Volatile Oil Composition, Phytochemical Screening and Insecticidal Activities of Indigofera tinctoria Against African Pink Worm Borer

doi:10.21203/rs.3.rs-3443596/v1

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Volatile Oil Composition, Phytochemical Screening and Insecticidal Activities of Indigofera tinctoria Against African Pink Worm Borer

https://doi.org/10.21203/rs.3.rs-3443596/v1

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The following study presents the constituents of the volatile oils found in the leaves and seeds of Indigofera tinctoria, as well as the secondary metabolites of its leaves crude fractions. The insecticidal activities of these fractions against Sesamia calamistis were also evaluated. The volatile oils from the leaf and seed of Indigofera tinctoria were extracted using the hydro-distillation method, utilizing an all-glass Clevenger apparatus. The extracted oils were then characterized using the Gas Chromatography-Mass Spectrometry (GC-MS) technique. The n-hexane, ethyl acetate and ethanol fractions of I. tinctoria were also investigated for secondary metabolites using standard qualitative phytochemical analysis methods. The insecticidal activities of these fractions against Sesamia calamistis were evaluated using a contact toxicity test procedure. The results of the study showed that Phytol (34.1%) and 1-octen-3-ol (63.8%) were the major constituents of the leaf and seed oil of I. tinctoria, respectively. Saponins were present in the hexane and ethanol fractions, while terpenoids and tannins were present in the ethyl acetate and ethanol fractions. The ethyl acetate fraction showed the highest percentage of S. calamistis larvae mortality of 70.0% at a 0.4% concentration compared to the n-hexane and ethanol fractions. Therefore, the ethyl acetate fraction of I. tinctoria leaf at a 0.4% concentration with the highest percentage of larvae mortality could be used to manage Sesamia calamistis on maize.

Indigofera tinctoria

GC-MS

Phytol

1-octen-3-ol

phytochemical

Sesamia calamistis

Plants have an essential role in sustaining life on earth. They convert simple substances into complex ones, resulting in vital chemicals that benefit human health. Because of this, plants are used worldwide as medicine [1]. Additionally, plants produce secondary metabolites that are responsible for antimicrobial and plant-insect interactions. These metabolites act as antifeedants, repellents, and contact poisons against insects on plants [2], making them useful as insecticides. Plant-derived insecticides are considered prototypes for industries that manufacture pest management products because they have advantages such as low persistence in the environment and little or no mammalian toxicity. They are widely accepted when used in food, cosmetics, pharmaceutical, and perfume industries [3].

Essential oils are highly concentrated substances extracted from various parts of plants such as flowers, leaves, seeds, roots, resins, barks, or fruit rinds [4]. They contain terpenes, terpenoids, and derivatives and are often used as flavors or for therapeutic purposes [5, 6]. The US Environmental Protection Agency has stated that these substances cause no adverse effects on human beings or the environment when used as food additives, insecticides, or repellents [7, 8]. The essential oil from Wrightia tinctoria (indigo plant) compose of hydroquinone, 2-cyclopentene-1-one, caryophyllene, caryophyllene oxide, indole just to mention a few [9]. Likewise, research conducted Indigofera microcapa belonging to the same family with Indigofera tinctoria shows the presence of β-caryophyllene, α-humilene as the major components [10].

Gas Chromatography Mass Spectroscopy (GC-MS) is one of the key techniques used for screening, identification and quantification of many groups of non-polar and semi-polar volatile components present in plant extracts. This is attainable using a wide variety of detectors [11].

Some bioactivities such as anti-hyperglycemic, anti-bacteria, anti-inflammatory, cytotoxic effect, anti-hepatoprotective, anti-diabetes, and anti-convulsive have been reported in the plant [12]. I. tinctoria has been reported to have insecticidal activities in a study conducted on the effect of retinoids extracted from I. tinctoria on Callosobruchus chinensis and Trogoderma granarium. It was observed that the methanol extract demonstrated maximum mortality at 1.0% and 2.0% concentrations [13]. Several reports exist in the literature on the efficacy of some plant essential oils and extracts against pestiferous organisms [14, 15, 16, 17, 18]. In a separate study on the efficacy of I tinctoria extract on Callosobruchus chinensis and Anopheles stephensi, the result shows that the extract has effect on the both insect but more on Anopheles stephensi [19]

Sesamia calamistis Hampson, commonly known as the African pink worm borer, is a stem borer that damages maize (Zea mays) in Africa. Larval infestation of the stem borer on maize causes deadhearts, which is the destruction of growing points. This interference with translocation of metabolites and nutrients results in malformation of the grain, impairing maize production. Yield loss ranges from 10–100% (total yield loss) in several parts of Africa [20, 21, 22]. Farmers rely on synthetic chemicals to control S. calamistis, but they leave undesirable effects on the environment. However, only a few plant species are effective against S. calamistis. Therefore, this study reports the secondary metabolites from the crude extract, the larvicidal activities of the crude fractions against S. calamistis, and the volatile oil constituents of I. tinctoria.

2.1 Plant Collection and Identification

Indigofera tinctora was collected from Ibadan southwest Nigeria in November 2018, identified and authenticated at the herbarium unit of Forestry Research Institute of Nigeria (FRIN), Ibadan, with the herbarium number FHI 111235.

2.2 Extraction of Essential Oils from Indigofera tinctoria Leaf and Seed

Hydro-distillation method was used to distill essential oils from fresh leaf (1,200 g) and seed (400 g) of I. tinctoria. The plant was collected and separated into leaves and seeds hence the difference in the mass of the plant parts hydro-distilled. An all-glass Clevenger apparatus was used to perform the hydro-distillation process. Essential oils were trapped in n-hexane for about 2–3 h. Chemical components of oils were analyzed by Gas Chromatography-Mass Spectrometry (GC-MS) analysis at Dipartmento di Farmacia, Università di Pisa, Italy.

2.3 GC-MS Analysis of Volatile Oils

Due to volatile nature of the essential oil, GC-MS analysis was proposed for the analysis of the essential oil composition. The hydro-distilled essential oils were diluted to 0.5% in HPLC-grade n-hexane and injected into GC-MS equipment. Gas chromatography-electron impact mass spectrometry (GC–EIMS) analyses were performed with an Agilent 7890B gas chromatograph (Agilent Technologies Inc., Santa Clara, CA, USA). The GC had an Agilent HP-5MS (Agilent Technologies Inc., Santa Clara, CA, USA) capillary column (30 m×0.25 mm; coating thickness 0.25 µm) and an Agilent 5977B single quadrupole mass detector (Agilent Technologies Inc., Santa Clara, CA, USA). Analytical conditions were as follows: injector and transfer line temperatures 220 and 240°C, respectively; oven temperature programmed from 60 to 240°C at three °C/min; carrier gas helium at 1 mL/min; injection of 1 µL (0.5% HPLC grade n-hexane solution); split ratio 1:25. The acquisition parameters were as follows: full scan; scan range: 30–300 m/z; scan time: 1.0 sec. The constituents' identification was based on comparing the retention times with the authentic samples' linear retention indices relative to the series of n-hydrocarbons. Computer matching was also used against commercial and laboratory-developed mass spectra library built from pure substances and components of known oils and MS literature data [23, 24, 25, 26, 27, 28]

2.4 Extraction and Phytochemical Analysis of Indigofera tinctoria Leaf

The dried leaf of I. tinctoria of about 1,500 g was extracted through a process of cold maceration using a mixture of 95% ethanol for 72 hours, as described by Dettweiler et al. (2020) [29]. The resulting extract was then partitioned using both n-hexane and ethyl acetate, which yielded their respective fractions. These fractions were then screened for the presence of various secondary metabolites, such as terpenoids, flavonoids, alkaloids, steroids, cardiac glycosides, tannins, saponins, and reducing sugars, using standard methods as outlined by Geetha (2016) [30].

2.5 Evaluation of I. tinctoria Leaf Fractions against Sesamia calamistis Larvae

2.5.1 Preparation of Treatments

To prepare different concentrations of leaf fractions of I. tinctoria, 0.12 g of the fractions were dissolved in 120 mL of appropriate solvents such as n-hexane, ethyl acetate, and ethanol, to obtain a 0.1% concentration. The second concentration of 0.2% was achieved by dissolving 0.24 g of each fraction in 120 mL of the solvent, while the third concentration of 0.4% was obtained by dissolving 0.48 g of each fraction in 120 mL of the solvent. The synthetic insecticide, Dichlorvos, was tested at 1000 g/L as per the manufacturer's recommendation.

2.5.2 Larvae Collection

The third instar larvae of Sesamia calamistis was collected at the Insect Rearing Unit of the International Institute of Tropical Agriculture (IITA) Ibadan, Nigeria

2.5.3 Contact Toxicity of Leaf Fractions against Sesamia calamistis Larvae

The effectiveness of I. tinctoria leaf extract fractions - n-hexane, ethyl acetate and ethanol - as insecticides against third instar larvae of Sesamia calamistis was tested. The larvae were fed a standard artificial diet containing varying concentrations 0.12 g/120cm³, 0.24 g/120cm³ and 0.48 g/120cm³ of the leaf fractions (0.1%, 0.2% and 0.4% respectively). A control treatment was also set up, which included an untreated diet, a diet with solvent, and a synthetic insecticide (Dichlorvos). The larvae were kept in plastic containers covered with fine mesh to allow aeration and maintained at a constant temperature of 27 ± 2 ^oC and RH of 75 ± 5% in the Insect Chemical Ecology Laboratory at the Department of Crop Protection and Environmental Biology, the University of Ibadan. The bioassay was carried out ten times in a completely randomized design. Larval mortality was assessed 48 hours after feeding, and the percentage larval mortality was calculated using Abbott's formula. The data was analyzed using ANOVA, and significant means were separated using the Least Significant Difference (LSD) at a 0.05 level of significance.

3.1 GC-MS analysis

During hydro-distillation of the leaf and seed of I. tinctoria, a colorless oil was obtained with a yield of 0.88 and 0.33 percent, respectively. The leaf and seed oils contained twenty and eleven compounds, respectively, accounting for 95.7% and 99.5% of the volatile oil. The chromatograms of the compounds eluted by the GC-MS from the volatile oils of the leaf and stem are shown in Fig. 1., Fig. 2, respectively Table 1.

The major constituent in the leaf volatile oil was phytol (34.1%), followed by 1-octen-3-ol (31.6%), 1-acetyl adamantane (4.71%), eugenol (4.1%), and 2-methyl-2-nonen-4-one. The dominant compound in the seed volatile oil was 1-octen-3-ol (63.8%), followed by linalool (17.9%), α-terpineol (4.2%), 2-octen-1-ol (4.2%), and geraniol (1.3%). Both leaf and seed volatile oils yielded 1-octen-3-ol (31.6%, 63.8%), linalool (2.3%, 17.9%), and 2-octen-1-ol (4.7%, 4.2%). This coincides with the report from Beena and Joji 2014, and Arriaga et al 2008 that shows the presence of compounds similar to the compounds reported above. The leaf and seed volatile oils were rich in oxygenated diterpenes and oxygenated monoterpenes, respectively. These two classes of terpenoids have been found to possess insecticidal activities against stored product insect pests [31, 32].

Phytol and 1-octen-3-ol have also been reported to possess antimicrobial activity. The mechanism of the antimicrobial action could be related to the ability of lipophilic molecules to cross cell membranes and exert their inhibitory activity on multiple targets simultaneously. Phytol and 1-octen-3-ol can scavenge reactive oxygen or nitrogen species (ROS/RNS) produced by cellular stress and metabolism, hence possessing antioxidant activity [33, 34, 35, 36].

Therefore, the volatile oil from the leaf and seed of I. tinctoria could be useful as an antimicrobial and antioxidant agent.

Table 1

Volatile Oil Composition of *Indigofera tinctoria* Leaf and Seed.
S/N	Compound	Retention Index	Leaf Relative Aboundance %	Seed Relative Aboundance %
1	1-Hexanol	871	0.9
2	1-Octen-3-ol	980	31.6	63.8
3	1,8-Cineole	1034	0.6
4	2-Octen-1-ol	1067	3.7	4.2
5	Linalool	1101	2.3	17.9
6	Tetrahydro-geraniol	1182	0.4
7	2-Methyl-2-nonen-4-one	1215	4.0
8	Nerol	1230	0.1	0.9
9	2-Phenyl-2-butenal	1279	2.2
10	Eugenol	1358	4.1	0.2
11	α-Copaene	1376	0.4	0.4
12	β-Caryophyllene	1420	0.3	0.7
13	1-Acetyladamantane	1462	4.7
14	Precocene1	1477	2.2
15	Dihydroactinidiolide	1531	0.7
16	Caryophyllene oxide	1581	1.2
17	Humulene epoxide	1607	0.3
18	Pentadecanal	1712	1.0
19	Hexahydrofarnesyl acetone	1845	0.8
20	Phytol	2114	34.1
21	(E)-3-Hexen-1-ol			1.6
22	α-Terpineol	1189		4.2
23	Geraniol	1257		1.3
24	Indole	1309		0.3
	Class of Compounds
1	Oxygenated monoterpenes		3.5	24.3
2	Sesquiterpene hydrocarbons		0.7	1.0
3	Oxygenated sesquiterpenes		1.5
4	Oxygenated diterpenes		34.1
5	Apocarotenoids		1.6
6	Phenylpropanoids		4.1	0.2
7	Non-terpene derivatives		50.3	73.7
8	Nitrogen compounds			0.3
	Total		95.7	99.5

3.2 Extraction and Qualitative Phytochemical Analysis

The crude extract about 33.46 g with percentage yield 2.23% was fractionated using n-hexane and ethyl acetate. Table 2 presents the results of a qualitative phytochemical analysis of secondary metabolites found in different fractions of I. tinctoria leaf. All fractions of the leaf contain varying amounts of flavonoids, alkaloids, steroids, and cardiac glycosides. Hexane and ethyl acetate fractions have steroids, while n-hexane and ethanol fractions have saponins. Terpenoids and reducing sugars are only present in the ethyl acetate fraction, while tannins are only present in the ethanol fraction. This list of phytochemicals is consistent with previous research by Saraswathi et al. (2012) [37], Motamarri et al. (2012) [38], Balamurugan and Selvarajan (2009) [12], and Pejin et al. (2014) [34]. Overall, the crude extract from TI contains Terpenoids, Flavonoids, Alkaloids, Steroids, Cardiac Glycosides, Tannins, Saponins, and Reducing Sugar, as shown in Table 3. This report is in line with the research conducted on plants of the same family by Rajaperuma et al. (2013) [39], Geetha et al. (2016) [30], and Aziz-Ur-Rehman et al. (2004) [40].

Table 2

Qualitative phytochemical analysis of *I. tinctoria* leaf fractions
Phytochemicals	Fractions
	n-Hexane	Ethyl Acetate	Ethanol
Terpenoids	-	+	-
Flavonoids	+	+	+
Alkaloids	+	+	+
Steriods	+	+	-
Cardiac glycosides	+	+	+
Tannins	-	-	+
Saponins	+	-	+
Carbohydrates (Reducing sugar)	-	+	-
+=Present; - = Absent

3.3 Larvicidal activities of different fractions from I. tinctoria leaf

Table 3 displays the mortality of Sesamia calamistis larvae when fed diets containing different concentrations of Indigofera tinctoria leaf fractions. The ethanol fraction showed little effect on the larvae, with mortalities ranging from 3.13% (0.2%) to 7.19% (0.1%), which were not significantly different from the control (0.0%). The n-hexane fractions, on the other hand, had a higher impact, with mortality rates increasing with concentration: 5.0% (0.2%) < 10.0% (0.1%) < 30.0% (0.4%).

The ethyl acetate fractions caused significant mortalities (p < 0.05) comparable to Dichlorvos. Mortality rates were dose-dependent, increasing as the fraction concentrations increased. Diets incorporating 0.2% and 0.4% ethyl acetate fractions caused 60.0% and 70.0% larval mortalities, respectively. These values were not significantly different from Dichlorvos-treated diet (100.0%). The Dichlorvos-treated diet had a total mortality rate of 100.0%.

Parvin et al. (2013) [41] reported different mortality rates for n-hexane and ethyl acetate of Calotropis gigantea methanol extract on Tribolium cataneum (red flour beetle). The ethyl acetate fraction of crude at 50 mg/mL in 48 hours caused an 80% mortality rate of T. castaneum, while the counterpart n-hexane fraction of the same concentration had only 40% mortality. This report coincides with the findings of this research that the ethyl acetate fraction had the highest mortality rate (70.0%).

It is worth noting that the insecticidal activity of the ethyl acetate fraction (0.2%) was comparable to the synthetic insecticide Dichlorvos (1000 g/L). The highest larval mortality of 70.0% was recorded from the 0.2% concentration ethyl acetate fraction, which was not significantly different from Dichlorvos (100.0%). Substantial mortalities of 57.5% were observed in larvae fed a 0.4% ethyl acetate-treated diet, which was not significantly different from mortality observed in 0.2% (70.0%). Lower mortality of 37.5% was recorded on larvae fed a 0.4% hexane-treated diet. Mortality rates of S. calamistis larvae in diets with ethanol fraction at 0.1% (37.8%) and 0.2% (37.5%) were similar, and it increased to 50.3% in diets with 0.4% ethanol fraction (p < 0.05). Diet coated with ethanol (solvent) caused the death of 12.5% of S. calamistis larvae.

As the fractions were incorporated into the diets of S. calamistis larvae, leading to their death after feeding, the mode of action of the fractions was stomach poison. Further studies are required to determine if the fractions have contact toxicity against the larvae. It is essential to determine the chemical constituents of the three fractions since mortality rates differ significantly among them. The varied mortality rates suggest that Indigofera tinctoria leaf has active principles with potent larvicidal properties similar to the synthetic insecticide Dichlorvos. Further investigations into the active compounds responsible for mortality would add to the pool of chemical compounds that are environmentally safe and friendly.

Table 3

Mean Percentage Mortality of Sesamia *calamistis* Larvae Fed with Artificial Diet **Mixed with Different Concentrations of Indigofera** **tinctoria** **Leaf Extract and Dichlorvos.**
Treatments	Larval Mortality (%)
	Hexane fraction	Ethanol fraction	Ethyl Acetate fraction
0.1%	10.0 ± 8.2 b	7.19 ± 6.7 a	32.5 ± 34.0 ab
0.2%	5.0 ± 5.8 ab	3.13 ± 3.6 a	60.0 ± 25.8 bc
0.4%	30.0 ± 5.0 c	4.69 ± 3.1 a	70.0 ± 18.3 c
Solvent	4.69 ± 6.0 ab	3.75 ± 4.3 a	10.0 ± 8.2 a
Dichlorvos (1000 g/L)	100.0 ± 0.0 d	100.0 ± 0.0 b	100.0 ± 0.0 c
Control (untreated)	0.0 ± 0.0 a	0.0 ± 0.0 a	0.0 ± 0.0 a

Means in the same column having the same letters are not significantly different at p < 0.05. Means were separated with Students Newman Kuels Test.

The report of essential oil from Indigofera tinctoria has not been reported before. The qualitative phytochemical analysis of the leaf fractions shows the presence of some secondary metabolites which determines the bioactivities.

The 0.4% ethyl acetate fraction can be used as larvicide against the pink worm. This coincides with the SDG on food security.

The next line of action in the research is the bioassay guided isolation of the active components of the ethyl acetate fraction responsible for the larvicidal activities of the plant and finally synthesis of the most active compound.

Author Contribution

O.O.O. supervised the research in the Department of Chemistry, University of IbadanA.O.Y. supervised the research in the Department of Crop Protection and Environmental Biology, University of Ibadan, and also did the statistical calculations M.I. conducted extraction of the plant in the Department of Chemistry and tested the extract on the insect in the Department of Crop Protection and Environmental BiologyM.A.A. breeds the insect used in insecticidal activities at the International Institute of Tropical AgricultureF.G. Conducted the GC-MS at the Dipartmento di Farmacia, Universita di Pisa.T.O.M. conducted the extraction of the plant in the Department of Chemistry University of Ibadan.

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Volatile Oil Composition, Phytochemical Screening and Insecticidal Activities of Indigofera tinctoria Against African Pink Worm Borer

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Abstract

Figures

1.0 INTRODUCTION

2.0 MATERIALS AND METHODS

2.1 Plant Collection and Identification

2.2 Extraction of Essential Oils from Indigofera tinctoria Leaf and Seed

2.3 GC-MS Analysis of Volatile Oils

2.4 Extraction and Phytochemical Analysis of Indigofera tinctoria Leaf

2.5 Evaluation of I. tinctoria Leaf Fractions against Sesamia calamistis Larvae

2.5.1 Preparation of Treatments

2.5.2 Larvae Collection

2.5.3 Contact Toxicity of Leaf Fractions against Sesamia calamistis Larvae

3.0 RESULTS AND DISCUSSION

3.1 GC-MS analysis

3.2 Extraction and Qualitative Phytochemical Analysis

3.3 Larvicidal activities of different fractions from I. tinctoria leaf

4.0 Conclusion

Declarations

Author Contribution

References

Status:

Version 1