Hedranthera barteri, inhibition, phytochemical.

GSJ: Volume 7, Issue 7, July 2019 ISSN 2320-9186

878

GSJ© 2019 www.globalscientificjournal.com

1. Introduction Skin is a multi-functional organ of human body that forms a

barrier for protection against infection. The body normally

hosts a variety of microorganisms including bacteria, mycelial

fungi and yeast fungi (such as Candida). Some of these are

useful to the body. Others may under appropriate conditions

multiply rapidly and cause infection. Fungal skin infections

are caused by microscopic fungi that flourish on the human

skin.

Medicinal plants are sources of bioactive compounds used to

treat many diseases. Many plants possess antimicrobial agents

and exhibit antifungal activity [1]. Several in-vitro and in-vivo

studies using plant products traditionally used in

ethnomedicine have demonstrated promising antifungal

activity without any side effects [2].

Herbal medicines have been used for thousands of years in

many parts of the world. Herbs include leaves, fruits, seeds,

stems, wood, bark, roots, rhizomes or other plant parts,

which may be entire, fragmented or powdered.

Hedranthera barteri is a typical example. It is a shrub of up to

2 meters high, in the under storey in damp situations of the

closed-forest in Ghana, Northern and Southern Nigeria,

Western Cameroons, Congo Brazzaville and other parts of the

world [3]. The large white tubular flowers with fragrant scent

make the plant decorative and worthy of cultivation. The plant

contains a white latex that does not coagulate [4]. The leaf-

decoction is drunk by the Igbo speaking people of Southern

Nigeria for dizziness, and the leaf is applied to tumour [4].

The fruit is taken in Nigeria for the treatment of gonorrhea and

as a vermifuge [3]. The antidepressant, antimicrobial, anti-

inflammatory and anti-nociceptive activities of the leaves

have been reported as well as the antiulcer and antioxidant

activities of the roots [5]; [6]; and [7].

The plant has also been reported to possess anti-inflammatory,

antimalarial, antibacterial and antinociceptive properties [8]

and [5]. This stimulated the interest to further investigate these

plants with a view of determining the antifungal properties and

phytochemical composition of the leaf and fruit extracts of this

plant.

2. Methodology

2.1 Plant Specimen Collection and Identification

Fresh samples of leaves and fruits of H. barteri were collected

from a bush in Federal University of Technology, Owerri and

were identified by a taxonomist Dr. M. C. Duru in the

Department of Biological Science, Federal University of

Technology, Owerri.

2.2 Plant Sample Preparation and Extraction The samples were washed with distilled water and dried at

room temperature to avoid heat destruction of the active

components. The dried leaves and fruits were separately

ground into powder with the aid of a blender and stored in

sterile containers. One hundred gram (100 g) of the powdered

fruits and leaves each were weighed with a weighing balance

and subjected to extraction in 250 ml of ethanol (99%) and in

250 ml of hot distilled water using maceration method as

described by [9]. 2.3 Collection, Preparation and Confirmation of

Test/Challenge Fungi Six known fungi; five molds and one yeast obtained from the

skin infections were collected from the Biotechnology

department of Federal University of Technology, Owerri.

These included; Microsporum canis, Trychophyton rubrum,

Trychophyton mentagrophytes, Trychophyton violaceum,

Epidermophyton floccosum and Candida albicans. These were

sub-cultured and reconfirmed using standard microbiological

procedures including both morphological and direct

microscopic method as described by [10] and [11].

2.4 Determination of Antifungal Activities This was done using the disc diffusion method to detect Zones

of inhibition and macro - broth dilution technique to assay the

Minimum Inhibitory Concentration (MIC) and Minimum

Fungicidal Concentration (MFC).

2.4.1 Disc Diffusion Method Disc diffusion method as recommended by [12] was adopted.

Discs were prepared from Whatman filter paper NO.1 and

were sterilized at 60°C for 5 minutes using a hot air oven. The

leaf and fruit extracts (ethanolic and hot distilled water) were

weighed using a weighing balance to obtain 50 mg, 100 mg,

150 mg and 200 mg respectively. These extracts were diluted

in 1 ml of distilled water to give 50 mg/ml, 100 mg/ml, 150

mg/ml and 200 mg/ml. Fluconazole discs were also prepared

by crushing the fluconazole (200 mg) tablet into powdered

form and the resulting powder was weighed as described

above to obtain the following concentrations (50, 100, 150 and

200 mg/ml). The sterile paper discs were then soaked in 1 ml

of each prepared concentrations for twenty minutes.

Sabouraud dextrose agar (SDA) was prepared according to the

manufacturer’s instruction and 20 ml of the prepared SDA was

poured into 25 sterile disposable Petri dishes and allowed to

solidify. The agar surface of each plate was streaked with a

sterile cotton swab with each of the test isolates and the

impregnated discs obtained from the ethanolic leaf and fruit

extracts, hot distilled water leaf and fruit extracts, fluconazole

and distilled water were then placed using a sterile forceps

onto the SDA plates freshly inoculated with the test isolates

respectively. The plates were sealed with parafilm tape to

avoid contamination and any possible drying up and incubated

at 30°C for 14 days. Discs soaked in distilled water served as

negative controls while the ones soaked in fluconazole served

as the positive control. The resulting zones of inhibitions

were measured using a ruler calibrated in millimeters as

recommended by [13]. The crude extracts that exhibited wide

zones of inhibition were further analyzed for MIC and MFC.

2.4.2 Determination of Minimum Inhibitory

Concentration (MIC) The minimum inhibitory concentration was determined using

macro-broth dilution technique by [14]. Five concentrations

(100 mg/dl, 50 mg/dl, 25 mg/dl, 12.5 mg/dl and 6.25 mg/dl) of

the respective extracts were used.


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Standardized suspension of the test organisms (1.0 x 105) were

inoculated into series of sterile test tubes of SDA broth

containing varying concentrations of the fluconzole, ethanolic

leaf and fruit extracts respectively and incubated at 270C for

14 days. The MICs were read as the least concentration that

inhibited the growth of the test organisms.

2.4.3 Determination of Minimum Fungicidal

Concentration (MFC) This was done according to the procedure described by [14].

The minimum fungicidal concentration was determined by

first selecting tubes that showed no growth during MIC

determination. A loopful from each tube was sub-cultured

onto drug/extract free agar plates and incubated for further

7 days at 30°C. The least concentration, that showed no

growth in the subculture was observed and noted as the

minimum fungicidal concentration (MFC).

2.5 Qualitative and Quantitative Phytochemical

and Food Component Analysis of the Plant Extracts. The qualitative analysis was carried out according to methods

described by [15] while the quantitative analysis was done

according to the gravimetric processes described by [16].

2.6 Statistical Analysis The data obtained were statistically analyzed using T-test for

comparative purposes.

3 Results The fluconazole, ethanolic, hot distilled water leaf and fruit

extracts of H. barteri exhibited different zones of inhibition on

the organisms tested at various concentrations (TABLE 1).

The ethanolic leaf extract inhibited the growth of all the

isolates giving diameter of inhibition within the ranges

between 3 - 12 mm for M. canis, 4 - 15 mm for T. rubrum, 3 -

14 mm for T. mentagrophytes, 2 - 12 mm for T. violaceum, 2 –

11 mm for E. floccosum and 1– 8 mm for C. albicans. The

ethanolic fruit extracts gave the diameter of inhibition of the

ranges 3 – 10 mm for M. canis, 2 - 8 mm for T. rubrum, 1 - 6

mm for T. mentagrophytes, 1 - 3.5 mm for T. violaceum, 1 - 5

mm for E. floccosum and 1 - 4 mm for C. albicans. The hot

distilled water leaf extracts gave the diameter inhibition range

of between 1 – 3 mm for T. rubrum, 0.5 - 2 mm for T.

mentagrophytes but did not inhibit the growth of T. violaceum,

M. canis, E. floccosum and C. albicans while the hot distilled

water of the fruit extracts did not inhibit the growth of any of

the isolates. The fluconazole also inhibited the growth of all

the isolates giving the zones of inhibition ranging between 5 -

19 mm for M. canis, 6 - 21 mm for T. rubrum, 6 - 20 mm for

T. mentagrophytes, 5 - 19 mm for T. violaceum, 3 - 16 mm for

E. floccosum and 2 - 10 mm for C. albicans.


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Table 1: Zones of Growth Inhibition Exhibited By the Extracts

Extracts Zones of Inhibition (mm) by varying concentrations (mg/ml)

Isolates 50 100 150 200

Ethanolic leaf

extract

Mc

Tr

Tm

Tv

Ef

Ca

3

4

3

2

2

1

6

7

6.5

5

4

3

8

10

9

6.5

7

6

12

15

14

12

11

8

Ethanolic fruit

extract

Mc

Tr

Tm

Tv

Ef

Ca

3

2

1

-

1

-

5

3

2

1

2

1

7

5

3.5

2

4

2

10

8

6

3.5

5

4

Hot distilled water

leaf extract

Mc

Tr

Tm

Tv

Ef

Ca

-

-

-

-

-

-

-

-

-

-

-

-

-

1

0.5

-

-

-

1

3

2

1

0.5

1

Hot distilled water

fruit extract

Mc

Tr

Tm

Tv

Ef

Ca

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

1

-

-

-

0.5

+Ve control Mc

Tr

Tm

Tv

Ef

Ca

5

6

6

5

3

2

8

10

9

8.5

6

4

11

15

14.5

13

9

8

19

21

20

19

16

10

Key:

Mc = Microsporum canis Tr = Trichophyton rubrum Tm = Trichophyton mentagrophytes

Tv = Trichophyton violaceum Ef = Epidermophyton floccosum Ca = Candida albicans

+ve control = Fluconazole discs (50, 100, 150, 200 mg/ml) - = No inhibition


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3.1 Minimum Inhibitory Concentration (MIC) Of Fluconazole, Ethanolic Leaf and Fruit Extracts of H. barteri

The results of the minimum inhibitory concentration of the fluconazole, ethanolic leaf and fruit extracts at different concentrations

showed that the higher the concentration, the greater the efficacy. The MIC of the fluconazole tested against the test isolates was

observed to be 25, 25, 25, 25, 25, 50 mg/ml for M. canis, T. rubrum, T. mentagrophytes, T. violaceum, E. floccosum and C. albicans

which was same for the ethanolic leaf extracts of H. barteri except for E. floccosum where fluconazole is more potent (MIC was

lower). The lower the MIC, the more the potent the crude extracts and vice versa. The ethanolic fruit extract gave MIC values of 50,

100, 100, 100, 50 and 100 mg/ml against M. canis, T. rubrum, T. mentagrophytes, T. violaceum, E. floccosum and C. albicans as

shown in TABLE 2.

Table 2: Minimum Inhibitory Concentration (MIC) of Fluconazole, Ethanolic Leaf and Fruit Extracts of H. barteri

Extracts 1solates Concentrations of extracts (mg/ml)

100 50 25 12.5 6.25

MIC

Fluconazole

M. canis

T. rubrum

T. mentagrophytes

T. violaceum

E. floccosum

C. albicans

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

+

+

+

+

+

+

+

+

+

+

+

+

+

25

25

25

25

25

50

Ethanolic leaf

extracts

M. canis

T. rubrum

T. mentagrophytes

T. violaceum

E. floccosum

C. albicans

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

+

+

+

+

+

+

+

+

+

+

+

+

+

+

25

25

25

25

50

50

Ethanolic fruit

extracts

M. canis

T. rubrum

T. mentagrophytes

T. violaceum

E. floccosum

C. albicans

-

-

-

-

-

-

-

+

+

+

-

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

50

100

100

100

50

100

Key: - = No Growth observed, + = Growth observed

The fluconazole gave MFC of 25, 25, 25, 25, 50 and 50 mg/ml for M. canis, T. rubrum, T. mentagrophytes, T. violaceum, E.

floccosum and C. albicans while the ethanolic leaf extracts of H. barteri gave MFC of 25, 25, 25, 50, 50 and 100 mg/ml for M. canis,

T. rubrum, T. mentagrophytes, T. violaceum, E. floccosum and C. albicans and the ethanolic fruit extracts gave MFC of 50, 50, 50, 50,

100, 100 mg/ml for M. canis, T. rubrum, T. mentagrophytes, T. violaceum, E. floccosum and C. albicans. (TABLE 3)

The ethanolic leaf extracts also exhibited greater efficacy than the ethanolic fruit extracts as seen in their various MFCs when

compared with that of the Fluconazole. The lower the MFC, the more potent the crude extracts and vice versa.


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Table 3: Minimum Fungicidal Concentration (MFC) of Fluconazole, Ethanolic Leaf and Fruit Extracts of H.

barteri.

Extracts 1solates Concentrations (mg/ml)

100 50 25 12.5 6.25

MFC

Fluconazole

M. canis

T. rubrum

T. mentagrophytes

T. violaceum

E. floccosum

C. albicans

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

+

+

+

+

+

+

+

+

+

+

+

+

+

+

25

25

25

25

50

50

Ethanolic leaf

extract

M. canis

T. rubrum

T. mentagrophytes

T. violaceum

E. floccosum

C. albicans

-

-

-

-

-

-

-

-

-

-

-

+

-

-

-

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

25

25

25

50

50

100

Ethanolic fruit

extract

M. canis

T. rubrum

T. mentagrophytes

T. violaceum

E. floccosum

C. albicans

-

-

-

-

-

-

-

-

-

-

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

50

50

50

50

100

100

KEY: - = No Growth observed + = Growth observed

The food components and qualitative phytochemical analysis of H. barteri leaves and fruits showed the presence of flavonoids,

phenols, glycosides, alkaloids, saponin, reducing sugar, resins, protein, oils and carbohydrates while tannins was not detected in the

leaves. The fruit analysis revealed presence of flavonoids, saponins, glycosides, alkaloids, reducing sugar, oils and resins; and absence

of protein, carbohydrates, phenols and tannins (TABLE 4).

The gravimetric analysis for total alkaloid, flavonoid and saponin contents in leaf and fruit parts of H. barteri exhibited that higher

alkaloid, flavonoid and saponin contents were present in leaf powder than that of the fruit powder.

In the leaves sample of H. barteri, the percentage yield for alkaloids content was 8.89%, flavonoids content was 20.5%, saponins

content was 15.74%, phenols content was 30.6, carbohydrates content was 7.2, protein content was found to be 6.2 and reducing sugar

content was found to be 5.3. In the fruits sample the percentage yield of phenols content was 20.7, alkaloids content was found to be

5.2%, flavonoids content was 12.8%, saponins content was 7.49%, reducing sugar content was 4.5, while carbohydrates and proteins

were not detected. The phenols content of the leaves and fruits were found to be in highest quantity than the other phtytochemicals

(TABLE 5).


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Table 4: Qualitative Phytochemical and Food

Components Analysis of The Leaf and Fruit Extracts

of H. barteri.

TEST LEAF

INFERENCE

FRUIT

INFERENCE

Saponins + +

Flavonoids + +

Resins + +

Phenols + +

Glycosides + +

Alkaloids + +

Tannins - -

Proteins + -

Oils + +

Reducing

sugar

+ +

Carbohydrates + -

Key: - Not detected, + Detected

Table 5: Quantitative Phytochemical and Food

Component Analysis of the Leaf and Fruit Extracts

PHYTOCHEMICA

LS

LEAVE

S

FRUIT

S

Alkaloids (%) 8.89 5.2

Flavonoids (%) 20.5 12.8

Saponins (%) 15.74 7.49

Phenols (abs)

Carbohydrates (abs)

30.6

7.2

20.7

-

Proteins (abs)

6.2 -

Reducing Sugars (abs) 5.3 4.5

Key: - = Not detected. abs = absorbance.

4. Discussion

The results obtained in this study showed the antifungal

efficacy of the ethanolic and hot distilled water leaf and fruit

extract of H. barteri on the test isolates. Comparative analysis

of the efficacy of the crude extracts and already known

conventional antifungal agent (fluconazole), revealed that the

ethanolic leaf extracts exhibited greater efficacy than the

ethanolic fruit extracts with wider zones of inhibition similar

to that of the fluconazole. The ethanolic leaf extract was most

efficacious against T. rubrum as seen in the zone of growth

inhibition while the ethanolic fruit extract was most

efficacious against M. canis. The hot distilled water leaf and

fruit extracts showed no efficacy against the test isolates. This

may imply that the ethanol extracted more active ingredients

that have antifungal efficacy than the distilled water. It

corroborates the findings of [17], who documented alcohol as

the best solvent for the extraction of plant active ingredients of

medical importance.

Statistical comparative analysis of the fluconazole, ethanolic

leaf and fruit extracts at varying concentrations revealed that P

< 0.05 which implies that there was no significant difference

between the zones of inhibition of the fluconazole and the

ethanolic leaf and fruit extracts. This also implies the crude

extracts can act as potent antifungal agents against

dermatophytes if the doses are fully determined. The

qualitative and quantitative phytochemical analysis done for

the leaves and fruits of H. barteri revealed the presence of

alkaloids, flavonoids, phenols, resins, glycosides, saponins,

reducing sugar, tannins, oils and carbohydrates. The study

revealed that the leaves and fruits of H. barteri possessed a

considerable high level of phenols, flavonoids, alkaloids and

saponins. This is in line with the reports of [6] and [18] on the

presence of alkaloids, flavonoids, polyphenols, glycosides and

saponins in the leaves of the plant.

5. Conclusion This study has shown that the leaf and fruit extracts of H.

barteri have active ingredients/phytochemicals which were

able to inhibit the fungal isolates tested. Generally, the plant

extracts especially the leaf extract can be said to have broad

spectrum antifungal properties. The observed antifungal effect

of this medicinal plant on the isolates appears interesting and

promising and may be effective as a source of novel antifungal

drug. These properties if harnessed appropriately can lead to

novel antifungal formulations that are cheap, easily absorbable

by the body, readily available as well as efficacious in their in-

use concentration.

6. Acknowledgment

My immense gratitude goes to my supervisor, Dr. (Mrs.) S.O.

Anyadoh-Nwadike for her committed effort to see the

completion of this work.

Special thanks to my loving husband, Mr. Chinedu Nwankwo,

for his support, understanding and words of encouragement

especially when the journey seemed bumpy. I am grateful for

his standing by me and never letting me to give up. May God

enlarge his coast Amen.


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I remain equally grateful to my parents, Sir Alex & Lady

Ngozi Ofochebe, my mother in-law Mrs Peace Nwankwo; my

lovely sister and her husband Mr. Ndubuisi & Mrs.

Ogochukwu Udemezue, my brothers Chukwuemeka,

Kenechukwu and Ugochukwu, and my Sisters in-law

Chinyere, Chidinma, Ogochukwu, and Ujunwa for all their

advice, care and generous support to see the success of this

work.

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Appendix

Fig. 1. Hedranthera barteri


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Documents

A.C. Ofochebe , S.O. Anyadoh-Nwadike 1 · sub-cultured and reconfirmed using standard microbiological procedures including both morphological and direct microscopic method as described