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Product Code: 00006596

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Several genera of fungi cause different important diseases of plants around the world. Plant diseases are mostly controlled by chemical pesticides and in some cases by cultural practices. The use of plant extract for the control of plant pathogenic fungi reduce the risk of food poisoning and increased the market value of crops. The antifungal activities of the leaf extract of Hyptis spicigera was screened against four species each of Aspergillus (A. flavus, A. fumigatus, A. niger and A. parasiticus) and Fusarium (F. oxysporum, F. graminearum, F. proliferatum and F. verticilloides). The powdered leaf was extracted with hexane, ethyl- acetate, methanol and water following an increase in polarity of these solvents. The qualitative and quantitative phytochemical screening was carried out on the extracts and fractions using standard procedures and the antifungal activities of the hexane, ethyl-acetate, methanol and aqueous extract with their terpenoids and flavonoids fractions was carried out to determine the zone of inhibition, minimum inhibitory concentration and minimum fungicidal concentration. The qualitative phytochemical screening indicates the presence of anthraquinones, unsaturated sterols and triterpenes, cardiac glycosides, saponins, flavonoids, tannins and alkaloids in all the extracts (n-hexane, ethyl-acetate, methanol and aqueous) except anthraquinones which was absent in the methanol and n-hexane extracts. The quantitative phytochemical screening indicates that saponins has the highest concentration of 920 mg/g/DE in the ethyl-acetate extract, phenolics was found to be highest in n-hexane extract with a quantity of 880 mg/g/GAE, flavonoid was higher in the aqueous extract with quantity of 220 mg/g/GAE. Alkaloids was found to be highest in n-hexane extract with a concentration of 170 mg/g/AE and tannins was having the highest concentration of 50 mg/g/QE as observed in the aqueous extract. The TLC chromatographic profile of terpenoids fraction indicates the presence of 7 terpenoids spots on the TLC plates while that of flavonoids fraction indicates 2 yellow and 2 red bands of flavonoids on the TLC plates when viewed under the UV light at 360nm. Ethyl-acetate extract recorded wider diameter of inhibition against A. parasiticus, A. fumigatus and A. flavus with diameter of 24.67±0.88 mm, 21.33±0.67mm and 22.67±0.88 mm respectively. This inhibition zones were significantly higher than the control fungicide (mancozeb) and the other extracts. Methanol extract showed the highest zone of inhibition on F. graminearum (21.00±0.00 mm) which was also significantly higher than the control fungicide (18.33±1.45). The terpenoids fraction from ethyl-acetate extract showed the highest zone of inhibition on F. oxysporum with a diameter of 19.33±0.33mm while there was no inhibition zone against this fungus by the control fungicide (mancozeb) and the other extracts on this fungus. In the case of F. proliferatum, the control fungicide showed higher inhibition zone (21.00±0.00 mm) which was significantly higher than the extracts with their corresponding terpenoids and flavonoids fractions. The aqueous extract was the only extract that showed zone of inhibition on A. niger with a diameter of 16.00±0.58 mm. All the extracts, terpenoids and flavonoids fraction did not show any activity on F. verticilloides while the fungus was sensitive to the control fungicide (22.00±0.58) but A. parasiticus and F. oxysporum were resistance to the control fungicide. The MIC of the extracts and fractions on all the fungi species ranged from 3.13 to 12.5 mg/ml and their MFCs was between 6.25 to 25 mg/ml. The result from this research indicates that the leaf extract of Hyptis spicigera contains phytochemicals which exert a broad range of fungistatic and fungicidal activity on some species of Aspergillus and Fusarium.

Contents pages

1.1 Background of the Study 1
1.2 Statement of Research Problem 4
1.3 Justification 5
1.4 Aim 6
1.5 Objectives of the research 6
1.6 Research Hypotheses 6

2.1 Fungi 7
2.2 Pathogenic Fungi 7
2.3 Types of plant pathogenic fungi 8
2.4 The Genus Aspergillus 8
2.4.1 Classification of Aspergillus 9
2.5 The Genus Fusarium 10
2.5.1 Classification of Fusarium 11
2.6 Economic importance of Aspergillus and Fusarium 11
2.7 Control of Aspergillus and Fusarium species 13
2.7.1 Mancozeb 14
2.8 Hyptis spicigera Lam 15
2.8.1 Classification, Habitat and Distribution of Hyptis spicigera Lam 15
2.8.2 Uses of Hyptis spicigera Lam 15
2.8.3 Chemical constituent present in the genus Hyptis 16
2.9 Uses of Plants Secondary Metabolites as fungicides 16
2.10 Chemical composition and mode of action of plant secondary metabolites 18
2.11 Plant Secondary Metabolites 19
2.11.1 Alkaloids 20
2.11.2 Flavonoids 21
2.11.3 Glycosides 22
2.11.4 Phenolic Compounds 22
2.11.5 Saponins 23
2.11.6 Tannins 23

3.1 Study Area 25
3.2 Source of Plant Materials 25
3.3 Preparation of Plant Materials 25
3.4 Extraction of Plant Materials 27
3.5 Determination of Percentage Yield of Extracts 27
3.6 Isolation of Terpenoids from Ethyl-Acetate Leaves Extract of Hyptis spicigera Lam 29
3.6.1 Thin Layer Chromatographic Profile and chemical test for Terpenoids 31
3.7 Isolation of Flavonoid from Methanol Leaves Extract of Hyptis spicigera Lam 31
3.7.1 Thin Layer Chromatographic Profile and chemical test for Flavonoids 32
3.8 Preliminary Qualitative Phytochemical Screening 33
3.8.1 Test for Carbohydrates 33
3.8.2 Test for Glycosides 35
3.8.3 Test for Anthraquinones Derivatives 35
3.8.4 Test for Unsaturated Sterols and Triterpenes 36
3.8.5 Test for Cardiac Glycoside 36
3.8.6 Test for Saponins 37
3.8.7 Test for Tannins 37
3.8.9 Test for Alkaloids 38
3.9 Quantitative Phytochemical Screening 38
3.9.1 Determination of Total Phenolic Compound (TPC) 38
3.9.2 Determination of Total Flavonoid Content (TFC) 39
3.9.3 Determination of Total Alkaloids Content (TAC) 39
3.9.4 Determination of Total Tannin Content (TTC) 40
3.9.5 Determination of Total Saponin Content (TSC) 40
3.10 Source of Fungal Isolates 41
3.11 Media Preparation 42
3.12 Culturing of Fungal Isolates 42
3.13 Preparation of Fungal Inoculum 42
3.14 Sensitivity Test 42
3.15 Minimum Inhibitory Concentration (MIC) 43
3.16 Minimum Fungicidal Concentration (MFC) 43
3.17 Data Analysis 44

4.0 RESULTS 45
4.1 Percentage Yield of the Leaf Extracts of Hyptis spicigera Lam.
using different Extraction Solvent 45
4.2 Qualitative Phytochemicals contents 45
4.3 Quantitative phytochemicals constituents 45
4.4 Chemical confirmation and TLC chromatographic Profile of
Terpenoids fraction from Ethyl-acetate leaf extract of Hyptis spicigera 50
4.5 Chemical confirmation and TLC chromatographic Profile of
Flavonoid from Methanol leaf extract of Hyptis spicigera Lam 50
4.6 Zones of Inhibition of Extracts, Terpenoids and Flavonoids from
Hyptis spicigera on Aspergillus and Fusarium species 53
4.7 Minimum Inhibitory Concentration (MIC) of the leaf extracts and
fractions from Hyptis spicigera on Aspergillus and Fusarium species 54
4.8 Minimum Fungicidal Concentration (MFC) of the leaf extracts and
fractions from Hyptis spicigera on Aspergillus and Fusarium species 57


6.1 Conclusion 66
6.2 Recommendations 67


3.1 Schematic diagram of extraction procedure with modification… 28

3.2 Terpenoids extraction procedure 30

3.3 Flavonoid extraction procedure 34


4.1 Percentage yield of the leaf extracts of Hyptis spicigera using different extraction solvents… 47

4.2 Qualitative phytochemical contents of Hyptis spicigera leaf extracts 48

4.3 Quantitative phytochemical constituents of Hyptis spicigera leaf extracts. 49

4.4 Inhibition zones (mm) of the leaf extracts and fractions from Hyptis spicigera Lam. on Aspergillus and Fusarium species… 56

4.5 Minimum inhibitory concentration (MIC) of extracts and fractions from Hyptis spicigera on Aspergillus and Fusarium species 56

4.6 Minimum fungicidal concentration (MFC) of extracts and fractions from Hyptis spicigera on Aspergillus and Fusarium species 58


3.1 Hyptis spicigera Lam… 26

4.1 Thin layer chromatographic profile for terpenoids from ethylacetate leaf extracts of Hyptis spicigera showing the calibrations (cm) of spots 51

4.2 Thin layer chromatographic profile for Flavonoids for methanol leaf extracts of Hyptis spicigera under UV light 52


I Fungicidal properties of some plant products… 81

II Mechanism of action of phytochemicals/secondary metabolites from plants 82

III Standard curves used for quantitative phytochemical analyses… 83

III a Gallic acid standard curve 83

III b Quercetin standard curve 83

III c Diosgenin standard curve 84

III d Atropine standard curve 84

IV Plates showing zones of inhibition zones of n-hexane leaf extract on Aspergillus and Fusarium species… 85

V Plates showing zones of inhibition of ethyl-acetate leaf extract on Aspergillus and Fusarium species… 86

VI Plates showing zones of inhibition of methanol leaf extract on
Aspergillus and Fusarium species… 87

VII Plates showing zones of inhibition of aqueous leaf extract on
Aspergillus and Fusarium species… 88

VIII Thin layer chromatographic profile of terpenoids and flavonoids 90

IX Isolation procedure for terpenoids and flavonoids extracted from ethyl-acetate and methanol leaf extract from Hyptis spicigera Lam… 90


1.1 Background of the Study
Fungi are ubiquitous in nature and vital for recycling of nutrients contained in organic matter. The vast majority of the known fungal species are strictly saprophytes, although there are a few capable of causing disease in humans (Bennett and Klich, 2003). However, there are several fungal genera containing species that cause diseases to plants and animals. These fungi can be categorized into two groups with regards to infection; saprophytic fungi which can be opportunistic pathogens that enter via wounds or due to a weakened state of the host and true pathogens that may depend on living plant or human tissues for nutrients but can also survive outside of the hosts (Anthony, 2007).

Several genera of fungi cause many important plant diseases around the world (Anderson et al., 2004; Strange and Scott, 2005). Plant diseases are of paramount importance to humans because they damage plants and plants products on which humans depend on for food, clothing, furniture and many other economical values. The kinds and amounts of losses caused by plant diseases on crop plants varies from species to species and also the type and nature of the pathogen, the locality, the environment, the control measures practiced and combination of these factors (Agrios, 2005).

Cereals and other agriculturally derived products represent an important nutrient source for mankind world-wide. In addition, they are the most important dietary food for most African populations (Riba et al., 2010). Unfortunately, agricultural products are naturally contaminated with fungi in the field, during drying, processing, transportation and subsequent storage and it may be difficult to completely prevent mycotoxins formation in contaminated commodities, particularly those that are produced in tropical and subtropical climates, in countries where high temperature and humidity promote the growth and proliferation of fungi (Kumar et al., 2008). Thus, they are often colonized by fungi, including species from the genus Aspergillus, Penicillium and Fusarium, which cause significant reductions in crop yield, quality and safety due to their ability to produce mycotoxins (Alkenz et al., 2015).

Fungal infections which causes as great as 25-50% loss and still remain an important challenge in sustainable food production (Chuang et al., 2007; Zaker, 2014) with Fusarium being one of the most economically important genus of phyto-pathogenic fungi. Several Fusarium species can infect agricultural produce such as millets, guinea corn, maize, tomatoes and several other vegetables, the predominant species can vary according to crop species involved, geographic region and environmental conditions (Longrieco et al., 2002; Vander-lee et al., 2015).

Aspergillus mold fungus is a large genus consist of over 200 species to which humans are constantly exposed. Only few of these species are pathogenic among which more than 95% of the infections are caused by three species of Aspergillus including A. fumigatus, A. flavus, A. niger (Anaissie et al., 2009). Aspergillus spp. are among the pathogenic fungi causing infection through spores entering human body and these infections are invasive and very serious in individuals with deficient immune systems. Even in healthy people, Aspergillus may cause local infections in lungs, sinuses, and other organs of the body (Teles and Seixas, 2015). Aflatoxins are among the most important mycotoxins which are produced by species assigned to the Aspergillus genus. Among the numerous aflatoxins identified are; Aflatoxin B1 which is the most toxic aflatoxin being a potent genotoxic carcinogen in laboratory animals and there is strong evidence for its liver carcinogen in humans (Backhouse, 2014). The International Agency for Research on Cancer has classified aflatoxin B1 as a group I carcinogen. The most important producer of Aflatoxin B1 is Aspergillus flavus, it is also an important pathogen of various cultivated plants including maize, cotton, cowpea and peanut, and cause serious yield losses throughout the world. Since aflatoxin production is favoured by moisture and high temperature, A. flavus is able to produce aflatoxins in warmer, tropical and subtropical climates (Varga et al., 2009).

Fusarium diseases that affect most crops are caused by several individual Fusarium or more commonly, co-occurring species. Fusarium spp. can cause indirect losses resulting from seedling blight or reduced seed germination, or direct losses such as seedling foot and stalk rots; however, the most important diseases in cereals due to severe reduction in yield and quality are head blight of small cereals such as wheat, barley, oat and ear rot of maize (Nganje et al., 2000; Munkvold, 2003). The coexistence of different Fusarium spp. in the field is a normal situation and although the number of detectable species can be high (Longrieco et al., 2007), only some of them are pathogenic, especially under suitable climatic conditions. The composition of species involved in Fusarium disease complex is dynamic (Kohl et al., 2007), the species comprises of a Fusarium community associate with each other and this cohabitation is particularly affected by climatic factors such as temperature and moisture.
Plant extracts act as contact fungicides by disrupting cell membrane integrity at different stages of fungal development, while others inactivate key enzymes and interfere with metabolic process (Rey et al., 2018). Crops treated with plant extract produce and accumulate elevated levels of specialized protein and other compound which inhibit the development of fungal diseases (Gebore et al., 2013).

Hyptis (Hyptis spicigera) is a genus of flowering plant in the Lamiaceae family. Hyptis spicigera is an erect, aromatic, annual or perenial herb growing up to 1meter tall, the plant is frequently grown as a food crop for its seeds in parts of tropical Africa (Ladan et al., 2014). The plant is commonly known as bushmint (due to the aromatic nature of their leaves), they are widespread in tropical North and South America, as well as parts of West Africa. There are 300 to 400 species, which may be annual or perennial, small or large shrub (Parak and Chanda, 2007). Hyptis spicigera is an important medicinal plant used in treatment of gastrointestinal disturbances, wounds, skin infections and insect bites (Esquivel-Ferrino et al., 2014). The member of this genus are usually being used traditionally as mosquitoes and other insect repellent, leaves of this plants are normally kept at the edge of rooms to repel mosquitoes. However, studies have shown that the plant in this genus contain some major bioactive compounds among which includes carbohydrates, saponin glycosides, alkaloids and flavonoids (Ladan et al., 2011).

1.2 Statement of Research Problem

Fungi are predominant inducers of severe diseases causing huge economic loss. Most of these fungi are known to release mycotoxins which destroy the quality and nutritive value of food. In agricultural sectors, a crop is susceptible to fungal contamination (a stage of making the crop unhealthy or changes from natural state) at various stages, right from the sowing of seeds to post harvesting periods. Approximately 25- 40% cereals and other storage agricultural produce worldwide are contaminated with fungi (Singh et al., 2010). These fungi produce different types of mycotoxins that can be mutagenic, teratogenic or carcinogenic causing feed refusal and emesis in human and animals (Shukla et al., 2012). Hence to suppress these pathogenic fungi different synthetic fungicides are being commercialized. These synthetic chemicals are bound with various adverse effects (Wang and Jeffers, 2010). For many years’ synthetic fungicides have been used for plant protection however their extensive use has led to resistance by most of the pathogenic fungi (Wang et al., 2014).

Death due to aflatoxins has been reported in humans, animals and birds (Gugnani, 2003; Agrios, 2005). Fumonisin produced by Fusarium species is also one of the toxin known globally to affect agricultural food and feed crops. Processing of infected grains and other agricultural produce from farms results in the release of airborne particulate matter that is contaminated with aflatoxins, thereby exposing the lungs of agricultural workers to these toxins (Yiannikouris and Jouany, 2014). In humans, inhaled aflatoxin can cause inflammation and eventually irreversible pulmonary interstitial fibrosis which is the scarring of the lung tissues between the air sacs (Lougheed et al., 2018)

1.3 Justification

The use of most synthetic fungicides have been restricted because of high acute toxicity, long degradation period, pathogen resistance, side effect on human, plants, animals and the ecology of most living organisms (Wuyep et al., 2017). Due to high usage of chemical fungicides on farm land near water bodies, there is an increase release of toxins in water bodies which will invariable enter into the food chains (Avasthi et al., 2010).

The development of non-toxic, safe and effective biodegradable alternative source to synthetic fungicides has in recent years puzzled researchers to screening of plants for bioactivity on plant pathogenic microbes (Onifade, 2000). Plants generally contain a wide variety of free radical scavenging molecules including phenols, flavonoids, vitamins and terpenoids that are rich in antifungal activity (Cai et al., 2014). Furthermore, the use of fungicides generates health concerns due to their carcinogenic and teratogenic properties (Sharma et al., 2009).

Also, the use of plant extract for the control of plant pathogenic fungi will virtually reduce the risk of food poisoning as some species of fungi such as Fusarium and Aspergillus produces toxins on crop plants (Davide et al., 2016). Increasing pathogen resistance to key fungicides, lack of replacement of fungicides, consequent restrictions on fungicide use and high cost of chemicals requires alternative methods which are safer and eco-friendly. The activity of Hyptis spicigera as an antifungal agent on plant pathogenic fungi has not been scientifically proven as claimed by the locals and as such the need for the research.
1.4 Aim

This research is to evaluate the in vitro antifungal activities of Hyptis spicigera leaf extracts on Aspergillus and Fusarium species.

1.5 Objectives of the research

The objectives of the study are to:

1. Determine the comparative qualitative and quantitative phytochemical components of Hyptis spicigera leaf extracts (n-hexane, ethyle-acetate, methanol and aqueous)

2. Evaluate the antifungal activities of Hyptis spicigera leaf extracts (n-hexane, ethyl- acetate, methanol and aqueous) on some species of Aspergillus and Fusarium

3. Evaluate the antifungal activities of terpenoids and flavonoids fractions against the selected species of Aspergillus and Fusarium

1.6 Research Hypotheses

1. There are no significant phytochemicals in Hyptis spicigera leaf extracts (n-hexane, ethyle-acetate, methanol and aqueous) with antifungal activities

2. Hyptis spicigera leaf extracts (n-hexane, ethyle-acetate, methanol and aqueous) have no antifungal activity on Aspergillus and Fusarium species

3. Terpenoids and flavonoids fractions from Hyptis spicigera leaf extracts have no antifungal activity on Aspergillus and Fusarium species

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