Phytochemical Constituents and Antimicrobial Activity of Crude Extract of Scent Leaf (Ocimum Gratissimum) on Fungal Phytopathogens

Emmanuel Sunday Olorunfemi, Kehinde Abraham Odelade, Rebecca Funke Olayiwola, Mansurat Omotayo Adesokan, Bassey Jecintha, Boluwatife Comfort Olawuyi,

doi:10.5281/zenodo.17732418

Research Paper | Open Access
Volume 02 | Issue 11 | Article Id IJSRT/250311131

Phytochemical Constituents and Antimicrobial Activity of Crude Extract of Scent Leaf (Ocimum Gratissimum) on Fungal Phytopathogens
Emmanuel Sunday Olorunfemi* ¹ Kehinde Abraham Odelade ¹ Rebecca Funke Olayiwola ¹ Mansurat Omotayo Adesokan ¹ Bassey Jecintha ² Boluwatife Comfort Olawuyi ¹
¹Federal Polytechnic Ayede, Ayede, Oyo State, Nigeria.
²Prescious Cornerstone University, Ibadan, Oyo state, Nigeria

Abstract

Chemical fertilizers and pesticides have long been used to enhance crop and fruit production. However, their excessive use has led to degradation of soil quality, promoting the rapid growth of pathogenic microorganisms which significantly contribute to fruit spoilage. Studies have shown that Ocimum gratissimum contains bioactive compounds with antifungal activity against plant pathogens. This study investigated the phytochemical composition and antimicrobial activity of the methanol extracts obtained from Ocimum gratissimum leaves. The antimicrobial activity of this medicinal plant extract was evaluated using the agar well diffusion method. Using this method, the extract was tested against a range of fungal species including Aspergillus flavus, Rhizopus oryzae, Trichoderma Harzianum, Rhizopus stolonifer, Aspergillus carbonarius, and Aspergillus niger. The outcome of the phytochemical analysis of the O. gratissimum leaves extract indicate that secondary metabolites such as alkaloids, anthraquinones, tannins, reducing sugars, and phenolic compounds were present. The extract showed varying levels of inhibitory zones against the tested fungi, with statistically significant differences at p values of <0.001, <0.01, and <0.05. The maximum zone of inhibition (27mm) was observed against Aspergillus niger at a concentration of 500mg/ml. R oryzae showed a slightly lesser maximum inhibition zone (26.75mm) at 250mg/ml. At a significance level of p < 0.001, Rhizopus oryzae (26.75mm), Aspergillus flavus (26mm), and Aspergillus niger (27mm) displayed the highest zones of inhibition for the extract at concentrations of 250mg/ml, 250mg/ml, and 500mg/ml, respectively. The methanolic extract performed favorably when compared to the standard antifungal Nystatin. This result supports the use of this O. gratissimum for disease management and prevention in plants.

Keywords

Ocimum gratissimum, biological activities, phytochemical constituents, methanolic extract, fungal phytopathogens

Introduction

Plant diseases are estimated to cause 5-10% loss of the world’s major crops annualy, resulting in economic losses up to $100billion. The fungal pathogens account for 70-80%and significantly affect crop growth and yield (Li et al 2017). In recent years, the prevalence of fungal plant diseases has increased due to large scale agricultural practices that reduce crop quality and increase plant susceptibility to infection (Marin-Menguiano et al. 2019), posing a major challenge for sustainable agriculture. These diseases are often caused by the interaction of multiple pathogens rather than one single organism (Chatterjee et al., 2016). Since ancient times, humans have relied on medicinal. Plants to prevent, manage or treat many diseases (Chaachouay et al., 2022). Traditional medicines have witnessed a rising popularity over conventional drugs due to the effectiveness of the bioactive compounds present in the them, as well as the ease of access and affordability (Ijioma et al., 2021). As stated by WHO (2019), in recent years, there has been a significant increase in research on medicinal plants and their bioactive compounds, highlighting their potential for combating and managing plant pathogenic diseases. Ocimum gratissimum L. (Lamiaceae), referred to as ‘scent leaf ‘, ‘tea bush” or ‘fever plant ‘is a medicinal plant with antimicrobial properties and potential use as a natural fungicide. In Nigeria, it is known by local names such as Nchuanwu,” “Shinji,” “Achigbu” (Igbo), “Efirin” (Yoruba), “Ihiri eziza” (Bini), “Dai doya tagida” (Hausa), or “Ntion” (Efik). This medicinal plant belongs to the Lamiaceae family and can be found in South America, Asia and Africa (Ugbogu et al., 2021). It used as a vegetable, spice or seasoning in cooking. Studies have shown that O. Gratissimum contains bioactive compounds including flavonoids, polyphenols, and essential oils which exhibit a range of beneficial properties (Irondi et al., 2016). The phytochemicals present in this plant are associated with the ability to inhibit the growth of fungal pathogens. Therefore, this study aimed to evaluate the effectiveness of O. Gratissimum leaf extract against fungal phytopathogens

MATERIALS AND METHOD

Collection and Preparation

Leaves of Ocimum gratissimum were collected from Irewolede Area, Ogbomosho, Ogbomosho south local government area of Oyo state, Nigeria. The leaves were thoroughly washed under running water, air dried for weeks, ground into a fine powder, and sieved. The powdered material was stored in a dry, cool place until further use.,

Plant Extraction Procedure

A total of 150 g of O. Gratissimum leaf powder was macerated in 450 mL of methanol for 48 hours. The solution was filtered through sterile muslin cloth, and the filtrate was allowed to settle in a dry, cool place to concentrate by evaporation. The resulting crude extract, which formed a colloidal substance, was stored at –4°C for subsequent phytochemical and antimicrobial analyses.

Phytochemical Screening of Plant Extract

Qualitative phytochemical analysis of the methanolic crude extract was performed using the methodology reported by Oladeji et al. (2020).

Preparation of Different Concentrations of the Crude Extracts

Solutions of crude extract were prepared at varying concentrations (25-500mg/ml)) following the procedure described by Oladeji et al. (2019).

Antifungal Susceptibility Analysis of the Methanolic Extract of O. Gratissimum

Potato Dextrose Agar (PDA) was prepared according to manufacturer instructions and aseptically inoculated with fungal suspensions. After inoculation, excess inoculum was removed. Sterile Whatman No. 1 filter paper discs (6 mm diameter) were oven-sterilized at 120°C for 2 hours. The discs were then impregnated with the plant extract solutions of different concentrations, while separate discs impregnated with methanol served as controls. The discs were placed on the surface of the inoculated PDA plates. Plates were incubated at 25°C for 2–3 days. Antifungal activity was assessed by measuring the zones of inhibition (mm) around each disc (Oladeji et al., 2019).

Pathogenicity Test

The fresh healthy oranges were double sterilized in sterile distilled water. The fruits were sterilized using 70% ethanol and washed with sterile water. Fruits were placed on sterile paper towels and dried under laminar airflow hood for 12 mins. Holes were created in the healthy oranges with the help of a sterile 10 mm cork borer. Test tubes containing the fungal isolates (pure culture) were inoculated in the cork borer hole. Finally, after inoculation, the inoculated oranges were sealed with sterile blue seal Vaseline to prevent contamination and the oranges were labeled accordingly. A control test without any isolate was conducted and sealed using the sterile blue seal Vaseline. After inoculation of each of the test isolates into their respective healthy oranges, the oranges were incubated at 25 °C in the humid chamber for 5 days. The fruits were observed daily for rotting characteristics like softening, color, and odor. The fruits were opened on the fifth day to expose internal portions of the fruits which were examined for rot. Circumference and diameter of the rotten and intact fruit of the rotten area and intact fruit were taken using transparent scale and recorded.

Statistical Analysis

The data obtained from the methanolic crude extract at different concentrations were analysed using Graph pad Prism 5. A two-way ANOVA test was carried out to check for the significance of the extract at different concentrations. Comparisons were made between the crude extract and the control as well as the extracts overall antimicrobial activity and that of the standard Nystatin. Values of P < 0.05; P < 0.01 and P < 0.001 were considered statistically significant and used to reject the null hypothesis. Graph pad Prism 5 was used in the analysis of methanolic crude extract at different concentrations. Two-way ANOVA test was used in the determination of the significance level of the crude extract at different concentrations. Statistical comparison was made between the methanolic crude extract and the control, and also the overall antimicrobial activities of the extracts with nystatin. P < 0.05; P < 0.01 and P < 0.001 was used to reject the null hypothesis

RESULTS AND DISCUSSION

The qualitative phytochemical screening of Ocimum gratissimum showed the presence and absence of various bioactive compounds as presented in Table 1 below.

Table 1: Phytochemical analysis of Ocimum gratissimum crude extract

S/N	Phytochemical compound	Methanolic extract
1	Saponins	-
2	Terpenoids	-
3	Reducing sugars	-
4	Quinones	-
5	Anthraquinones	++
6	Flavonoids	++
7	Phlobatannins	-
8	Steroids	-
9	Tannins	+++
10	Phenolic compounds	+++
11	Alkaloids	+++

KEYS: ‘-, ++, and +++’ indicates absent, present, very present.

As shown in the table above, the methanolic crude extract contained anthraquinones, tannins, phenolic content and alkaloids and flavonoid while saponins, phlobatannins, quinones, reducing sugars, terpenoids and steroids were not detected. The graphs (Figure 1-6) below show the antifungal susceptibility test (AFST) results showing the level of significance of Ocimum gratissimum crude extracts compared with the control across different phytofungal isolates. Values marked with (*, **, and ***) indicate results significantly higher than the control at P <0.05; P < 0.01 and P < 0.001. Respectively;(ns) indicate no significant difference.

Figure 1: AFST of Ocimum gratissimum extract against Aspergillus flavus

Figure 2: AFST of Ocimum gratissimum against Rhizopus oryzae

Figure 3: AFST of Ocimum gratissimum against Trichoderma harzianum

Figure 4: AFST of Ocimum gratissimum against Rhizopus stolonifer

Figure 5: AFST of Ocimum gratissimum against Aspergillus carbonarius

Figure 6: AFST of Ocimum gratissimum against Aspergillus niger

Plate 4.5 Zone if inhibition (in mm) of Ocimum gratissimum extract against Aspergillus Niger

Plate 4.7 Zone if inhibition (in mm) of Ocimum gratissimum extract against Trichoderma harzianum

The statistical analysis of antimicrobial activities of methanolic crude extract, of Ocimum gratissimum with respect to their zone of inhibition (mm) on the fungal isolates is presented in Figures 1-6. All concentrations of the extract showed significant antimicrobial activities against the fungal isolates; Aspergillus flavus, Rhizopus oryzae, Trichoderma harzianum, Rhizopus stolonifer, Aspergillus carbonarius and Aspergillus niger at P<0.05, P<0.01, P<0.001. All methanolic crude extract concentrations (25-500mg/ml) significantly inhibited majority of the tested fungal isolates. The statistical analysis showed that the methanolic crude extract of Ocimum exhibited a significant performance than the control at P < 0.001. Overall, the extract demonstrated inhibition zones of 13mm to 27mm across concentrations C1 to C5, whereas the control recorded only 8mm to 10mm. The Methanolic extract at concentration C1 (25 mg/ml) showed a high to average zone of inhibition against A. flavus (17.5mm) (Figure 1), R. Oryzae (18.5mm) (Figure 2), T. harzianum (15.5mm) (Figure 3), A. carbonarius(14.5mm) (Figure 5), and A.niger(14mm) (Figure 6) respectively, while the lowest zone of inhibition at concentration 25 mg/ml was recorded against R. stolonifer with a diameter of 13mm (Figure 4). Therefore, the statistical analysis of methanolic crude extract and control at C1 shows that methanolic crude extract is significantly higher than the control at P<0.001 as indicated in Figures 1-6. Methanolic crude extract at concentration C2 (50 mg/ml) exhibited consistently high zones of inhibition against A. flavus (21mm), R.oryzae (20.75mm), T. harzaianum (20mm), R.stolonifer (15.7mm), A.carbonarius(17.5mm), and A. niger (16mm). These values were also higher than the control which recorded inhibition zones between 8mm and 10mm. Therefore, the statistical comparison of methanolic extract and control at C2 indicates that the extract produced significantly greater antimicrobial activity than the control at P < 0.001 as shown in Figures 1-6. At concentration C3 (100 mg/ml), the extract also produced high inhibition zones across all fungal isolates: A. flavus (23.85 mm), R. oryzae (24.25 mm), T. harzianum (20.75 mm), R. stolonifer (18.60 mm), A. carbonarius (20.50 mm) and A. niger (20 mm). Although the lowest zone of inhibition at concentration 100mg/ml was recorded against R. stolonifer with the diameter of (18.60 mm), it was still substantially higher than the performance of the control with a record range of 8 mm to 10 mm. Therefore, the statistical analysis comparing the methanolic extract and the control at C3 shows that methanolic extract produced significantly higher antimicrobial activity than the control (P < 0.001) as indicated in Figures 1-3 and Figures 4-6. Methanolic crude extract at C4 (250 mg/ml) and C5 (500 mg/ml) also show a consistent high zone of inhibition against A. flavus (25.50 mm and 25.40 mm), R.oryzae (26.75 mm and 26 mm), T. harzianum (24 mm and 25.25 mm), R. stolonifer (22.75 mm and 25.15 mm), A. carbonarius (20.65 mm and 23.35 mm) and A. niger (23 mm and 27 mm), respectively. All of these values significantly higher than those of the control which consistently ranged between 8mm and 10mm. Consequently, statistical analysis at C4 and C5 confirms that the methanolic crude extract was more effective than the control at P < 0.001 as shown in figures 1-6. The highest zones of inhibition of methanolic crude extract were recorded against A. Niger (27mm) (Figure 6) at 500 mg/ml. R. Oryzae (26.75mm) (Figure 2) at 250mg/ml, A. flavus (25.50mm) (Figure 1) at 250 mg/ml, T. harzianum (25.25mm) (Figure 3) at 500 mg/ml, R. stolonifer (25.15) (Figure 4) at 500mg/ml, and A. carbonarius (23.35mm) (Figure 5) at 500mg/mg/ml which is statistically significant than the control at P<0.001. Furthermore, antimicrobial effect of crude extracts of Ocimum gratissimum leaves demonstrated notable antifungal activity against all tested fungal isolates. The methanolic crude extracts showed activity at all the concentrations evaluated (25 mg/ml, 50 mg/ml, 100 mg/ml, 250 mg/ml and 500 mg/ml). The performance of methanolic crude extract was statistically compared with antifungal agent, Nystatin as seen in table 2. The antifungal activity was analysed at signifance levels of P < 0. 05, P < 0.01 and P < 0.001. To minimize experimental errors, the mean ±SD for each concentration was calculated.

Table 2: Comparative activity of Ocimum gratissimum extract against Nystatin

			Methanolic crude extract (mm)
Aspergillus flavus	Nystatin	15.00	±0.000
	C1	17.75	±1.060 **
	C₂	21.00	±1.414 ***
	C₃	23.85	±0.212 ***
	C4	25.50	±0.707 ***
	C₅	25.40	±0.565 ***
Rhizopus oryzae	Nystatin	17.00	±0.000
	C1	18.50	±0.707 **
	C₂	20.75	±0.353 ***
	C₃	24.25	±1.060 ***
	C4	26.75	±1.767 ***
	C₅	26.75	±1.414 ***
Trichoderma harzianum	Nystatin	15.00	±0.000
	C1	15.50	±0.707 **
	C₂	18.50	±2.121 ***
	C₃	20.75	±1.060 ***
	C4	24.00	±1.414 ***
	C₅	25.25	±1.767 ***
Rhizopus stolonifer	Nystatin	12.00	±0.000
	C1	13.00	±1.414
	C₂	15.35	±0.494 ***
	C₃	18.60	±0.848 ***
	C4	22.75	±2.474 ***
	C₅	25.15	±0.212 ***
Aspergillus carbonarius	Nystatin	12.00	±0.000
	C1	14.50	±0.707 **
	C₂	16.75	±1.060 ***
	C₃	20.50	±2.121 ***
	C4	20.65	±0.919 ***
	C₅	23.35	±1.909 ***
Aspergillus niger	Nystatin	11.00	±0.000
	C1	14.00	±1.414**
	C₂	16.00	±0.707***
	C₃	20.00	±0.707***
	C4	23.00	±0.707***
	C₅	27.00	±1.414 ***

C1 – 25mg/ml, C2 -50 mg/ml, C3 – 100 mg/ml, C4 – 250 mg/ml, C5 – 500 mg/ml. Values are expressed as mean ± SD for n=2 for each concentration. Values with (*, **, ***) are significantly higher than the standard at P < 0.05, P < 0.01, P < 0.001 respectively.

At concentrations 25mg/ml, 50mg/ml, 100 mg/ml, 250 mg/ml, and 500 mg/ml, the zone of inhibition methanolic crude extract produced on A. flavus are 17.75mm, 21mm, 23.85mm, 25.50mm and 25.40mm. These values were all higher than those produced by the antifungal standard Nystatin which recorded a zone of inhibition of 15mm. The difference between methanolic extract and Nystatin at all concentrations is at P < 0.001 as indicated in Table 2. Similarly, at concentration 50mg/ml, 100 mg/ml, 250 mg/ml, and 500 mg/ml, the methanolic crude extract produced zones of inhibition of 20.75mm, 24.25mm, 26.75mm and 26mm against R. oryzae respectively. Nystatin produced a zone of inhibition of 17 mm against the organism. Statistical analysis shows that the extract exhibited significantly greater antifungal activity than Nystatin (P <0.001) as indicated in the Table 2. The methanolic crude extract produced zones of inhibition of 15.50mm, 18.50mm, 20.75mm, 24mm and 35.25mm against T. harzianum at concentrations 25 mg/ml, 50mg/ml, 100 mg/ml, 250 mg/ml, and 500 mg/ml respectively. In contrast, Nystatin produced a zone of inhibition of 15.0 mm. As shown in table 2, the methanolic crude extract exhibited significantly higher antifungal activity than Nystatin at all concentrations (P < 0.001). Against stolonifer, the methanolic crude extract produced inhibition zones of 13mm, 15.35mm, 18.60mm, 22.75mm and 25.15mm at concentration 25mg/ml, 50 mg/ml, 100 mg/ml, 250 mg/ml, and 500 mg/ml respectively, whereas the inhibition zone for Nystatin is 12mm. Statistical analysis reveals that the extract showed significant difference between methanolic crude extract and Nystatin on R. stolonifer. At concentration 25mg/ml, the methanolic crude extract was significantly higher than Nystatin at P<0.01, while at concentrations 50mg/ml, 100mg/ml, 250mg/ml and 500mg/ml, the crude extract is significantly higher at P<0.001 as seen in the table 2. The methanolic crude extract showed clear antifungal activity against A. carbonarius at all tested concentrations. The zones of inhibition were 15mm, 17.5mm 22 mm, 21.3mm, and 24.7 mm at 25mg/ml, 50mg/ml, 100 mg/ml, 250 mg/ml, and 500 mg/ml are 15mm, 17.5mm 22 mm, 21.3mm, and 24.7 mm respectively, while the zone of inhibition for Nystatin produced a zone of 15mm. Statistical analysis confirmed that the methanolic extract was significantly more effective than Nystatin at all concentrations (P < 0.001). The Zone of inhibition of methanolic crude extract produced on A. niger at concentrations 25 mg/ml, 50 mg/ml, 100 mg/ml, 250 mg/ml, and 500 mg/ml is 15mm, 17 mm, 20.5 mm, 24 mm and 27.5 mm respectively, while the zone of inhibition for Nystatin is 17 mm. Statistical analysis shows the significant difference between methanolic crude extract and Nystatin on A. niger. At concentration 25mg/ml, the methanolic crude extract is significantly higher at P<0.05, at concentration 50mg/ml, methanolic crude extract is significantly higher at P<0.01 while at concentrations,100mg/ml, 250mg/ml and 500mg/ml, the methanolic crude extract is significantly higher at P<0.001 as revealed in Table 2.

DISCUSSION

This study highlights the potential of plant extracts as an alternative approach for controlling plant diseases, particularly due to the presence of phytochemicals found in Ocimum gratissimum plant extracts, which exhibit high efficacy against fruit pathogens. Secondary metabolites such as phenolics, steroid and terpenoids have significant value as they are considered sustainable choices for controlling phytopathogens (Jiménez-Reyes et al. 2019). These compounds are produced by plants in response to environmental conditions and disease pressure serving as a potent innate defence against pathogens. Nevertheless, poor stability and low absorption when compared to synthetic chemicals pose challenges emphasizing the importance of developing strategies to improve their effectiveness. Tannins and alkaloids have also been found in phytochemical screening of Ocimum gratissimum leaf extract (Olumide et al. 2019). Tannins offer a characteristic taste and provide the plant with its antimicrobial properties and antioxidant properties as well. The antioxidant properties have potential to increase the shelf life of fruits by reduction of oxidative stress and delay the spoiling of fruits. Reports from Kpètèhoto et al. (2019) also indicated the presence of tannins, alkaloids as well as terpenoids and steroids, the latter of which were absent as seen in Table 1. This research confirmed the antimicrobial activity of the plant which is attributed to the presence of bioactive constituents. These compounds exhibit potent antibacterial, antifungal and antioxidant properties justifying the traditional medicinal use of Ocimum gratissimum. Moreover, the bioactive compounds in O. Gratissimum could be used as an alternative treatment for plant diseases or as building blocks for the development of synthetic antimicrobial agents. The crude extract displayed strong antifungal activity against Rhizopus oryzae (26.75mm) and Aspergillus niger (27mm) at concentrations of 250 mg/ml and 500mg/ml respectively. The results showed that the methanolic crude extract of Ocimum gratissimum inhibited the growth of all tested fungi; Aspergillus flavus, Rhizopus oryzae, Trichoderma harzianum, Rhizopus stolonifer, Aspergillus carbonarius and Aspergillus niger. Table 2 demonstrates the antifungal activity of the crude extract of O gratissimum compared with Nystatin. The methanolic crude extract consistently produced clear zones of inhibition demonstrating notable antifungal activity against all tested fungal isolates. Overall, the inhibition zones ranged from 13mm to 27mm. According to Adeeyo et al., (2018), zones between 12mm and 18mm indicate low activity, 19mm and 22mm indicate moderate activity and 23mm to 38mm represent strong activity. Therefore, the extract showing inhibitory zones between 18mm and 20mm against most fungi can be regarded as having meaningful antimicrobial strength. The antifungal activity maybe be attributed to several mechanisms including disruption or cell membranes (Eze et al., 2018) and alterations to the cell wall and membrane permeability (Jung et al., 2021). Such disruptions can destabilize microbial membranes, cause leakage of cell contents, alter membrane potential, redistribute lipids, enable peptide entry, block anionic cell components, activate autolytic enzymes, and ultimately lead to cell death (Hassan et al., 2022). Similar antifungal activity of plant extracts has been reported in earlier studies involving Ocimum gratissimum and Carica papaya L. (Onaebi et al., 2019), as well as Acorus calamus, Allium sativum, Mucuna pruriens, and Sesamum indicum L (Arasu et al., 2019).

CONCLUSION

This study demonstrates that Ocimum gratissimum lead extract possess string antifungal properties highlighting it’s potential as a natural fungicide. The extract showed notable inhibitory activity against all tested fungi. A result likely linked to the presence of alkaloids, steroids, phenolic and rates metabolites. This study emphasizes the effectiveness of crude extract it’s suitability as an eco-friendly and safe alternative to conventional drugs overall, the ability of the plant to suppress fungal growth highlights its value for sustainable plant management.

FUNDING:

This research was funded by Institution Based Research (IBR) intervention 2023 Tertiary Education

Trust fund (TETFUND).

ACKNOWLEDGMENTS:

The authors acknowledged the grant given Institution Based Research (IBR) intervention 2023 Tertiary Education Trust fund (TETFUND) and the management of Federal Polytechnic Ayede, Oyo State, Nigeria.

Conflicts of Interest: The authors declare no conflicts of interest.

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