EVALUATION OF ANTI-FUNGAL PROPERTIES OF

Artemisia annua L.,

Author Name and Affiliate: Zainab Hammajoda, Modibbo Adama University of
Technology,Yola, Nigeria
Department of Plant Science,
Modibbo Adama University of Technology Yola, Adamawa State, Nigeria.
hammanjodaz@yahoo.com

ABSTRACT
Aspergillus brasiliensis (A. niger) and Penicillium chrysogenum are an important plants pathogenic
fungus that causes many diseases. Challenges to agriculturist is losses due to fungal pathogens, fungal
losses can be 100% if a susceptible cultivar is planted and fungicides used and good husbandry
employed, to carry out a control trial on the effect of Artemisia annua L., extracts on these organisms;
A.brasiliensis and Penicillium chrysogenum, hence these attempt. To find a promising method for the
control of these pathogens, extracts of Artemisia annua L. parts (leaf, stem, and root) were screened in
the study presented using ethanol and aqueous. 2mls of the extracts were amended with PDA in a petri-
dish and allowed to cooled, before inoculations. The organisms were incubated for 10 days. The result
showed that stem ethanol extracts and root aqueous extracts had the highest inhibitory activity with 100%
effectiveness against Penicillium chrysogenum, other parts only suppressed the growth of pathogen.
All datas were analyzed using Analysis Of Variance (ANOVA), Significant means were separated using
Least Square Differences (LSD) at P=0.05

Key word: Evaluation, Anti-fungal, Artemisia annua L.

INTRODUCTION
Artemisia (Artemisia annua L.) is commonly known as annual wormwood, sweet worm wood or
sweet annie, which is a highly aromatic annual herb of Asiatic and Eastern European origin widely
dispersed throughout the temperate region (Simon, 1990). Artemisia, one of the largest genera of the
Asteraceae family belongs to a useful group of aromatic and medicinal plants comprising about 300
species which are distributed throughout the world (Bertea, et al., 2005).
Artemisia annua otherwise known as Qinghao by Chinese has been used for many centuries in
the treatment of fever and malaria (Brown, et al., 2003). The plant species is a source of both essential oil
(1.4-4.0%) and other substances such as Sesquiterpene lactones, Flavonoids, Polyalkenes and Coumarins
(Botsaris, 2007).
Scientific Classification of A. annua L.
Kingdom - Plantae
Division - Angiosperms
Unranked - Eudicots
Class - Asterids
Order - Asterales
Family - Asteraceae
Genus - Artemisia
Species - A. annua
Statement of the Problem
There has been an increase in the re-evaluation of traditional medicinal plants worldwide, with
extensive research on various therapeutic properties being carried out on Artemisia annua. One challenge
of agriculturalist is losses due to infection by fungal pathogen. There is need to carry out control trial on
fungal isolates with this plant
Justification
Artemisia annua species is an important herb which is grown in Jigawa and Kaduna States of
Nigeria. However, there has been little research on crop losses that can be directly attributed to fungi. So
far the researchers have not come across any control trial on fungi using this plant. Hence these attempt.
Aim
To evaluate the anti-fungal properties of Artemisia annua extracts on Aspergillus brasiliensis and
Penicillium chrysogenum.
In 1971, scientists demonstrated the plant extracts had anti-malaria activity in primate models and
in 1972, the active ingredient, artemisinin (formerly referred to as arteannuin), was isolated and its
chemical structure described. (^Duke S.O, Paul, R.N. 1993).

In 2015, artemisinin was shown to bind to a large number targets suggesting that it acts in a non-selective
manner (Wang et al.; 2015).
Apart from the active compound Artemisinin, recent studies show that A. annua is one of the four
medicinal plants with the highest Oxygen Radical Absorbance Capacity (ORAC) level (^Brisibe et al.,
2009). Artemisia annua possess the capacity to produce high phenolic compound which results in high
antioxidant activity. Five major groups (Coumarins, Flavones, Flavonoids, Phenolic acids and
Miscellaneous). Containing over 50 different phenolic compounds were identified analyzing A. annua
(Ferreira et al., 2010).
It is proven, that Artemisinin has anti-cancer activity as well, because it contains an endo-peroxide group.
Artemisinin has high anti-cancer activity due to its interaction with iron complexes (Efferth et al., 2004).
Recent investigations linked the influence of flavonoids with different enzymes involved in drug
metabolism and in chemical carcinogenesis process. This induces to a therapeutic potential (^Kale et al.,
2008). Many studies show anti-cancer result analyzing different flavonoids, such as flavones and
flavonols. In general it has been shown that specific flavonoid compounds can inhibit specific cancer cell
growth as well as cell proliferation. Furthermore, these flavonoids induce cell apoptosis (Ferreira et al.,
2010).
The earliest record of the herbs clinical use dates back at least 2,000 years to the Wushi Er Bring tang
(Prescription for fifty two diseases) which was unearthed from the Ma Wang Diu tomb at Changsha,
Hunan in 1973. In the 4th century, the medicinal use of the herb for fever was prescribed in Chinese
Handbook of prescriptions for emergency treatment (Dhingra et al., 2000). Artemisinin, the most studied
derivatives and its semi-synthetic derivatives; arteether, artemether, and artesunate, have been clinically
evaluated and are the only anti-malarial drugs to which clinical resistance has never been documented
(Moore et al., 1995).

MATERIALS AND METHOD

Study Area
This study was conducted in the laboratory of Plant Science Department of Modibbo Adama
University of Technology (MAUTECH) Yola, Nigeria. Located at latitude 90 14N and 120 27E
(Adebayo 1999), where the isolation, identification and control was carried out.

Sources of Samples
Artemisia annua was collected from the Institute for Agricultural Research; IAR, ABU-Zaria.
The sample was conveyed to the laboratory in a sterile polythene bag, where 309g of the sample were
dried and pounded.

Sterilization
Sterilization of the laboratory environment was carried out to avoid contamination using 100%
ethanol, UV light switched on for 30 minutes before carrying out inoculations. The inoculation needle
was also sterilized by flaming and deeping into methylated spirit to cool. The inoculation of the
organisms was carried out in the inoculating chamber.
Preparation of Potato Dextrose Agar (PDA)
Twenty (20g) of Potato Dextrose Agar (PDA) was dissolved in 500mls of distilled water and was
autoclaved at 121oc pressure for 15 minutes. One capsule of chloramphenicol was added to the sterilized
media, just before pouring into petri-dishes, to prevent bacterial growth (Sulaiman and Michael, 2013).

Pouring of media
The prepared media was poured in an inoculating chamber; 20ml of the PDA was poured in 9cm
sterile petri-dishes, Each plates was poisoned with 2mls of plant extracts and allowed to cool and
solidify (Smith and Onion, 1994) for the isolation.

Preparation of Plant Extract

Leaf, stem, and root of A. annua was dried under shade to maintain its composition and powdered
with mortar and pistil. The powdered sample was extracted with ethanol and distilled water.
Aqueous extract
The dried leaf, stem and root parts of A. annua in powdered form was Dissolved in sterile water
for 14 days and was filtered with filter paper.
Ethanol extract
The above listed parts were dissolved in Ethanol for14days and were filtered
Control Trial In vitro
In vitro control was carried out using food poisoning method as suggested by Suleiman and
Michael, (2013). The Potato Dextrose Agar (PDA) was amended with extracts of Artemisia annua parts
before inoculation. Inoculating the pathogen, singular method was made to serve as control all in replicate
of three and incubated at room temperature for ten (10) days.

Experimental Design and Statistical Analysis

Completely Randomized Design of three (3) treatments which include leaf, stem and root of A.
annua in two (2) solvents (Ethanol and Distilled water). There were three replications alongside control.
All the data was analyzed using Analysis of Variance (ANOVA) according to Gomez and Gomez (1984).
Significant means was separated using least square differences (LSD) at P = 0.05 with statistical software
SAS according to (Ogbulu, 2009).
RESULTS

Table 1: Mean table for Aspergillus brasiliensis ethanol (extract)

Plant material Mean

Day 1 Day 3 Day 5 Day 10
Leaf ethanol 0.00 0.00 49.33 59.67

Root ethanol 0.00 0.00 62.00 81.33

Stem ethanol 0.00 0.00 51.67 71.00

Control 14.67 31.00 68.33 90.00

LSD(P=0.05) 0.58 1.73 4.42 4.67

The above table showed that leaf ethanol extract had the highest and root ethanol extract had the lowest
inhibitory activity against A. brasiliensis.
Table 2: Mean table for Aspergillus brasiliensis aqueous (extract)
Plant material Mean
Day 1 Day 3 Day 5 Day 10
Leaf aqueous 4.67 9.67 41.67 51.67

Root aqueous 5.00 9.67 60.33 83.00

Stem aqueous 4.33 9.67 9.67 13.67

Control 15.00 30.00 69.00 90.00

LSD (P = 0.05) 0.94 0.58 3.25 4.80

The table above showed that stem aqueous extract had the highest and root aqueous had the lowest
inhibitory activity against A. brasiliensis.
Table 3: Mean table for Penicillium chrysogenum ethanol (extract)
Plant material Mean
Day 1 Day 3 Day 5 Day 10
Leaf ethanol 0.00 0.00 41.67 61.67

Root ethanol 0.00 0.00 18.33 30.67

Stem ethanol 0.00 0.00 0.00 0.00

Control 15.00 31.00 69.00 90.00

LSD (P = 0.05) 0.00 0.10 4.81 1.67

The above table showed that stem ethanol extract had greater and leaf ethanol extract had lowered
inhibitory activity against P. chrysogenum.
Table 4: Mean for Penicillium chrysogenum aqueous (extract)
Plant material Mean
Day 1 Day 3 Day 5 Day 10
Leaf aqueous 3.00 9.33 9.00 14.67

Root aqueous 0.00 0.00 0.00 0.00

Stem aqueous 3.00 9.00 9.33 14.00

Control 15.00 31.00 69.00 90.00

LSD (P = 0.05) 1.30 1.73 1.29 1.15

The table showed that root aqueous extract had the highest and leaf, stem aqueous extracts had the lowest
inhibitory activity against P. chrysogenum.
Table 5: Mean Table for Aspergillus brasiliensis combined
Plant material Mean
Day 1 Day 3 Day 5 Day 10
Leaf ethanol 0.00 0.00 49.33 59.67

Stem ethanol 0.00 0.00 51.67 71.00

Root ethanol 0.00 0.00 62.00 81.33

Leaf aqueous 4.67 9.67 41.67 51.67

Stem aqueous 4.33 9.67 9.67 13.67

Root aqueous 5.00 9.67 60.33 83.00

Control 15.00 30.00 69.00 90.00

LSD (P = 0.05) 0.60 0.55 3.44 4.43

It is evident that among all the extracts; stem aqueous had greater inhibitory activity and root aqueous had
the lowest inhibitory activity against A. brasiliensis.
Table 6: Mean Table for Penicillium chrysogenum combined
Mean
Plant material Day 1 Day 3 Day 5 Day 10
Leaf ethanol 0.00 0.00 41.67 61.67

Stem ethanol 0.00 0.00 0.00 0.00

Root ethanol 0.00 0.00 18.33 30.67

Leaf aqueous 3.00 9.33 9.00 14.67

Stem aqueous 3.00 9.00 9.33 14.00

Root aqueous 0.00 0.00 0.00 0.00

Control 15.00 31.00 69.00 90.00

LSD (P = 0.05) 0.91 1.10 3.11 1.55

The table above showed that root aqueous and stem ethanol extracts had greater inhibitory activity, while
leaf ethanol had the lowest inhibitory activity against P. chrysogenum.
Discussions
The result showed that extracts of Artemisia annua; stem (aqueous) inhibit the growth of
Aspergillus brasiliensis by 85%, root (aqueous) by 20%, leaf (aqueous) by 50%, and stem (ethanol)
inhibit the growth of Aspergillus brasiliensis by 30%, root (ethanol) by 20%, leaf (ethanol) by 40%.
The extracts of Artemisia annua; stem (aqueous) inhibit the growth of Penicillium chrysogenum
by 85%, root (aqueous) by 100%, leaf (aqueous) by 85%, and stem (ethanol) by 100%, root (ethanol) by
70%, leaf (ethanol) by 40%.
It is evident that among all the extracts, stem (ethanol) and root (aqueous) had the highest
controlling and inhibiting properties on the growth of fungal pathogen by 100%. Whereas, others
suppressed the growths of pathogens at different percents or levels.
Also, the result showed that the extracts of Artemisia annua had significantly greater inhibitory
activity against Penicillium chrysogenum compared to Aspergillus brassiliensis.
Control of Penicillium chrysogenum at 100% using extracts of A. annua stem (ethanol) and root
(aqueous) showed that the two extracts had a highly significant impact on the growth of fungus, leading to
complete inhibition of growth.

Conclusion
The plants parts extracts that better controlled the growth considering Penicillium chrysogenum
were stem (ethanol) and root (aqueous)
For the leaf (ethanol), root (ethanol), stem (aqueous) and leaf (aqueous) extracts of A. annua
although it has not had satisfactory or complete inhibitory result, but were able to suppressed the growths
of pathogens.
The result of the different parts of Artemisia annua extracts, i.e. stem (ethanol) and root (aqueous)
demonstrated strong antifungal potential overall.

Considering the scarcity and effectiveness of these medicinal plants in controlling the growth of
fungal pathogens, researchers needed to grow this plant in abundance and further studies to explore the
potentials of these plants.

Based on the results of this research, the stem (ethanol) and root (aqueous) can be useful to farmers
for complete control of disease of plants associated with Penicillium chrysogenum. And other extracts of
Artemisia annua can effectively suppress disease of plant associated with Aspergillus brasiliensis.
Effective control of plant diseases associated with Aspergillus brasiliensis and Penicillium
chrysogenum can be achieved by spraying with stem, leaf and root extracts of ethanol to diseased plants at
three (3) days intervals, as the growth of fungal isolates were fully controlled by the extracts at three (3)
days of incubation.
Acknowledgement
I am immeasurably grateful to Almighty Allah for His grace and mercy upon me. I wish to express my
gratitude to Dr F. K. Channya, Mr Zakariya, Hashim Murtala Tukur and Ahmed Usman.
My gratitude also goes to staffs of Agronomy department, Institute for Agricultural Research, ABU-Zaria.
Especially Mal Bala.
My profound gratitude to my Parents Mr and Mrs Hammajoda Ibrahim for their support. I want to express
my gratitude to those who have been of assistance to me in accomplishing this work.
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 APPENDICES

Appendix i. Effect of Artemisia annua on Aspergillus brasiliensis growth

Days Stem Leaf Root Control Stem Leaf Root Control
Ethanol Ethanol Ethanol (mm) Aqueous Aqueous Aqueous
(mm) (mm) (mm) (mm) (mm)
1 0 0 0 15 5 4 5 15
1 0 0 0 14 4 5 5 15
1 0 0 0 15 4 5 5 15
3 0 0 0 30 10 10 10 30
3 0 0 0 33 9 9 9 30
3 0 0 0 30 10 10 10 30
5 50 47 60 69 10 40 60 69
5 55 50 61 68 9 45 60 70
5 50 51 65 68 10 40 61 68
10 70 57 80 90 15 50 84 90
10 75 60 81 90 12 55 80 90
10 68 62 83 90 14 50 85 90
Appendix ii. Effect of Artemissia annua on Pennicillium chrysogenum growth
Days Stem Leaf Root Control Stem Leaf Root Control
Ethanol Ethanol Ethanol (mm) Aqueous Aqueous Aqueous (mm)
(mm) (mm) (mm) (mm) (mm) (mm)
1 0 0 0 15 3 4 0 15
1 0 0 0 15 4 3 0 15
1 0 0 0 15 2 2 0 15
3 0 0 0 30 10 9 0 15
3 0 0 0 31 9 10 0 15
3 0 0 0 32 8 9 0 15
5 0 40 20 70 10 9 0 64
5 0 45 15 69 9 10 0 68
5 0 40 20 68 9 8 0 70
10 0 60 30 90 15 15 0 90
10 0 63 32 90 14 14 0 90
10 0 62 30 90 13 15 0 90
Appendix iii. ANOVA Table for Aspergillus brasiliensis ethanol
Mean

Source DF Day1 Day 3 Day 5 Day 10

Treatment 5 96.83** 432.75** 144.87** 311.23**

Plant 2 161.3** 720.75** 237.89** 515.22**

Rep 2 0.08ns 0.75ns 5.33ns 5.25ns

Error 6 0.08 0.75 4.89 5.47

Appendix iv. ANOVA Table for Aspergillus brasilensis aqueous
Mean

Source DF Day1 Day 3 Day 5 Day 10

Treatment 5 48.18** 186.35** 1243.17** 2186.65**

Plant 2 80.31** 310.08** 2070.56** 3644.53**

Rep 2 0.000ns 0.75ns 2.08ns 0.33ns

Error 6 0.22 0.08 2.64 5.78

Appendix v. ANOVA Table for Penicillium chysogenum ethanol
Mean

Source DF Day1 Day 3 Day 5 Day 10

Treatment 5 101.25** 432.55** 1603.88** 2719.75**

Plant 2 168.75** 720.75** 2672.97** 4531.86**

Rep 2 0.000ns 0.25ns 0.25ns 1.58ns

Error 6 0.00 0.25 5.80 0.69

Appendix vi. ANOVA Table for Penicillium chrysogenum aqueous
Mean

Source DF Day1 Day 3 Day 5 Day 10

Treatment 5 79.95** 312.43** 1813.83** 2994.53**

Plant 2 132.75** 520.67** 3022.33** 4990.67**

Rep 2 0.75ns 0.08ns 1.08ns 0.33ns

Error 6 0.42 0.75 0.42 0.33

Appendix vii. ANOVA Table for Aspergillus brasiliensis combined
Mean

Source DF Day1 Day 3 Day 5 Day 10

Treatment 7 63.90** 256.25** 866.36** 1534.26**

Plant 5 85.21** 341.52** 1152.75** 2045.00**

Rep 2 0.00ns 0.43* 7.19ns 2.05ns

Error 11 0.11 0.09 3.75 6.21

Appendix viii . ANOVA Table for Penicillium chrysogenum combined
Mean

Source DF Day1 Day 3 Day 5 Day 10

Treatment 7 67.61** 293.05** 1062.77** 2585.43**

Plant 5 540.00** 390.71** 1950.16** 3447.10**

Rep 2 0.86ns 0.05ns 0.61ns 0.43ns

Error 11 0.26 0.38 3.06 0.76

Highly Significant (**)

Significant (*)
ns
Non-Significant ( )
Biography
Zainab Hammanjoda is a graduate of Modibbo Adama University of Technology (MAUTECH) Yola,
Nigeria with a Bachelor of Technology in Botany