Chemical composition and antifungal potential of Vinca rosea leaf essential oil and extracts from Northern India

Abstract

Advancing understanding of the diverse properties of essential oils has spurred a significant increase in both their production and application. The current research aimed to study the chemical, physical, and antifungal characteristics of Vinca rosea leaves’ essential oil compared to its extract. The leaf’s essential oil and extract composition were examined using GC-MS and further tested for antifungal activity against Fusarium graminearum and Bipolaris sorokiniana by poisoned food technique. 34 compounds were identified in leaves essential oil predominantly composed of butylated hydroxytoluene (32.74%). The methanolic extract showed the presence of alkaloids, mainly vindoline, and the non-polar extract showed the presence of cholesterol as the primary compound. The methanolic extract demonstrated stronger antifungal activity against F. graminearum, with ED₅₀ and ED₉₀ of 80 and 240 µg/mL, compared to the essential oil, which had higher ED₅₀ and ED₉₀ of 98 and 865 µg/mL, respectively. In the case of B. sorokiniana, ED₅₀ values of methanol extract, essential oil, and hexane extract were 76, 100, and >1000 µg/ml, respectively, and ED₉₀ were observed to be 290, 875, and >1000 µg/ml. Vinca rosea essential oil can potentially be used to develop natural antifungal agents. For the first time, the chemical composition of the essential oil of V. rosea cultivated in the Northern Indian region is being reported.

Graphical abstract

Keywords:

• Catharanthus roseus
• extraction
• GC–MS analysis
• phytochemical
• Fusarium graminearum
• Bipolaris sorokiniana

REVIEWING EDITOR:

• Manuel Tejada, Universidad de Sevilla, Spain

SUBJECTS:

• Agriculture & Environmental Sciences
• Chemistry
• Biochemistry
• Biology

1. Introduction

The use of synthetic fungicides is restricted in agriculture due to the increasing resistance of fungi against them and awareness about their harmful effects, which leads to the inclination to use organic fungicides. Various natural substances derived from plants, including extracts and essential oils, serve crucial roles in medicine and agriculture due to their versatility. Different plant parts offer a wealth of phytochemicals with a wide range of biological activities, which the agricultural and pharmaceutical sectors have utilized to develop numerous drugs and chemicals.

Vinca rosea (also known as Catharanthus roseus) belongs to the Apocynaceae family, is native to Madagascar, and is cultivated worldwide for its medicinal properties. In India, it is commonly known as Sadabahar and Nityakalyani, mainly in Punjab, Kerala, Gujarat, Uttar Pradesh, and Assam. Vinca rosea has been traditionally used to treat high blood pressure, diabetes, wasp stings, and various other treatments (Das et al., 2020). This plant’s terpenoid indole alkaloid pathway synthesizes over one hundred thirty alkaloids (Almagro et al., 2015). Various alkaloids found in this plant are of commercial importance. Vincristine and vinblastine are used as anticancer drugs, and ajmalicine is used as an antihypertensive drug approved by the World Health Organization for their clinical usage (Almagro et al., 2015). In previous studies, dry and fresh leaves were used to extract the essential oil of V. rosea. Leaves showed the presence of esters, fatty acids, alkanes, terpenoids, aldehydes, alcohols, ketones, and various unidentified volatile components in essential oil, some of which were isolated for commercial purposes (Pandey et al., 2006; Cavaleiro et al., 2006). Various categories of compounds, like phenols, flavonoids, anthocyanins, volatile compounds, alkaloids, etc., were also reported in the extracts of V. rosea plants. It has also been gaining acceptance for its diverse application in various agricultural and pharmacological properties such as anthelmintic (Agarwal et al., 2011), antioxidant (Barkat & Mujeeb, 2013), antimicrobial (Prajakta & Ghosh, 2010), antibacterial (Ibrahim et al., 2011), antifungal (Balaabirami & Patharajan, 2012), antidiarrheal (Rajput et al., 2011), to name a few. Vindoline, vincristine, vinblastine, catharanthine, leurosine, syringic acid, petunidin, quercetin, para-coumaric acid, heneicosane, tricosane, and palmitic acid are some of the known crucial constituents of this plant responsible for its applications in agronomic and therapeutic practices (Pandey et al., 2006; Renault et al., 1999; Hisiger & Jolicoeur, 2007).

Vinca rosea has been established as an essential plant for pharmaceutical purposes, but its usage has yet to be explored much when it is categorized in agriculture. According to the literature, these plants could vary in their constituents based on their location, resulting from their geographical and climatic conditions. Little work has been reported on the V. rosea leaves essential oil obtained from the Northern Indian region and their antifungal activity against phytopathogenic fungi. Fusarium graminearum is a fungal pathogen causing billions of dollars of economic losses worldwide (Bai & Shaner, 2004; De Wolf et al., 2003). This infection alters the amino acid composition in wheat, leading to shrivelled kernels and contaminating the remaining grains with mycotoxins, primarily deoxynivalenol, which also hinders protein synthesis (Beyer & Aumann, 2008; Cavaleiro et al., 2006). Bipolaris sorokiniana is the fungal agent that causes a wide variety of cereal diseases, which cause millions of tons of wheat loss each year (Kumar & Rai, 2018).To study the relationship between the active ingredients and biological activity, the present study evaluated the antifungal activity of leaves essential oil and extracts against pathogenic wheat fungi, i.e. F. graminearum, which causes Fusarium head blight and B. sorokiniana, causing spot blotch and root rot in wheat.

2. Material and methods

2.1. Chemicals and equipment

All solvents, reagents, and absorbents employed in the experiments were of high analytical grade. The analysis of V. rosea essential oil was performed using Gas Chromatography-Mass Spectrometry (GC-MS) at the Advanced Instrumentation Research Facility (AIRF) located at Jawaharlal Nehru University, New Delhi. The specific GC-MS equipment used was a Thermo Focus-DSQ model.

2.2. Materials

2.2.1. Plant collection and authentication

Vinca rosea leaves were harvested in the spring from the botanical gardens of Punjab Agricultural University (PAU) in Ludhiana, Punjab. The precise location of the collection is at coordinates 30.900965°N latitude 75.857277°E longitude. The plant species was formally identified and authenticated by the Principal Scientist and Head of the Department of Floriculture and Landscaping at PAU, with an authentication certificate provided for reference (refer to SI).

2.2.2. Pathogen cultures

Cultures of two phytopathogenic fungi, Fusarium graminearum and Bipolaris sorokiniana, were obtained from Dr. Jaspal Kaur, a Senior Plant Pathologist at PAU. These fungal strains were maintained on slants of potato dextrose agar (PDA) and stored at 4 °C to preserve viability.

2.3. Isolation of essential oil

Freshly harvested Vinca rosea leaves were washed thoroughly under running water and rinsed twice with sterilized water to remove soil and debris. A batch of 250 g of leaves was soaked overnight in 2.5 L of water in a 5-liter round-bottom flask. The essential oil was extracted using the steam distillation method with a Clevenger apparatus for a duration of 4 h (Pandey et al., 2006). This process was repeated 20 times to gather a sufficient quantity of essential oil, which could be further used to study bioactive compounds as well as for antifungal study. The collected essential oil was then dried over anhydrous sodium sulfate and stored at 4 °C to maintain its integrity.

2.4. Physical properties of essential oil

The optical rotation of the essential oil was measured using a polarimeter, and the refractive index was determined with a refractometer (Perfit India). All measurements were conducted at a controlled room temperature of 25 °C (Kaur et al., 2021).

2.5. Gas Chromatography-mass spectrometry (GC-MS) analysis of Vinca rosea leaf essential oil

The essential oil from the leaves of V. rosea was analyzed to determine its chemical composition using a Shimadzu QP2010 Plus gas chromatography-mass spectrometry (GC-MS) system. During the analysis, a single injection was made while keeping the split valve closed for the initial minute to enhance the introduction of the sample. The injector was maintained at a temperature of 280 °C. Helium served as the carrier gas at a steady pressure of 69 kPa. The oil’s components were separated using an Rtx-5 MS capillary column, measuring 30 m in length, 20 mm in internal diameter, and with a film thickness of 0.25 μm. The temperature of the column was programmed to start at 50 °C, held steady for 2 minutes, then increased at a rate of 3 °C per minute to 180 °C, and finally raised to 280 °C at a rate of 10 °C per minute. In the mass spectrometry stage, conditions were set to electron ionization at 70 eV, with an interface temperature also at 280 °C. The scan range was from 40 to 600 amu, providing a detailed examination of the sample’s components. Retention indices of the detected peaks were calculated using a series of n-alkanes (C9-C33), and reference values were calculated under the same conditions using the ADAM RI system (Adams, 2007). The identification of compounds was further supported by comparing mass spectra with those in databases like NIST08, WILEY8, and specialized libraries focused on fragrance and flavor compounds. The calculated retention indices were cross-verified with data from the NIST Chemistry WebBook, ensuring accurate identification of the oil’s components. This meticulous, analytical approach was essential for assessing the antifungal properties of the essential oil.

The chemical composition of Vinca rosea leaf extracts was examined using a Shimadzu QP2010 Plus GC-MS system. The analysis entailed injecting the sample in single-injection mode while keeping the split valve closed for the first minute to ensure optimal sample introduction, with the injector temperature set to 280 °C. Helium gas was utilized as the carrier, maintaining a steady pressure of 69 kPa. The components of the extracts were separated using an Rtx-5 MS capillary column, which measured 30 meters in length, had an internal diameter of 20 mm, and a film thickness of 0.25 μm. The oven’s temperature program started at 50 °C for two minutes, then gradually increased to 180 °C at a rate of 3 °C per minute, and finally rose to 200 °C at 10 °C per minute. During mass spectrometry, electron ionization was performed at 70 eV, with an interface temperature of 200 °C, and scanning was done over a mass range from 40 to 600 amu. The samples were injected in split mode (120:1 ratio) and diluted in methanol (1/100 v/v). The chemical constituents were quantified by normalizing the peak areas observed in the chromatogram (Hashmi et al., 2013).

2.6. Preparation of extract

Extract of the leaves was prepared using the maceration technique (Kapoor & Rani, 2019). Dried leaf powder of V. rosea (50 g) was soaked with 100 ml of polar solvent (methanol) and was kept at room temperature for 72 h with occasional stirring. The extracted solution was filtered using Whatman filter paper no. 2 and the excess solvent was removed under rotary evaporation. The extract was then stored at 4 °C. The same procedure was used to prepare the non-polar extract, i.e. hexane.

2.7. Yield calculation

The yield of essential oil and various extracts was calculated using the following formula: $$\text{Yield\hspace{0.17em}}\left(\text{\%}\right)=\frac{\text{Weight\hspace{0.17em}of\hspace{0.17em}extract/essential\hspace{0.17em}oil}}{\text{Weight\hspace{0.17em}of\hspace{0.17em}leaves\hspace{0.17em}sample\hspace{0.17em}taken}}\hspace{0.17em}\times \hspace{0.17em}100$$

2.8. Phytochemical analysis

Extracts of leaves of V. rosea were subjected to phytochemical analysis to detect bioactive compounds. These phytochemical tests were carried out by taking into consideration the standard procedure as described by Kapoor and Rani (Kapoor & Rani, 2019).

2.8.1. Alkaloids

To test for alkaloids, combine 1.0 ml of the extract with 2.0 ml of concentrated hydrochloric acid, followed by a few drops of Mayer’s reagent. The formation of yellow precipitates indicates the presence of alkaloids.

2.8.2. Amino acids

For amino acid identification, 2.0 ml of extract is treated with 2% copper (II) sulfate drop and 1.0 ml of 95% ethanol. Adding excess potassium hydroxide pellets results in a pink layer in the ethanolic solution, confirming the presence of amino acids.

2.8.3. Carbohydrates

To detect carbohydrates, boil 1.0 ml of the extract with 1.0 ml of each of Fehling’s solutions A and B. The presence of carbohydrates is confirmed by the formation of a red precipitate.

2.8.4. Flavonoids

Add 2.0 ml of dilute sodium hydroxide to 1.0 ml of extract to test for flavonoids. Adding 3.0 ml of dilute hydrochloric acid turns the yellow solution colourless, indicating flavonoids.

2.8.5. Glycosides

Glycosides were detected by adding 1.0 ml of extract to 2.0 ml of chloroform and 2.0 ml of concentrated sulfuric acid. A reddish-brown ring indicates glycosides.

2.8.6. Phenols

Add 3–4 little drops of ferric chloride to 2.0 ml of the extract for phenols. A dark green colour signifies the presence of phenols.

2.8.7. Saponins

Mix 1.0 ml of extract with 5.0 ml of distilled water to test for saponins and shake vigorously. Stable foam formation confirms the presence of saponins.

2.8.8. Tannins

Detect tannins by treating 2.0 ml of extract with 10% alcoholic ferric chloride solution. The appearance of a dark blue or greenish-grey colour indicates tannins.

2.8.9. Terpenoids

Mix 1.0 ml of extract with 1.0 ml of chloroform and layer with concentrated sulfuric acid for terpenoids. A reddish-brown interface indicates terpenoids.

2.8.10. Cardiac glycosides

Mix 5.0 ml of the extract with 2.0 ml sodium picrate reagent to test for cardiac glycosides. A yellow-orange colour indicates their presence.

2.9. Antifungal activity

The poisoned food technique was employed to test the antifungal efficacy of V. rosea essential oil and extracts against Fusarium graminearum and Bipolaris sorokiniana. Potato dextrose agar (PDA) was prepared by combining 250 g of potato, 20 g of agar, and 20 g of dextrose in 1 L of distilled water. The mixture was sterilized in an autoclave at 15 psi for 30 minutes at 121 °C (Sharma et al., 2019). In this technique, the concentration was 20 times higher than the required concentration, which caused the solution to be diluted to the required concentration. The stock solution was prepared in water along with Tween 80 and then added to molten PDA to obtain concentrations of 1000, 500, 250, and 125 μg/ml. The solutions of varying concentrations were poured into sterilized 90 mm Petri dishes and cooled. A 5 mm mycelial disk, taken from a seven-day-old culture, was placed in the center of each PDA plate, sealed with paraffin tape, and incubated in darkness at 25 ± 1 °C for seven days in triplicate. A Petri plate containing an unamended PDA served as a positive control. The percentage inhibition was recorded using formula (Gupta & Tripathi, 2011). $$\text{Percent\hspace{0.17em}Mycelial\hspace{0.17em}growth\hspace{0.17em}inhibition\hspace{0.17em}}=\hspace{0.17em}\frac{\text{AC}-\text{AT}}{\text{AC}}\hspace{0.17em}\times \hspace{0.17em}100$$

AC = Average increase in mycelial growth in control

AT = Average increase in mycelial growth in treatment

The result obtained from F. graminearum and B. sorokiniana was compared with standard fungicide propiconazole (Tilt 25 EC), which served as a negative control and represented in terms of ED₅₀ and ED₉₀ values (effective doses that inhibit 50 per cent and 90 per cent mycelial growth) (Gupta & Tripathi, 2011).

2.10. Statistical analysis

All experiments for the antifungal analysis on both fungi were conducted in triplicates. The data obtained was expressed as mean ± standard deviation. Statistical analysis was performed using the Tukey multiple range test with SPSS 16.0 and one-way ANOVA via CPCS 1 software (Software package for social studies, Version 16.0, Armonk, NY). A P-value of less than 0.05 was considered statistically significant.

3. Results and discussion

The gas chromatography-mass spectrometry (GC-MS) analysis identified and quantified thirty-four compounds in the essential oil extracted from V. rosea leaves. Table 1 provides detailed information on these compounds, and the GC-MS chromatogram of the essential oil is provided in the Supporting Information (SI). The essential oil extracted from the leaves of V. rosea was light yellow in colour and had an average yield of 0.055% (w/v) based on the dry weight of the leaves. The oil exhibited a strong and unpleasant odor, consistent with previous literature reports (Pandey et al., 2006; Lawal et al., 2015; Aziz et al., 2015). Physicochemical properties of the oil include a density of 0.84 g/cm³ at 20 °C and a refractive index of 1.38. Its optical rotation was measured to be 84°6’. Solubility tests showed that the essential oil is soluble in methanol and ethanol, sparingly in hexane, and insoluble in water (Aziz et al., 2015).

Table 1. Chemical composition of essential oil of Vinca rosea leaves.

Peaks	Compound name	Molecular formula	Retention time	Area%	m/z	RI (Lit.)	Adam RI (cal.)
1.	2,4-Dichlorophenol	C6H4Cl2O	17.857	2.64	163	1144	1282
2.	4-vinylguaiacol	C9H10O2	23.84	1.13	150	1313	1356
3.	2,6-Di-tert-butyl-4-hydroperoxy-4-methyl −2,5-cyclohexadien-1-one	C15H24O3	25.166	1.23	236	1478	1373
4.	Tetradecane	C14H30	27.321	0.92	198	1400	1400
5.	2,6-Di-tert-butyl-P-benzoquinone	C14H20O2	28.401	0.44	220	1443	1504
6.	2,6-Di-butyl-2,5-cyclohexadiene-1,4-dione	C14H20O2	29.769	6.43	220	1460	1523
7.	2,5-cyclohexadien-1-one, 2,6-bis(1,1-dimethylethyl)-4-hydroxy-4-methyl-	C15H24O2	29.924	9.97	236	1478	1525
8.	4-Phosphatricyclo[6.1.1.0(3,7)]dec-3(7)-ene,4,9,9-trimethyl	C12H19P	30.926	0.56	194	1442	1538
9.	Butylated hydroxytoluene	C15H24O	31.591	32.74	220	1503	1548
10.	1,2,4,5-Tetramethyl-6-methylenespiro [2.4]heptane	C12H20	32.267	0.8	164	1460	1557
11.	2-Methyl-2-(3-methyl-2-oxobutyl) cyclohexanone	C12H20O2	33.394	1.22	196	1470	1572
12.	2-Butyl-5-isobutylthiophene	C12H20S	34.37	0.26	196	1416	1586
13.	Pseudodiosphenol	C10H16O2	34.852	1.54	168	1267	1592
14.	Hexadecane	C16H34	35.376	2.47	226	1600	1600
15.	1,3-Pentadiene, 2,4-di-t-butyl-	C13H24	36.668	0.19	180	1097	1374
16.	Butan-2-one, 1-[3-(3,3-dimethyl-2-oxo-butylidene)piperazin-2-ylidene]-3,3-dimethyl-	C16H26N2O2	37.922	4.58	278	2277	1464
17.	2,6-di-tert-butyl-4-methylphenyl methylcarbamate	C17H27NO2	39.447	0.21	277	1846	1572
18.	2,6-Di-tert-butyl-4-methylene-2,5-cyclohexadienone	C15H22O	39.659	0.42	218	1483	1578
19.	Cyclopropanecarboxylic acid, 1-hydroxy-, (2,6-di-t-butyl-4-methylphenyl) ester	C19H28O3	40.106	0.36	304	2178	1619
20.	Leaf acetal	C10H20O2	41.178	0.81	172	1111	1695
21.	3-Methylheptadecane	C18H38	41.615	0.29	254	1746	1726
22.	2,4-Diphenyl-4-methyl-1-pentene	C18H20	41.808	0.48	236	1846	1739
23.	Octadecane	C18H38	42.654	1.79	254	1800	1800
24.	Nonadecane	C19H40	44.062	0.75	268	1900	1900
25.	1-Cyclohexenyl phenyl ketone	C13H14O	45.677	10.36	186	1687	1931
26.	Octacosane	C28H58	46.099	0.17	394	1461	1996
27.	Docosane	C22H46	47.648	1.34	310	2200	2200
28.	3-Indazolinone, 2-phenyl-	C13H10N2O	48.083	0.87	210	2087	2077
29.	Anthraquinone	C14H8O2	48.51	5.64	208	1956	2079
30.	Eicosane	C20H42	49.266	2.06	282	2000	2000
31.	Tricosane	C23H48	51.009	0.23	324	2300	2300
32.	3-Methyleicosane	C21H44	51.468	0.34	296	2074	2106
33.	Phytol	C20H40O	52.922	4.75	296	2100	2200
34.	Oxacyclononadec-10-en-2-one	C18H32O2	53.28	1.64	280	2158	2245
35.	Unidentified compounds	—	55.62	0.37	—	—	—
	Total area percentage			100

Compounds are categorized according to classification.

Hydrocarbons: Alkane (4,14,23,24,26,27,30,31); Alkene (10,22); Branched Alkene (21,32).

Aromatic hydrocarbon (1,2,3,5,6,7,8,9,11,12, 13, 15,17,18).

Oxygenated hydrocarbons: Alcohol (33); Ketone (20,34); Phenols (2,18); Quinones (3).

Thiophens (12); Phosphorus compounds (8); Carboxylic acid esters (19); Heterocyclic compounds (16); Acetal (20); Ketone (25,28,29,35).

RI (ref) = Kovats retention indices from literature (Adams, 2007).

RI (cal) = = Kovats retention indices from literature (Adams 21; NIST web book) (Adams 2007).

Among the compounds identified, none exceeded a concentration of 32.74% of the total essential oil. The three major constituents were: butylated hydroxytoluene (Figure 1a) (32.74%), 1-cyclohexenyl phenyl ketone (Figure 1b) (10.36%), and 2,5-cyclohexadien-1-one, 2,6-bis(1,1-dimethylethyl)-4-hydroxy-4-methyl- (Figure 1c) (9.9%); together, these three compounds accounted for 53.0% of the essential oil.

Figure 1. Major compounds in the essential oil of leaves.

The essential oil also contained several minor compounds, contributing to 39% of the characterized components. These included: 2,6-di-butyl-2,5-cyclohexadiene-1,4-dione (Figure 2a) (6.43%), anthraquinone (Figure 2b) (5.64%), phytol (Figure 2c) (4.75%), 1-[3-(3,3-dimethyl-2-oxo-butylidene)piperazin-2-ylidene]-3,3-dimethyl-butan-2-one (Figure 2d) (4.58%), 2,4-dichlorophenol (Figure 2e) (2.64%), hexadecane (Figure 2f) (2.47%) eicosane (Figure 2g) (2.06%), octadecane (Figure 2h) (1.79%), oxacyclononadec-10-en-2-one (Figure 2i) (1.64%), pseudodiosphenol (Figure 2j) (1.54%), docosane (Figure 2k) (1.34%), 2,6-di-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadien-1-one (Figure 2l)(1.23%), 2-methyl-2-(3-methyl-2-oxobutyl)cyclohexanone (Figure 2m) (1.22%) and 4-vinylguaiacol (Figure 2n) (1.13%). The other identified components’ relative percentage was lower than 1%. Alkanes and aromatic hydrocarbons predominate the chemical composition of the essential oil.

Figure 2. Minor compounds in the essential oil of leaves.

In addition to the primary constituents identified in the V. rosea leaf essential oil, its composition has several noteworthy features. One significant finding is the presence of butylated hydroxytoluene (BHT), a compound known for its antioxidant properties (Huang et al., 2020). This underscores the potential of V. rosea leaf oil as a natural source of antioxidants and piques interest in its potential applications. Another notable compound in considerable concentration is phytol, which is involved in the biosynthesis of vitamins and is known for its biological activity (Islam et al., 2018). The essential oil composition suggests possible interference with the plant’s genetic regulation pathways, especially given the role of leaves in photosynthesis. Leaves, with their photosynthetically active chloroplasts, might influence the expression of terpenoid biosynthetic genes, leading to organ-specific production of these compounds (Islam et al., 2018; Li et al., 2020; Verdeguer et al., 2020).

Moreover, the volatile profile of the leaf oil shows considerable variation depending on its geographical origin. Comparative analysis of essential oils from V. rosea leaves collected from different regions such as France, India (Delhi), Portugal, Bangladesh, and Africa reveal significant differences in their chemical compositions (Lawal et al., 2015; Brun et al., 2001; Aziz et al., 2015). These variations underscore the impact of agro-climatic conditions and genetic differences on the essential oil profiles and emphasize these factors’ significance in our research (Islam et al., 2018; Li et al., 2020).

For instance, Lawal et al. reported that the major components of the essential oil from V. rosea leaves were linolenic acid ethyl ester (43.9%), phytol (7.3%), stearic acid (10.6%), and hexadecanoic acid (6.8%). Minor components in their study included oleic acid ethyl ester (5.8%), 2,6-bis(1,1-dimethylethyl)-2,5-cyclohexadiene-1,4-dione (6.2%), and linoleic acid (5.6%). These specific compounds were not detected in our analysis (Lawal et al., 2015). In contrast, essential oil from V. rosea leaves in France was found to contain high levels of palmitic acid (64.9%), methyl hexahydrofarnesyl acetone (4.0%), and palmitate (7.2%). Portuguese leaf oil was characterized by compounds such as 2,3-epoxy-α-ionone, β-ionone, benzaldehyde dihydroactinidiolide, trans-2-decen-1-ol, phenylacetaldehyde, ethyl hexanoate, and palmitic acid ethyl ester among others (Brun et al., 2001). Data from Bangladesh showed a predominance of phytol (57.47%) and geraniol (7.9%) as major components, with neophytadiene (3.37%), (E)-Octenal (2.30%), 3,4,4-trimethylcyclohexa-2-en-1-ol (2.28%), and menthol (2.07%) as minor compounds. Meanwhile, the essential oil from India (Delhi) had high concentrations of citronellol (7.9%), phytol (6.4%), pentadecanal (6.6%), (E, E)-2,4-hexadienal (7.7%), and geraniol (7.9%). It also contained a variety of other minor compounds, such as di-tert-butyl-p-cresol, hexadecenoic acid, and palmitic acid (Aziz et al., 2015).

Comparatively, our analysis identified phytol (4.75%) and hexadecane (2.47%) as minor components, and we did not detect butylated hydroxytoluene (BHT) in the previously mentioned studies. These discrepancies could be attributed to growth conditions or plant genetic variations. The sample preparation method (fresh vs dried leaves) could also affect the oil’s composition. For example, dried leaves were used for essential oil extraction in France and Africa, while fresh leaves were used in the study from India (Delhi) (Huang et al., 2020; Islam et al., 2018; Li et al., 2020). Overall, the variation in our findings compared to previously reported literature suggests that the composition of the essential oil of Vinca rosea leaves is highly influenced by environmental and genetic factors (Li et al., 2020). This variability allows the discovery of new compounds within the leaf oil that have not been previously identified (Li et al., 2020).

In addition to analyzing the essential oil from V. rosea leaves, extracts were prepared using methanol and hexane as solvents. The methanolic extract had the highest yield at 9.3%, followed by the hexane extract at 4.4%, presented in Table 2. This aligns with previous studies, which found that polar solvents like methanol generally extract more compounds from plant materials than non-polar solvents like hexane (Agarwal et al., 2011; Renault et al., 1999; Kapoor & Rani, 2019). For instance, earlier reports noted higher extraction yields from Vinca rosea leaves and Taxus wallichiana stem, bark, and needles when using polar solvents (Adhikari et al., 2018). The observed yield of 9.3% for the methanolic extract in this study closely matches the 8.8% yield reported in the literature (Agarwal et al., 2011; Renault et al., 1999; Kapoor & Rani, 2019).

Table 2. Percentage yield of extracts of leaves.

Extract	Yield (%)
Methanol	9.3
Hexane	4.4

Phytochemical analysis is crucial for identifying the bioactive profiles of plants. The Vinca rosea leaf extracts were examined for various phytochemicals, including alkaloids, terpenoids, tannins, saponins, phenols, glycosides, flavonoids, carbohydrates, amino acids, and cardiac glycosides (Table 3). Each class of compounds has distinct biological activities: Phenols exhibit antioxidant, antibacterial, and antiviral properties. Tannins contribute to the plant’s flavor, astringency, and colour. Alkaloids have antimicrobial properties by interacting with microbial DNA. Flavonoids are crucial for the plant’s colour and aroma, attracting pollinators. Saponins are known for their cholesterol-lowering, anti-inflammatory, and anticancer effects (Renault et al., 1999, Kapoor & Rani, 2019, Islam et al., 2018). The extracts were prepared in methanol and hexane to maximize the diversity of bioactive compounds extracted. The methanolic extract contained many bioactive constituents, including alkaloids, amino acids, flavonoids, phenols, saponins, tannins, carbohydrates, glycosides, cardiac glycosides, and terpenoids. In contrast, the hexane extract contained saponins, tannins, and terpenoids. Both extracts were found to have phenols and flavonoids (Pandey et al., 2006; Renault et al., 1999). The findings of this study are consistent with previously reported results, demonstrating similar profiles of bioactive compounds in V. rosea leaf extracts from various sources (Renault et al., 1999; Kapoor & Rani, 2019).

Table 3. Phytochemical identification test for extracts of leaves.

Chemical constituents	Methanol	Hexane
Alkaloids	+	–
Amino acid	+	–
Carbohydrates	+	–
Flavonoids	+	+
Glycosides	+	–
Phenol	+	+
Saponin	+	+
Tannin	+	+
Terpenoid	+	+
Cardiac Glycosides	+	–

(+) = Presence and (-) = Absence.

The GC-MS analysis of V. rosea leaves’ methanolic extract revealed several bioactive alkaloids and flavonoids (Table 4). Key alkaloids identified include isoindolinine (Figure 3a), coronaridine (Figure 3b), Desmethoxyvindoline (Figure 3c), and vindoline (Figure 3d). These compounds are noteworthy because vindoline is a commercially significant anti-cancer drug precursor synthesizing various pharmaceutical agents. In contrast, the GC-MS analysis of hexane extract (Table 5) showed the presence of lipid compounds, specifically cholesterol (Figure 4a), 26-Nor-5-cholesten-3á-ol-25-one (Figure 4b). This distinct lipid profile highlights the hexane extract’s potential effectiveness in antifungal applications and utility in isolating specific lipid compounds (Rajput et al., 2011; Renault et al., 1999; Daniel, 2006).The GC-MS chromatograms for both extracts are available in the Supporting Information (SI), providing detailed insights into their chemical compositions. The results show the different extraction efficiencies and compound profiles obtained with polar (methanol) versus non-polar (hexane) solvents, supporting the targeted extraction of specific bioactive molecules from V. rosea leaves (Rajput et al., 2011; Renault et al., 1999; Daniel, 2006).

Figure 3. Main compounds in methanolic extract of leaves.

Figure 4. Main compounds in hexane extract of leaves.

Table 4. The results of GC-MS of methanolic extract of leaves of Vinca rosea.

Peaks	Compound name	Molecular formula	Retention time	Area %	m/z
1	Glycerol	C3H8O3	16.006	17.58	92
2	4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-	C6H8O4	16.556	1.68	144
3	Sandalore	C14H26O	31.457	0.59	210
4	Dihydroactinidiolide	C11H16O2	32.574	0.43	180
5	1,2-Dimethyl-3,5-bis(1-methylethenyl) cyclohexane	C14H24	33.151	0.09	192
6	Megastigmatrienone A	C13H18O	36.278	0.17	190
7	Caryophyllene acetate	C17H28O2	38.869	0.28	264
8	Myristic acid	C14H28O2	41.843	0.13	228
9	17-Oxo-4-propyl-3,4-seco-4-androsten-3-oic acid methyl ester	C23H36O3	42.027	0.23	360
10	2,6,10-Trimethylundecan-(5E)-2,5,9-Trien-4-one	C14H22O	42.239	0.15	206
11	5-(2-Butenyl)-17-oxo-4-nor-3,5-seco-5.alpha.-androstan-3-oic acid, methyl ester	C23H36O3	42.542	0.14	346
12	3H-3,10a-Methano-1,2-benzodioxocin-3-ol, octahydro-7,7-dimethyl-, (3.alpha.,6a.beta.,10a.beta.)-	C13H22O3	43.537	0.31	226
13	alpha-Cedrene epoxide	C15H24O	43.763	0.08	220
14	Phytol	C20H40O	43.876	0.69	296
15	hexahydrofarnesyl acetone	C18H36O	44.082	0.15	268
16	Neophytadiene	C20H38	45.308	0.13	278
17	Inositol	C6H12O6	46.743	0.22	180
18	Methyl palmitate	C17H34O2	46.907	1.95	270
19	Butyl octyl phthalate	C20H30O4	47.804	0.31	334
20	Palmitic acid	C16H32O2	48.745	16.38	256
21	Methyl linoleate	C19H34O2	52.089	1.18	294
22	Methyl oleate	C19H36O2	52.319	3.88	296
23	Phytol	C20H40O	52.704	3.42	296
24	Methyl stearate	C19H38O2	53.181	0.4	298
25	(Z)-Tetradec-7-enal	C14H26O	54.093	19.61	210
26	Stearic acid	C18H36O2	54.707	1.44	284
27	4,8,12,16-Tetramethylheptadecan-4-olide	C21H40O2	59.582	0.27	324
28	7a-Methyl-1,2,3,6,7,7a-hexahydro-5H-inden-5-one	C10H14O	60.03	0.45	150
29	2-cyclohexen-1-one, 3-methyl-5-(1-methylethyl)-4-(2-methyl-1-propenyl)-	C14H22O	60.649	0.68	206
30	Hexanoic acid 2-ethyl-hexadecyl ester	C24H48O2	61.94	0.84	368
31	2-Methylhexacosane	C27H56	62.409	0.17	380
32	Isoindolinine	C21H24N2O2	63.262	0.59	336
33	Isovindolinone	C21H24N2O2	63.524	0.79	336
34	Dotriacontane	C32H66	63.807	0.12	450
35	Hexanoic acid 2-ethyl-hexadecyl ester	C24H48O2	64.689	0.35	368
36	Coronaridine	C21H26N2O2	65.019	0.57	338
37	cis-Cyclo(Leucyl-Tyrosyl)	C15H20N2O3	65.55	0.16	276
38	cis-Cyclo(Leucyl-Tyrosyl)	C15H20N2O3	65.832	0.36	276
39	Squalene	C30H50	66.219	1.03	410
40	alpha-Tocospiro A	C29H50O4	66.56	0.3	462
41	alpha-Tocospiro-B	C29H50O4	66.804	0.41	462
42	Desmethoxyvindoline	C24H30N2O5	68.134	1.24	426
43	Stigmast-5-en-3-ol, oleate	C47H82O2	69.851	0.67	679
44	Vitamin E	C29H50O2	70.541	1.64	430
45	Vindoline-d3	C25H32N2O6	71.842	5.5	459
46	Campesterol	C28H48O	72.477	0.89	400
47	Stigmasterol	C29H48O	73.034	0.6	412
48	beta.-Sitosterol	C29H50O	74.443	1.91	414
49	5H-3,5a-Epoxynaphth[2,1-c]oxepin, dodecahydro-3,8,8,11a tetramethyl-	C18H30O2	75.356	0.49	278
50	24-Norursa-3,12-diene	C29H46	75.667	1.03	394
51	24-Norursa-3,12-diene	C29H46	76.604	1.01	394
52	Olean-12-en-3-ol, acetate, (3beta)-	C32H52O2	77.641	0.69	468
53	γ-Sitostenone	C29H48O	77.852	0.27	412
54	alpha-Fernenol	C30H50O	78.139	0.51	426
55	Methyl Commate A	C32H52O4	79.121	3.57	470
56	beta-Simiarenol	C30H50O	79.631	1.25	426
				100

Compounds are categorized according to different classes.

Alcohol and derivatives (1,14,17,23,27,54); Aldehydes and ketones (2,4,10,11,25,28,29,53); Alkanes and hydrocarbons (5,16,31,34,39); Aromatic compounds and derivatives (3,12,13,32,33,36,42); Carboxylic acids and derivatives (8,9,20,26,30,35,18,21,22,24); Esters and lactones (7,27,55); Steroids and Terpenes (6,13,46,47,48,50,51,52,56); and Miscellaneous (19,37,38,40,41,43,44,45,49).

Table 5. The results of GC-MS of hexane extract of leaves of Vinca rosea.

Peaks	Compound name	Molecular formula	Retention time	Area %	m/z
1	n-Hexane	C6H14	3.03	0.15	86
2	3-methylpentane	C6H14	3.25	6.9	86
3	2,3-Dimethylpentane	C7H16	3.5	12.61	100
4	3-Ethyl-2,2-dimethylpentane	C9H20	3.78	2.67	128
5	Sulphuric acid dibutyl ester	C8H18O4S	4.08	8.02	210
6	2,4-Dimethylhexane	C8H18	5.74	1.31	114
7	1-Methyl-2-pyrrolidinone	C5H9NO	6.3	4.28	99
8	5,7-Dimethylundecane	C13H28	7.25	0.99	184
9	3-Methyldecane	C11H24	8.2	0.38	156
10	2,5,9-Trimethyldecane	C13H28	8.74	1.76	184
11	6-Ethyl-2-methyldecane	C13H28	8.92	0.33	184
12	trans-1-Butyl-2-methylcyclopropane	C8H16	9.09	0.24	112
13	1,14-Tetradecanediol	C14H30O2	9.38	0.19	230
14	2-Hexyl-1-octanol	C14H30O	9.5	0.24	214
15	2,3,6-Trimethyloctane	C11H24	9.76	0.92	156
16	4,7-Dimethylundecane	C13H28	10.17	1.83	184
17	2-Hexyl-1-octanol	C14H30O	10.84	0.24	214
18	10-Methylnonadecane	C20H42	10.96	0.11	282
19	2,6,10,14-Tetramethylheptadecane	C21H44	11.04	0.21	296
20	2,7,10-Trimethyldodecane	C15H32	11.2	0.33	212
21	2,4,6-Trimethyloctane	C11H24	11.52	1.46	156
22	Cedrene	C15H24	12.07	0.44	204
23	2,5,5,6,8a-Pentamethyl-trans-4a,5,6,7,8,8a-hexahydro-gamma-chromene	C14H24O	12.33	0.28	208
24	Dihydroactinidiolide	C11H16O2	13.29	0.42	180
25	O-Decylhydroxylamine	C10H23NO	14.03	0.52	173
26	Nojigiku acetate	C12H18O2	14.61	0.52	194
27	4-(3-Hydroxybutyl)-3,5,5-trimethylcyclohex-2-en-1-one	C13H22O2	15.28	0.24	210
28	Sulfurous acid, decyl 2-pentyl ester	C15H32O3S	16.28	0.37	292
29	6,10,14-Trimethylpentadecan-2-one	C18H36O	16.71	0.65	268
30	Methyl 14-methylpentadecanoate	C17H34O2	17.55	0.71	270
31	Palmitic acid	C16H32O2	18.01	8.9	256
32	Phytol	C20H40O	19.38	3.88	296
33	(6Z,9Z)-Pentadeca-6,9-dien-1-ol	C15H28O	19.71	12.32	224
34	1,2-Epoxyoctadecane	C18H36O	21.19	0.13	268
35	7-Methyl-Z-tetradecen-1-ol acetate	C17H32O2	21.77	0.23	268
36	Cholesterol	C27H46O	22.21	5.59	386
37	Palmitic acid, 2-(octadecyloxy)ethyl ester	C36H72O3	23.87	0.54	553
38	Phthalic acid, di(6-methylhept-2-yl) ester	C24H38O4	24.37	3.19	390
39	Benzyl oleate	C25H40O2	26.12	1.01	372
40	Butyl oleate	C22H42O2	28.02	0.39	338
41	2-methyltetracosane	C25H52	28.16	0.28	352
42	26-Nor-5-cholesten-3á-ol-25-one	C26H42O2	28.89	14.25	386
				100.03

Compounds are categorized according to different classes.

Alkanes (1,2,3,4,6,8,9,10,11,15,16,18,19,20,21,29,41); Alcohols and derivatives (13,14,17,33,32,35); Esters (5,25,26,28,30,37,38,39,40); Ketones (24,27,29); Carboxylic acids and derivatives (31,37,42); Cycloalkanes and derivatives (12,22,23,27); Ethers (7,34); and Steroids and Sterols (36,42).

In recent years, the medicinal and agricultural sectors have increasingly turned to natural products as alternatives to synthetic compounds. This shift is driven by environmental and health concerns and a growing consumer demand for natural ingredients. Despite the broad exploration of natural antifungal agents, the antifungal activity of V. rosea essential oil against wheat fungi such as F. graminearum and B. sorokiniana has yet to be extensively studied. These fungi are significant agricultural problems in India (De Wolf et al., 2003).

Given this context, we screened the antifungal potential of V. rosea leaf extracts and essential oil against these wheat pathogens using the poisoned food technique. Many plant species have demonstrated natural fungicidal properties, and we investigated whether V. rosea could serve a similar role. The percent mycelial inhibition for F. graminearum and B. sorokiniana under different treatments is presented in Figures 5 and 6, respectively. Additionally, the ED₅₀ (effective dose to inhibit 50% of mycelial growth) and ED₉₀ (effective dose to inhibit 90% of mycelial growth) values for the essential oil and extracts are detailed in Table 6.

Figure 5. Percent mycelial growth inhibition of different treatments against F. graminearum.

Figure 6. Percent mycelial growth inhibition of different treatments against B. sorokiniana.

Table 6. ED₅₀ and ED₉₀ value of different treatments against F. graminearum and B. sorokiniana.

	F. graminearum			B. sorokiniana
Components	Tukey test mean	ED50	ED90	TUKEY TEST MEAN	ED50	ED90
		(µg ml^-1)	(µg ml^-1)		(µg ml^-1)	(µg ml^-1)
Methanol Extract	91.472^ab	80	240	90.532^ab	76	290
Hexane Extract	21.508^d	>1000	>1000	18.519^d	>1000	>1000
Essential Oil	77.089^c	98	865	74.286^c	100	875
Tilt (25 EC)	100.00^a	62.5	110	100.00^a	62.5	110

CD (1 % interaction): Components (A): 0.477 Concentration (B): 0.477 (A) x (B): 0.954.

Percent inhibition of all the components was significantly different from each other with P-value of 0.01.

Based on the ED₅₀ and ED₉₀ values, we established a classification for the effectiveness of the plant materials. For Fusarium graminearum, the methanolic extract of leaves was the most effective (ED₅₀ = 80 µg/mL, ED₉₀ = 240 µg/mL), followed by the essential oil (ED₅₀ = 98 µg/mL, ED₉₀ = 865 µg/mL). These results were compared to the standard fungicide propiconazole (Tilt 25 EC), which inhibited fungal growth at 125 µg/mL and had the lowest ED₅₀ value of 62.5 µg/mL, making it the most effective treatment overall. The hexane extract was the least effective, with ED₅₀ and ED₉₀ values exceeding 1000 µg/mL.

A similar trend was observed for Bipolaris sorokiniana. The ED₅₀ values for the methanolic extract, essential oil, hexane extract, and standard fungicide were 76, 100, >1000, and 62.5 µg/mL, respectively, while the ED₉₀ values were 290, 875, >1000, and 110 µg/mL, respectively (Table 6). The order of effectiveness against both fungi, based on their ED₅₀ and ED₉₀ values, was as follows: $$\text{Methanolic\hspace{0.17em}leaf\hspace{0.17em}extract\hspace{0.17em}}>\text{\hspace{0.17em}Essential\hspace{0.17em}oil\hspace{0.17em}}>\text{\hspace{0.17em}Hexane\hspace{0.17em}leaf\hspace{0.17em}extract}$$

Our findings indicated that both the methanolic extract and the essential oil of V. rosea were highly effective in controlling the growth of both fungi. The strong antifungal activity of the methanolic extract is likely due to its high content of bioactive alkaloids, particularly vindoline, known for its anticancer properties, as well as phenols and flavonoids, which possess antimicrobial activities. The antifungal properties of the essential oil may be attributed to its components, such as butylated hydroxytoluene, known for its antioxidant effects, and other alkanes and aromatic hydrocarbons (Tyagi & Malik, 2010; Miron et al., 2014; Hashmi et al., 2013).

Alkaloids are nitrogen-containing compounds that disrupt fungal cell membranes, impairing cell function and inhibiting growth. Lipophilic terpenoids can similarly disrupt fungal cell membranes, inhibiting spore germination and mycelial growth (Tyagi & Malik, 2010; Miron et al., 2014; Hashmi et al., 2013). The antifungal activity of Vinca rosea extracts and essential oil may result from these compounds’ ability to alter cell wall synthesis, increase membrane permeability, and impair fungal enzymatic systems (Tyagi & Malik, 2010). This can cause leakage of intracellular contents, disrupt ion balance, and inhibit respiratory chain functions (Amber et al., 2010).

In summary, our antifungal data unequivocally demonstrate that the essential oil and methanolic extract of V. rosea hold significant promise as natural antifungal agents. These findings not only underscore the potential of V. rosea in the field of agriculture but also advocate for their use in formulating herbal solutions to combat fungal pathogens in agricultural settings.

4. Conclusion

This study provides the first detailed report on the chemical constituents of the essential oil derived from the fresh leaves of V. rosea cultivated in Ludhiana, Punjab. Through GC-MS analysis, thirty-four compounds were identified in the essential oil, with butylated hydroxytoluene being the most abundant. The essential oil and methanolic leaf extract demonstrated significant antifungal activity against the wheat pathogens F. graminearum and B. sorokiniana, outperforming the hexane extract. The strong antifungal properties of the methanolic extract are likely due to its high content of bioactive alkaloids, such as vindoline, and phenolic compounds, while the essential oil’s effectiveness can be attributed to its rich composition of hydrocarbons and aromatic compounds, including butylated hydroxytoluene. These findings highlight the potential of V. rosea extracts and essential oil as natural antifungal agents, offering a promising alternative to synthetic fungicides. Future research should focus on in vivo studies to justify and evaluate the commercial application of these natural products, paving the way for their potential integration into agricultural practices for sustainable disease management.

Authors’ contributions

Parul Sharma: Searched and worked on the project, extracted the data, wrote the original draft, given final approval of the version to be published. Ramandeep Kaur: Conceptualization, supervision, drafting and revising the manuscript, and giving the final approval of the version to be published. Urvashi Bhardwaj: Reviewed and final approval of the version to be published. Jaspal Kaur: Worked on getting the antifungal activity data, conceptualization, and final approval of the version to be published. All authors have read and approved the final version of the manuscript submitted for publication and are responsible for the final content.

Acknowledgments

The authors are thankful to Dr. Kiranjit Kaur Dhatt, Principal Scientist-cum-Head, Department of Floriculture and Landscaping, Punjab Agricultural University (Ludhiana), for the identification and authentication of the plant specimen and Head of the Department of Soil Science, Punjab Agricultural University, for providing the mineral analysis facility.

Disclosure statement

The authors confirm that they have no competing financial interests or personal relationships that could have influenced the work reported in this paper.

Data availability statement

We have carried out the research work and assure you that it can be provided whenever required from the corresponding author.

Additional information

Funding

There are no funders to report for this submission.

Notes on contributors

Parul Sharma

Parul Sharma earned her Master’s in Natural Product Chemistry from Punjab Agricultural University, Ludhiana, India. She is pursuing a Ph.D. in Organic Chemistry at Oklahoma State University, Stillwater, USA, and is currently working as a Graduate Research Assistant. Her research focuses on discovering new methods for synthesizing pharmaceutically important compounds.

Ramandeep Kaur

Ramandeep Kaur has been a Chemist at Punjab Agricultural University, Ludhiana, since May 2018. She received the Maulana Azad National Fellowship (JRF and SRF) from the University Grants Commission (UGC), India. With over 40 research and review articles published in high-impact journals, her interests include Natural Product Chemistry, Agro-chemistry, Organic Chemistry, and Green Chemistry.

Urvashi Bhardwaj

Urvashi Bhardwaj has also been a Chemist at Punjab Agricultural University, Ludhiana, since May 2018. She has published over 38 research and review articles in prestigious journals. Her research interests encompass Natural Product Chemistry, Agrochemistry, and Green Chemistry.

Jaspal Kaur

Jaspal Kaur is a Principal Plant Pathologist in the Department of Plant Breeding and Genetics at Punjab Agricultural University. She specializes in wheat and barley pathology, focusing on identifying resistance sources for these diseases, monitoring and surveying them, identifying new rust pathotypes using differentials and trap nurseries, and studying their epidemiology and management.