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A COMPARISON OF ANTIFUNGAL POTENTIAL OF
STANDARDIZED AQUEOUS AND ALCOHOLIC EXTRACTS OF
TREES OF CLASS MANGNOLIATAE
Shruti Sharma1, Tripti Bhatnagar2, Fanish Pandey. 1
School of Sciences, Noida International University, Greater Noida.
2
Corresponding author, Codon Biotech Pvt. Ltd, Noida.
ABSTRACT
The extracts of 9 different leaves of trees belonging to Class Mangnoliatae were screened for their antimicrobial activities against plant pathogenic fungal diseases. The aqueous and alcoholic extracts of Psidium guajava, Syzygium cumini, Aegle marmelos, Nyctanthes arbor-tristis, Callistemon lanceolatus, Citrus limonum, Combretum indicum, Calliandra haematocephala, Polyalthia longifolia were made from the leaves of the plants and were tested against a no. of plant pathogenic fungi namely, Alternaria alternata, Alternaria pori, Alternaria solani, Aspergillus Niger, Aspergillus flavus, Penicillium crysogenum. Among the tested extracts, the methanolic extract gave highest ie. (85%- 90% ) fungal inhibition as compared to ethanolic and aqueous extracts. The methanolic extracts were highly inhibitory to seed storage fungal pathogens like Aspergillus niger and Aspergillus flavus. Alternaria solanai and Alternaria alternate were also inhibited to an extent of 90% in many cases. Thus, antifungal effect depends on the type and concentration of the secondary metabolite extracted by different solvents.
Key words : Antifungal activity, Myrtacae, Rutacae, alcoholic extract, aqueous extract
I. Introduction
Pathogenic fungi are important infectious agents attacking plants all over the world. They are
the main cause of alterations starting from the development stages of the plants until storage,
causing various disorders and diseases related to appearance, nutritional value, organoleptic
characteristics, etc. (Agrios, 2004). Furthermore, phytopathogenic fungi development is also
responsible for food contamination with mycotoxins, one of the main representatives being
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several aflatoxins. Thus, a significant portion of the agricultural produce in the country and
the world over become unfit for human consumption due to mycotoxins contaminations of
grains, especially those produced by species of Aspergillus (Devi et al, 2001) Current control of phytopathogenic agents is done using synthetic fungicides which have adverse effects on
human health and the environment (Buttu et al, 2013). As a result there is a need for
development of biological methods that will work for inhibition of fungal growth, such as the
use of antifungal active ingredients extracted from plants (Quiroga et al., 2001)
Many researchers have found that plant extracts, root extracts, stem extracts, fruit
extracts etc. contain good activity against plant fungal pathogens. Plants have ability to
synthesize aromatic substances such as phenolic,( Khan et al. 2010) (e.g. phenolic acids,
flavonoids, quinones, coumarins, lignans, stilbenes, tannins), nitrogen compounds (alkaloids,
amines), vitamins, terpenoids (including carotenoids) which serve as plant defence
mechanisms against microbes, insects, herbivores (Bharathi et al., 2011). In addition to this,
the bioactivity of plant extracts depends on the water and ethanol concentration used in the
extraction process (Ganora, 2008, Kumar A., Bhatii V. 2011). Although a great amount of
research has been performed to determine the antifungal activity of medicinal plants, optimal
extraction of bioactive compounds has not been well established for most plants.
Thus, the objective of the study was to identify optimum type of alcohol, water and
alcohol ratio required to extract the secondary metabolites which provides highest level of
antifungal activity comparable synthetic chemical which are commonly used on plants. For
the present study different trees of four classes were taken including Myrtaceae, Annonaceae,
Fabaceae and Rutaceae.
II. Material and methods
A. Collection of Plant materials
Fresh parts of different nine plant leaves namely: Psidium guajava, citrus limonum, Nyctanthes arbor-tristis, Polyalthia longifolia, Callistemon lanceolatus, Calliandra haematocephala, Aegle marmelos, Syzygium cumini Combretum indicum were collected from different parts of Greater Noida & Noida city. The apparently healthy parts of these
plants were thoroughly times with tap water and once with sterile distilled water, and used for
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Table 1 - Test plants used for antifungal assay.
Name Family Plant part used
Psidium guajava Myrtaceae Leaf
Polyalthia longifolia Annonaceae Leaf
Calliandra haematocephalla Fabaceae Leaf
Callistemon lanceolatus Myrtaceae Leaf
Aegle marmelos Rutaceae Leaf
Syzygium cumini Myrtaceae Leaf
Nyctanthes orbortristis Oleaceae Leaf
Combretum indicum Myrtaceae Leaf
Citrus limonum Rutaceae Leaf
B. Preparation of extracts
Alcoholic Extracts and Aqueous Extracts
Collected plant parts were shade dried and ground to a fine powder using grinder mixer. For
the present study two different water and alcohol ratios were used for extraction of the
antifungal activity. A ratio water and alcohol ration of 1:1 and 1:4were used. The air dried
plant material was further grinded in 10 ml of respective solvent in pestle mortar and
incubated for overnight for proper extraction. The extract was then filtered with Whatman’s
filter paper no.1 and was then centrifugated at 5000rpm for 10 minutes. The supernatant was
treated as the extract and stored at 4°C in sterile tubes.
C .Test fungal pathogens
Fungal pathogens were maintained on Potato Dextrose Agar medium. The test fungal
pathogens taken were as follows: Aspergillus flavus, Aspergillus niger, Alternaria alternata, Alternaria solani, Penicillium crysogenum, Alternaria porri.
D. Antifungal activity testing
The antifungal activity was performed by agar plate method. The growth of pathogenic fungi
was observed in control and in plates containing the different plant extracts. Comparing the
growth in blank and test samples, the percentage inhibition of the fungus was calculated in
every case. The extracts and fungi were incubated for 2-3 days in BOD at 25°C and the observation were recorded as percentage Inhibition by comparing the growth of fungi on
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III. Result & Discussion
The present study involves a comparative analysis of aqueous and alcoholic [Methanolic and
Ethanolic] extract of leaves. The type of solvent and the ratio of water : alcohol concentration
is also important to assess the actual antifungal activity of the plants. This was important
because most of the plant secondary metabolites show toxic effect against microorganisms.
The secondary metabolites like alkaloids, flavonoids are normally soluble in non- polar and
alcoholic solvents but antifungal proteins and glycoproteins are soluble in aqueous solutions.
Thus, the concentration and type of secondary metabolite extracted in each case is also
important in the resulting antifungal activity. Different plant metabolites would affect and
cause toxicity to different fungal pathogens. In this study the In vitro testing was carried out
against plant pathogenic Fungi namely: Alternaria alternata, Alternaria porri, Alternaria solani, Aspergillus Niger, Aspergillus flavus, Penicillium crysogenum.
It is difficult to find compounds that selectively inhibit only fungal metabolism and not the
growth and metabolism of plants also. There is lack of effective and safe antifungal
chemicals and fungicides. Normally chemical fungicides are used during storage of seeds and
crops and also when crops are standing in the fields to prevent and protect them from fungal
disease (Pawar B. T .2011). The chemicals fungicides are toxins and when consumed in large
quantities cause harm to humans and animals and further pollute the environment.
The methanolic extract was more effective against almost all the fungal pathogens as seen in
Table :1. Since the 1: 4 ratio water and methanol extract showed more that 85% inhibition
against all the fungal pathogens taken for the study. The antifungal activity and toxicity
against Alternaria porri was less in all cases of plants taken but still showed higher than 50% inhibition. In the 1:4 extract it was observed that higher concentration of the antifungal
compounds was extracted. This is clearly ( Table no. 2 and 3 and Figure no. 1) depicted by
the fact that 1:1 ration extract gave lower levels of inhibition of fungus as compared to 1:4
ratio extract. Thus, higher concentration of methanol was effective in extracting higher
concentration of secondary metabolites from plant tissue. Similar results were also obtained
by other workers on various different fungal strains where they compared between the effect
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Table -2 Antifungal activity of Methanolic extracts (water: methanol ratio- 1:1) against different fungal pathogens.
PLANT NAMES PERCENTAGE INHIBITION (%)
A.Solani A.alternata A.pori A.niger A.flavus P.crysogenum
Psidium guajva 71 82 64 86 80 69
Syzigium cumini 73 88 55 83 83 63
Aegle marmelos 75 88 60 86 88 89
Nyctanthes arbor-tristis 79 85 64 85 85 84
Combretum indicum 74 83 46 79 83 75
Callistemon lanceolatus 71 77 57 73 78 88
Polyalthia longifolia 87 88 70 72 78 83
Citrus limonum 73 83 60 88 84 85
Calliamdra haematocephala
70 87 43 78 82 89
Table - 3 Antifungal activities of methanolic extracts (water: methanol ratio- 1:4) against different plant pathogens.
PLANT NAMES PERCENTAGE INHIBITION (%)
A.Solani A.alternata A.porri A.niger A.flavus P.crysogenum
Psidium guajva 75 89 68 89 89 75
Syzigium cumini 81 92 64 87 89 69
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Nyctanthes arbor-tristis 80 89 68 87 88 89
Combretum indicum 79 89 56 83 86 82
Callistemon lanceolatus 85 83 65 83 83 94
Polyalthia longifolia 89 92 73 83 91 88
Citrus limonum 76 87 65 92 89 88
Calliomdra haematocephala
[image:6.595.75.510.284.537.2]71 91 47 83 84 90
Figure 1:Graphical representation of the percentage inhibition of fungal pathogens by Plant extracts (Methanolic 1:4)
The Ethanolic extracts, on the other hand did not show effective antifungal activity against
the same test fungal strains. As seen in the Table No.4 and 5 and Figure: 2, the Ethanolic
extracts of Psidium guajava, Citrus limonum and Nyctanthes arbor-tristis showed 85% inhibition against Aspergillus niger. As an exception, all the nine tree leave extracts showed 80% or more inhibition only against Penicillium crysogenum. Only Polyalthia longifolia showed 87 % inhibition against A.solani.
Table -4 Antifungal activities of Ethanolic extracts (water: ethanol ratio - 1:1) against different plant pathogens.
0 10 20 30 40 50 60 70 80 90 100
A.Solani
A.alternata
A.pori
A.niger
A.flavus
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PLANT NAMES PERCENTAGE INHIBITION (%)
A.Solani A.alternata A.porri A.niger A.flavus P.crysogenum
Psidium guajva 62 60 54 67 60 87
Syzigium cumini 67 68 60 56 62 84
Aegle marmelos 72 68 62 74 64 84
Nyctanthes arbor-tristis 62 60 52 83 64 85
Combretum indicum 68.3 60 54 77 60 80
Callistemon lanceolatus 62 40 56 67 64 75
Polyalthia longifolia 83 70 70 67 60 76
Citrus limonum
67 60 60 61 72 81
Calliamdra haematocephala
[image:7.595.67.525.399.645.2]60 60 56 77 60 87
Table - 5Antifungal activities of Ethanolic extracts (water: ethanol ratio - 1:4) against different plant pathogens.
PLANT NAMES PERCENTAGE INHIBITION (%)
A.Solani A.alternata A.porri A.niger A.flavus P.crysogenum
Psidium guajva 67 64 60 83 70 94
Syzigium cumini 72 72 64 61 70 87
Aegle marmelos 75 72 70 78 70 89
Nyctanthes arbortristis 67 70 58 85 74 88
Combretum indicum 70 64 60 79 62 83
Callistemon lanceolatus
67 70 60 78 70 88
Polyalthia longifolia 87 78 76 78 70 83
Citrus limonum 72 68 70 74 80 83
Calliamdra haematocephala
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Figure 2:Graphical representation of the percentage inhibition of fungal pathogens by Plant extracts (Ethanolic 1:4)
In the present study when aqueous extracts of the same trees of Myrtaceae,
Annonaceae, Fabaceae and Rutaceae family were tested against the same fungal plant pathogens , it
was observed that the antifungal potential was better than the ethanolic extract . In the case of
Polyalthia longifolia aqueous extract, it gave a very high antifungal activity against all the fungal strains i.e Alternaria alternata, Alternaria pori, Alternaria solani. Inhitbition against seed storage disease caused by Aspergillus niger was also observed to a level of 83-85 % by extracts of Aegle marmelos, , Combretum indicum, , Polyalthia longifoli and Citrus limonum.
0 10 20 30 40 50 60 70 80 90 100
A.Solani
A.alternata
A.pori
A.niger
A.flavus
P.crysogenum
Plant name
A. porri
A. alternata
A. solani
A. niger
A. flavus
P. chrysogenum
Psidium guajava 75 57 73 47 79 78
Syzygium cumini 50 50 56 42 85 82
Aegle marmelos 78 62 67 75 77 75
Nyctanthes arbor-Tristis 69 50 47 67 79 82
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Table 6: Antifungal activities (Percentage inhibition) of Aqueous extracts (50%) against different plant fungal pathogens.
Plant name
A. porri
A. alternata
A. solani
A. niger
A. flavus
P. chrysogenum
Psidium guajava 91 67 89 67 81 81
Syzygium cumini 71 67 60 67 89 89
Aegle marmelos 86 75 78 83 84 88
Nyctanthes arbor-tristis 75 75 56 75 85 93
Combretum indicum 78 58 60 82 93 77
Callistemon lanceolatus 75 67 44 75 80 82
Polyalthia longifolia 86 91 91 83 80 86
Citrus limonum 89 70 67 83 93 86
Calliandra haematocephala
64 80 89 67 85 69
Table 7: Antifungal activities (Percentage inhibition) of Aqueous extracts (80%) against different plant fungal pathogens.
Callistemon lanceolatus 75 50 33 66 77 67
Polyalthia longifolia 75 83 76 72 71 75
Citrus limonum 67 58 50 82 84 75
Calliandra Hematocephala
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Figure 3:Graphical representation of the percentage inhibition of fungal pathogens by Aqueous extracts (80%)
These results suggest that different solvents used in the extraction process may result in the
retention of different bioactive components. Polysaccharides, proteins and some polyphenols
have been shown to be present in water extracts while alkaloids, flavanoids and phenols are
main component of alcoholic extract.
Plants have ability to synthesize aromatic substances of different functional groups, most of
which are phenols or their oxygen substituted derivatives. Many of these are secondary
metabolites, which show antimicrobial activity. In many cases, these substances serve as
plant defence mechanisms against predation by microorganisms, insects, and herbivores
(Khan and Tanveer 2013). Cowan 1999, showed that alcoholic extracts have higher
extraction ability for secondary metabolites and thus show better antimicrobial activity.
Another study by Ahmad et al 2013, showed that the secondary metabolites like phenols,
alkaloids, flavonoids present in various tree leaves and barks have inhibitory effect on fungal
cells. They cause fungal cell breakage, oxidation of cell surface compounds and oxidation of
intracellular enzymes thus causing toxicity to fungal pathogens and finally leading to death of
the pathogen. Another study (Khan and Nasreen, 2010) showed that aqueous extracts had
very less inhibitory effect which might be attributed to the extracting capacity of solvent and
0 10 20 30 40 50 60 70 80 90 100
A.porri
A. alternata
A.solani
A.niger
A.flavus
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the concentration of the active ingredients in the extracts and also most of the active
ingredients are dissolved better in alcoholic solvents than in water . Several other workers
have also reported that water extracts do not have much activity against fungi [Martin G
1995, Paz et al 1995, Vlietincket al 1995]. Myrtacae and Rutacae and Fabacae family
contains more bioactive compounds such as triterpenes, sterols and alkaloids thus methanolic
extracts showed more than 90% inhibition of most of the plant fungal pathogens
Thus, the present study and future research on different plants for extraction of their
secondary metabolites is an important aspect for developing higher number of herbal
antifungal formulations which could be then tried at field level and commercial level.
V. Conclusion
Fungal infections are very serious global problem for humans as well as and during seed
storage and in crop production. Fungi produce mycotoxins during storage which can be
harmful for everyone that why this study was undertaken to analysis antifungal activity found
in Myrtacae family. The methanolic extracts of Psidium guajava, Syzygium cumini, Aegle marmelos, Nyctanthes arbor-tristis, Callistemon lanceolatus, Citrus limonum, Combretum indicum, Calliandra haematocephala, Polyalthia longifolia were found to be highly effective as Antifungal compounds and they can be used for developing ecofriendly, herbal and plant
originated fungicide formulations.
Acknowledgements
We sincerely thank Dr.Tripti Bhatnagar [CEO of Codon Biotech Pvt Ltd.], Dr. Fanish Pandey for their kind support during my research work.
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