TX_1:ABS~AT/TX_2:ABS~AT


85 http://journals.cihanuniversity.edu.iq/index.php/cuesj CUESJ 2020, 4 (1): 85-89

ReseaRch aRticle

Effect of Isatis spp. Extraction on the Growth of Aspergillus 
niger and Candida albicans 
Zehra Odabaş-Serin1*, Ali H. Salim Agha2, Zhala B. Taha2 
1Department of Forest, Kahramanmaraş Sütçü Imam University, Faculty of Forestry, 46040, Kahramanmaraş, Turkey, 
2Department of Bioengineering and Science, Kahramanmaraş Sütçü İmam University, Faculty Bioengineering and Science, 46040, 
Kahramanmaraş, Turkey and Cihan University-Erbil, Department of Biology, Iraq

ABSTRACT

In this study, the crude extraction of Isatis spp. (Isatis tinctoria, Isatis busichaina, and Isatis lusitanica) was investigated for their 
antifungal activity. Each of methanol, ethanol, and H2O was used as a solvent. Due to find the most effective part of each species, 
the plant parts were used separately. The aim of this research was to determine the natural products effect on two common 
pathogenic fungi Aspergillus niger and Candida albicans. The results show that most of used plants have significant effect on 
both used fungal species. Extracted each of flower, I. tinctoria was showed the best results comparing to the other used species. 
Extracted leaf and flower of I. tinctoria by methanol were showed the best result on A. niger and C. albicans. The growth zone was 
around 90 mm for control and 61 mm at 75% concentration of methanolic and ethanolic extraction. The flower was for followed 
by the stem. Depend on the results, the methanol was showed highest number, then ethanol and the lowest inhibition zone area 
was for H2O extraction. 

Keywords: Antifungal activity, Aspergillus niger and Candida albicans, Isatis spp., pathogenic fungi

INTRODUCTION

A latest study presented that tertiary care clinics are consuming antimicrobial and antifungal agents improperly. This rising trend is an ever-increasing threat 
to the health of humans who are currently at risk of dying 
from previously fixable infections. Unluckily, drug resistance 
(adaptation of the drug target site, drug modification, or 
restricted drug penetration) improves at a pace quicker than 
new drug development.[1] This sensation is also happening in 
the pathogenic microorganism Candida albicans, an otherwise 
commensal kinds found in and on the human body.[2] Modern 
antifungal agents are less active against C. albicans now than 
when they were first introduced because Candida species have 
developed resistance to these agents. In addition, research by 
the major pharmaceutical companies in the development of 
antifungal agents has been on the decline due to low coming 
back on investment.[3]

MATERIALS AND METHODS

Plant Extracts 

Three methods were used to extract water, ethanolic, 
and methanolic, according to several studies, with some 
modification as needed and as follows:[4]

Aqueous Extraction

Hot water extraction was performed by mixing 40 g of 
plant form with 160 ml of distilled water twice (i.e., 1:4 
weight:volume), heated to 80–90°C, then stirred and left to 
cool down and mixed together then placed in refrigerator for 
24 h, which was then administered through several layers of 
medical gauze, and again administered by Buchner funnel 
using filter papers Whatman No.1 to dispose of the non-
powdered parts and fibers to obtain the raw liquid extract and 
then put it in the rotary evaporator device at a temperature 
not exceeding 40°C, working on the basis of evaporation under 
pressure and then put the extracted in the shaker incubator 
at a temperature of 30–35°C. After the drying of the extract, 

Corresponding Author: 
Zehra Odabaş-Serin, Kahramanmaraş Sütçü Imam University, Faculty 
of Forestry, 46040, Kahramanmaraş, Turkey. Phone: +90 3443001780. 
Fax: +90 3443001712. E-mail: zehra@ksu.edu.tr

Received: Jan 16, 2020 
Accepted: Apr 27, 2020 
Published: Jun 20, 2020

DOI: 10.24086/cuesj.v4n1y2020.pp85-89

Copyright © 2020 Zehra Odabaş-Serin, Ali H. Salim Agha, Zhala B. Taha. This is 
an open-access article distributed under the Creative Commons Attribution License.

Cihan University-Erbil Scientific Journal (CUESJ)

mailto:zehra@ksu.edu.tr
https://creativecommons.org/licenses/by-nc-nd/4.0/


Odabaş-Serin, et al.: Antifungal activity of Isatis spp

86 http://journals.cihanuniversity.edu.iq/index.php/cuesj CUESJ 2020, 4 (1): 85-89

it was kept in a sealed containers in the freezer (−4°C) and 
wrote the information on each until use. 

Ethanolic Extraction

Prepare the ethanol extract by mixing 20 g of plant powder 
in 200 ml of absolute ethanol with stirring (i.e., 1:10 
weight:volume) and then leave the mixture in the refrigerator 
for 24 h. Then, the extract was filtered through several layers 
of gauze and then filtered for 2nd time using Whatman No.1 
filter papers to remove the non-powdered plant parts and the 
remaining fibers, then put in the rotary evaporator device at 
40°C and after evaporation of the alcohol obtained a thick layer 
of the extract and then placed in the shaker incubator shaker 
incubator at 25–30°C. After drying the extract, it was kept in a 
sealed container in a freezer with writing the information on 
each until use.

Methanolic Extraction

Prepare the methanol extract by mixing 20 g of plant powder 
in 200 ml of absolute methanol with stirring (i.e., 1:10 
weight:volume) and then leave the mixture in the refrigerator 
for 24 h to soak. Then, the extract was filtered through several 
layers of gauze and then filtered for 2nd time using Whatman 
No.1 filter papers to remove the non-powdered plant parts and 
the remaining fibers, then put in the rotary evaporator device 
at 40°C and after evaporation of the methanol obtained a thick 
layer of the extract and then placed in the shaker incubator 
shaker incubator at 25–30°C. After drying the extract, it 
was kept in a sealed container in a freezer with writing the 
information on each until use. 

Media Preparation 

The above studied media were prepared according to the 
information on the package by the manufacturer company[5] 
then sterilized at 121°C and 15 lb/kg2 for 15 min. The medium 
was distributed in Petri dishes (diameter 9 cm), and the dishes 
were left under the temperature of the laboratory until the 
hardening of the medium in sterilizer hood. A quantity of 
distilled water was also sterilized for use as a substitute for 
extracts in control transactions (ddH

2
O).

Fungi 

Two types of fungus obtained from the media center in Erbil-
Iraq for diagnosing the disease with mention the strain type 
were used in this study.
• Aspergillus niger ATCC 16404
• C. albicans ATCC 10231

Preparation of Petri dishes to Test 
Extracts against Fungus

For this study, concentration method was used by mixing with 
the medium. The fungal isolates were planted in Petri dishes 
on the potato dextrose agar media, mixed with three different 
concentrations of plant extracts that mentioned in previously, 
then incubated the dishes in the incubator at 25 ± 2°C and 37 
± 2°C. For example, Candida species need a temperature of 
between 25 ± 2°C and 37 ± 2°C for optimal growth according 
to the nature of their growth to fungus or semi-yeast. The 

process of incubation continued for 10 days. The readings 
were taken every 3 days according to replicates, influenced by 
concentrations of plant extracts.[6,7]

Statistical Analysis

GLM-univariate analysis was applied to the arithmetic 
means obtained in SPSS with 95% confidence interval. If the 
difference was significant (P ≤ 0.05), the homogeneity groups 
were determined by Tukey B test. The analytical test was 
done separately for each solvent type (hot water, ethanol, and 
methanol) to show the effect of the samples part (flower, leaf, 
and stem) and extract concentration (25%, 50%, and 75%).

RESULTS AND DISCUSSION

The antifungal compound is one of the vital substances in 
pharmaceutical sciences that can find in the natural medicinal 
plants. This needs to identify the activity of the samples 
compound by different parameters. In this study, the antifungal 
activities of extracts obtained with solvents that vary from non-
polar to polar were investigated. The results were took after the 
growth was rich the optimum point of two types of used fungi 
in optimum incubation condition. The growth of the control 
Petri dishes was 90 mm for both. The following table is showed 
the effect of Isatis tinctoria on A. niger ATCC 16404 and C. 
albicans ATCC 10231 growth. The best results were with flower 
extraction by methanol with 75% concentration against A. niger 
ATCC 16404, following by the stem with methanolic extraction 
75% against A. niger ATCC 16404 too. In this table, all of the 
methanolic extraction had significant effect on both fungi A. 
niger ATCC 16404 and C. albicans ATCC 10231 [Table 1]. The 
second table is showed the effect of Isatis busichaina on A. niger 
ATCC 16404 and C. albicans ATCC 10231 growth. The best results 
were with leaf extraction by methanol with 75% concentration 
against C. albicans ATCC 10231, following by the flower with 
methanolic extraction 75% against A. niger ATCC 16404. In this 
table, all of the methanolic extraction had significant effect on 
both fungi A. niger ATCC 16404 and C. albicans ATCC 10231 
[Table 2]. However, Table 3 shows the effect of Isatis lusitanica 
on A. niger ATCC 16404 and C. albicans ATCC 10231 growth. 
The best results were with flower extraction by methanol with 
75% concentration against A. niger ATCC 16404, following by 
the flower with methanolic extraction 75% against C. albicans 
ATCC 10231. In this table, all of the methanolic extractions 
had significant effect on both fungi A. niger ATCC 16404 and 
C. albicans ATCC 10231 [Table 3].

Depend on the results, the significant results were 
obtained from the extraction of methanol and ethanol for I. 
tinctoria in all concentrations, plant parts, and both types of 
used fungi. The aqueous extraction has less effect in correlation 
with ethanol and methanol [Table 4]. The inhibition effects of 
the extracts of I. tinctoria obtained from leaf, stem, and flower 
parts using different solvents on the two different strains of 
pathogenic fungi. The results were compared with the each to 
find the correlation. The aqueous leaf extracts were showed 
significant results on A. niger ATCC 16404, also the flower 
was showed the significant results by ethanol extraction for 
C. albicans ATCC 10231, although the flower extracts by 
methanol were showed the significant results for A. niger ATCC 
16404. Other differences are shown in Table 4.



Odabaş-Serin, et al.: Antifungal activity of Isatis spp

87 http://journals.cihanuniversity.edu.iq/index.php/cuesj CUESJ 2020, 4 (1): 85-89

Table 1: Fungi test results of I. tinctoria. (Control=90 mm)

Plant Plant part Strains Growth of fungi (mm)

Water concentration Ethanol concentration Methanol concentration

25% 50% 75% 25% 50% 75% 25% 50% 75%

I. tinctoria Flower C. albicans 85.75 
(0.96)

82.75 
(0.50)

80.25 
(0.96)

81.00 
(0.82)

79.00 
(0.82)

72.50 
(1.29)

73.00 
(1.15)

71.75 
(0.50)

62.00 
(1.41)

A. niger 84.00 
(0.82)

82.50 
(1.29)

79.00 
(0.82)

81.25 
(0.96)

80.00 
(0.82)

78.00 
(1.41)

69.75 
(0.50)

68.00 
(0.82)

61.25 
(0.50)

Leaf C. albicans 84.50 
(2.38)

80.75 
(0.96)

79.50 
(1.29)

79.75 
(1.26)

78.75 
(0.96)

77.75 
(0.96)

70.5 
(0.58)

69.25 
(0.50)

62.00 
(1.41)

A. niger 84.00 
(0.82)

81.00 
(0.82)

79.25 
(0.96)

80.25 
(1.71)

78.75 
(0.96)

77.50 
(0.58)

70.25 
(0.50)

68.50 
(1.00)

62.75 
(0.50)

Stem C. albicans 84.75 
(0.96)

82.25 
(2.06)

80.50 
(0.58)

82.00 
(0.82)

80.50 
(0.58)

73.00 
(1.83)

73.50 
(1.29)

70.25 
(0.50)

63.25 
(1.50)

A. niger 84.50 
(1.29)

82.75 
(0.96)

80.00 
(0.82)

81.25 
(0.50)

79.25 
(0.96)

 78.50 
(0.58)

70.75 
(0.96)

69.50 
(1.29)

61.75 
(0.50)

I. tinctoria: Isatis tinctoria, C. albicans: Candida albicans, A. niger: Aspergillus niger

Table 2: Fungi test results of I. busichaina. (Control=90 mm)

Plant Plant part Strains Growth of fungi (mm)

Water concentration Ethanol concentration Methanol concentration

25% 50% 75% 25% 50% 75% 25% 50% 75%

I. busichaina Flower C. albicans 84.75 
(0.96)

80.25 
(1.26)

79.50 
(1.29)

80.00 
(0.82)

78.50 
(0.58)

76.00 
(1.41)

71.00 
(0.82)

70.00 
(1.63)

63.00 
(1.83)

A. niger 83.50 
(1.29)

79.75 
(0.96)

77.75 
(2.22)

80.00 
(0.82)

78.25 
(0.50)

76.00 
(0.82)

70.75 
(0.96)

68.00 
(0.82)

61.5 
(1.29)

Leaf C. albicans 83.25 
(0.96)

79.50 
(1.29)

77.00 
(1.83)

80.00 
(0.82)

79.00 
(0.82)

75.50 
(1.29)

71.00 
(1.15)

66.75 
(2.06)

61.00 
(0.82)

A. niger 83.25 
(0.96)

80.50 
(0.58)

77.25 
(1.71)

80.75 
(0.96)

78.50 
(0.58)

74.25 
(0.50)

70.75 
(0.50)

66.25 
(0.50)

63.00 
(1.83)

Stem C. albicans 84.00 
(0.82)

80.75 
(1.50)

77.25 
(1.50)

81.75 
(0.96)

80.00 
(0.82)

78.25 
(0.96)

72.5 
(2.38)

70.50 
(0.58)

68.50 
(0.58)

A. niger 84.75 
(1.50)

82.25 
(1.26)

79.25 
(0.96)

81.75 
(0.96)

80.50 
(0.58)

79.25 
(0.96)

71.75 
(0.96)

70.00 
(0.82)

69.00 
(1.41)

I. busichaina: Isatis busichaina, C. albicans: Candida albicans, A. niger: Aspergillus niger

Table 3: Fungi test results of I. lusitanica. (Control=90 mm)

Plant Plant part Strains Growth of fungi (mm)

Water concentration Ethanol concentration Methanol concentration

25% 50% 75% 25% 50% 75% 25% 50% 75%

I. lusitanica Flower C. albicans 83.25 
(0.96)

81.50 
(1.29)

79.00 
(0.82)

80.50 
(0.58)

79.50 
(1.29)

78.00 
(0.82)

70.00 
(0.82)

67.75 
(0.50)

64.50 
(1.29)

A. niger 83.25 
(0.96)

80.75 
(0.96)

79.00 
(0.82)

80.50 
(0.58)

78.25 
(1.71)

76.75 
(0.96)

68.50 
(1.29)

66.50 
(1.00)

63.75 
(1.71)

Leaf C. albicans 82.25 
(1.71)

81.75 
(1.71)

79.50 
(1.29)

80.00 
(0.82)

79.00 
(0.82)

76.75 
(0.96)

69.75 
(0.96)

67.50 
(0.58)

66.50 
(1.00)

A. niger 82.00 
(0.82)

80.25 
(0.96)

79.50 
(0.58)

80.25 
(0.50)

78.25 
(1.71)

77.50 
(1.29)

68.25 
(0.96)

65.75 
(0.96)

65.25 
(0.50)

Stem C. albicans 84.50 
(0.58)

82.25 
(0.50)

80.50 
(1.29)

81.50 
(1.29)

80.75 
(0.96)

79.00 
(0.82)

72.00 
(0.82)

70.50 
(0.58)

69.00 
(0.82)

A. niger 83.50 
(1.29)

80.75 
(0.96)

80.25 
(1.26)

81.50 
(0.58)

80.00 
(0.82)

79.00 
(0.82)

72.00 
(0.82)

70.25 
(0.96)

69.50 
(0.58)

I. lusitanica: Isatis lusitanica, C. albicans: Candida albicans, A. niger: Aspergillus niger

The inhibition effects of extracts of I. busichaina obtained 
from leaf, stem, and flower parts using different solvents on 

the two different strains of pathogenic fungi. The results were 
compared with the each to find the correlation. For C. albicans 



Odabaş-Serin, et al.: Antifungal activity of Isatis spp

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ATCC 10231, each of aqueous leaf extraction, ethanol 
extraction of flower and leaf, and leaf extraction by methanol 
was showed the significant result correlations. For A. niger 
ATCC 16404, the aqueous extraction of flower and leaf, the 
ethanol extraction of flower and leaf, and methanol extraction 
of flower and leaf too was showed the significant results. The 
total result statistics shown in Table 5.

The inhibition effects of the extracts of I. lusitanica 
obtained from leaf, stem, and flower parts using different 
solvents on the two different strains of pathogenic fungi. 
The results were compared with the each to find the 
correlation. For C. albicans ATCC 10231, each of flower 
and leaf of aqueous, ethanol, and methanol extraction was 
showed the significant results. For A. niger ATCC 16404, 
the ethanol and methanol extraction of flower and leaf was 
showed the significant results. The total result statistics is 
shown in Table 6. Some factors have to be measured to select 

a solvent for extracting herbal medicine. About 65–75% 
methanol extraction at room temperature for 15 h is practical 
for routine analytical study. Although, it is not fully active 
to extract other plants with same procedure because of 
dissimilarities in the biological compositions. This method is 
appropriate for controlling a large number of fixed samples 
and needs no special equipment.[8]

The present study proved that methanol and ethanol 
showed best results than water extracts. Our study was in 
accordance with several studies.[9] Tryptanthrin, indole-3-
acetonitrile, and p-coumaric acid methylester were identified 
by physical and spectral data (MS, NMR) and by comparison 
with authentic synthesized samples of these compounds. 
Tryptanthrin has already been isolated from I. tinctoria and 
characterized as an antidermatophytic.[10] Phenylpropanoids 
mainly comprised flavonoid and lignan metabolites, have 
been considered as the primary antiviral constituents, which 

Table 4: Fungi arithmetic values and Tukey B statistical analysis results of I. tinctoria

C. albicans concentration A. niger concentration Tukey B

Plant part 25% 50% 75% Tukey B 25% 50% 75%

Water Flower 85.75 (0.96) 82.75 (0.50) 80.25 (0.96) A 84.00 (0.82) 82.50 (1.29) 79.00 (0.82) AB

Leaf 84.50 (2.38) 80.75 (0.96) 79.50 (1.29) A 84.00 (0.82) 81.00 (0.82) 79.25 (0.96) B

Stem 84.75 (0.96) 82.25 (2.06) 80.50 (0.58) A 84.50 (1.29) 82.75 (0.96) 80.00 (0.82) A

Tukey B A B C A B C

Ethanol Flower 81.00 (0.82) 79.00 (0.82) 72.50 (1.29) B 81.25 (0.96) 80.00 (0.82) 78.00 (1.41) A

Leaf 79.75 (1.26) 78.75 (0.96) 77.75 (0.96) A 80.25 (1.71) 78.75 (0.96) 77.50 (0.58) A

Stem 82.00 (0.82) 80.50 (0.58) 73.00 (1.83) AB 81.25 (0.50) 79.25 (0.96)  78.50 (0.58) A

Tukey B A B C A B C

Methanol Flower 73.00 (1.15) 71.75 (0.50) 62.00 (1.41) B 69.75 (0.50) 68.00 (0.82) 61.25 (0.50) B

Leaf 70.50 (0.58) 69.25 (0.50) 62.00 (1.41) C 70.25 (0.50) 68.50 (1.00) 62.75 (0.50) A

Stem 73.50 (1.29) 70.25 (0.50) 63.25 (1.50) A 70.75 (0.96) 69.50 (1.29) 61.75 (0.50) A

Tukey B A B C A B C

I. tinctoria: Isatis tinctoria, C. albicans: Candida albicans, A. niger: Aspergillus niger

Table 5: Fungi arithmetic values and Tukey B statistical analysis results of I. busichaina

Plant part C. albicans concentration Tukey B A. niger concentration Tukey B

25% 50% 75% 25% 50% 75%

Water Flower 84.75 (0.96) 80.25 (1.26) 79.50 (1.29) A 83.50 (1.29) 79.75 (0.96) 77.75 (2.22) B

Leaf 83.25 (0.96) 79.50 (1.29) 77.00 (1.83) B 83.25 (0.96) 80.50 (0.58) 77.25 (1.71) B

Stem 84.00 (0.82) 80.75 (1.50) 77.25 (1.50) AB 84.75 (1.50) 82.25 (1.26) 79.25 (0.96) A

Tukey B A B C A B C

Ethanol Flower 80.00 (0.82) 78.50 (0.58) 76.00 (1.41) B 80.00 (0.82) 78.25 (0.50) 76.00 (0.82) B

Leaf 80.00 (0.82) 79.00 (0.82) 75.50 (1.29) B 80.75 (0.96) 78.50 (0.58) 74.25 (0.50) B

Stem 81.75 (0.96) 80.00 (0.82) 78.25 (0.96) A 81.75 (0.96) 80.50 (0.58) 79.25 (0.96) A

Tukey B A B C A B C

Methanol Flower 71.00 (0.82) 70.00 (1.63) 63.00 (1.83) B 70.75 (0.96) 68.00 (0.82) 61.5 (1.29) B

Leaf 71.00 (1.15) 66.75 (2.06) 61.00 (0.82) C 70.75 (0.50) 66.25 (0.50) 63.00 (1.83) B

Stem 72.5 (2.38) 70.50 (0.58) 68.50 (0.58) A 71.75 (0.96) 70.00 (0.82) 69.00 (1.41) A

Tukey B A B C A B C  

I. busichaina: Isatis busichaina, C. albicans: Candida albicans, A. niger: Aspergillus niger



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Table 6: Fungi arithmetic values and Tukey B statistical analysis results of I. lusitanica

Plant part C. albicans concentration Tukey B A. niger concentration Tukey B

25% 50% 75% 25% 50% 75%

Water Flower 83.25 (0.96) 81.50 (1.29) 79.00 (0.82) B 83.25 (0.96) 80.75 (0.96) 79.00 (0.82) A

Leaf 82.25 (1.71) 81.75 (1.71) 79.50 (1.29) B 82.00 (0.82) 80.25 (0.96) 79.50 (0.58) A

Stem 84.50 (0.58) 82.25 (0.50) 80.50 (1.29) A 83.50 (1.29) 80.75 (0.96) 80.25 (1.26) A

Tukey B A B C A B C

Ethanol Flower 80.50 (0.58) 79.50 (1.29) 78.00 (0.82) B 80.50 (0.58) 78.25 (1.71) 76.75 (0.96) B

Leaf 80.00 (0.82) 79.00 (0.82) 76.75 (0.96) B 80.25 (0.50) 78.25 (1.71) 77.50 (1.29) B

Stem 81.50 (1.29) 80.75 (0.96) 79.00 (0.82) A 81.50 (0.58) 80.00 (0.82) 79.00 (0.82) A

Tukey B A B C A B C

Methanol Flower 70.00 (0.82) 67.75 (0.50) 64.50 (1.29) B 68.50 (1.29) 66.50 (1.00) 63.75 (1.71) B

Leaf 69.75 (0.96) 67.50 (0.58) 66.50 (1.00) B 68.25 (0.96) 65.75 (0.96) 65.25 (0.50) B

Stem 72.00 (0.82) 70.50 (0.58) 69.00 (0.82) A 72.00 (0.82) 70.25 (0.96) 69.50 (0.58) A

Tukey B A B C A B C

I. lusitanica: Isatis lusitanica, C. albicans: Candida albicans, A. niger: Aspergillus niger

probably contribute to the outstanding pharmacological 
activity of Radix isatidis.[11,12]

It is distinguished that a solvent system for extraction 
is selected according to the purpose of extraction such as 
preparation or analysis, the nature of interested components, 
the physicochemical properties of the matrix, the availability 
of reagents and equipment, cost, and safety concerns.[13] 
The solvent systems included complete ethanol at 25°C by 
different methods, 95% ethanol, ethanol–water for 10 min,[14] 
methanol–water, methanol–HCl (1.0 N, 85:15, v/v), and H

2
O. 

In another study that done by in 2012,[15] using the ethanol 
under pressure, extraction condition may be a well choice 
for viable productions, meanwhile, ethanol may be reused 
and fewer solvent left over is generated through production. 
However, extraction with ethanol may be a blameless means to 
make effective extracts for analysis and research from a minor 
number of samples. 

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