Phytochemical and antibacterial assessment of essential oils extracted from aerial parts of cordia dichotoma G. Forst

ABSTRACT

This study involved the exploration of hydro-distilled essential oil (EsO) from the aerial parts of Cordia dichotoma fruits, stems, seeds and leaves via gas chromatography‒mass spectrometry. Approximately 33 volatile components were detected throughout the study. Almost 27 and 23 compounds (comprising 64.83% and 31.55%, respectively, of the total components) were detected in the fruits and stems, whereas 25 compounds (57.12% of the total components) were detected in the seeds, and 25 compounds (52.48% of the total components) were found in the leaves. The main components of the fruit oil were Nonanal (5.77%), Oleic acid (4.30%), β-Damascenone (4.12%), 3, 4-Dehydro-β-ionone (4.06%), Methyl jasmonate (3.32%), Sabinene (3.31%), Phytol (3.22%) and Vitispirane (3.11%). From stem oil, the major components were Nonanal (6.52%), Nonadecane (3.22%) and 2, 3-Octanedione (2.16%). Whereas, the major components of the seed oils were Nonanal (12.14%), Oleic acid (5.13%), Dihydroactindiolide (3.35%), 2-Methoxy-4-vinylphenol (3.18%), Dodecanoic acid (3.16%) and Methyl jasmonate (3.11%). Similarly, the major components of the leaf oils were 2, 4-di-t-Butylphenol (10.25%), 3-Methylnonane (4.73%), 2-Undecanol (4.64%), 3, 4-Dehydro-β-ionone (3.52%) and Nonanal (3.16%). These EsO were used for antibacterial activity against two bacteria i.e. E. coli (G-ve) and the S. aureus (G+ve) by disc diffusion method to determine the MIC (minimum inhibitory concentration) and MBC (maximum bactericidal concentration). However, among the four parts of C. dichotoma, fruit and seed EsO were highly effective against E. coli (MIC and MBC were 125 μg/mL, 1000 μg/mL respectively) compared to S. aureus (MICs of 250 and 500 μg/mL with an MBC of 1000 μg/mL)

KEYWORDS:

• Cordia dichotoma
• essential oils
• chemical composition
• GC-MS
• antibacterial activity

Introduction

Essential oils (EsO) are aromatic and volatile liquids distilled from different plant materials.^[1] These are low-molecular-mass compounds of complex mixtures that generally include monoterpenes, sesquiterpenes and hydrocarbons. Likewise, oxygenated constituents consist of phenols, aldehydes, ethers, ketones, alcohols and esters obtained from these hydrocarbons.^[2] Numerous studies have shown that EsO possesses antioxidant, antibacterial, antifungal, and nematicidal activities, and its chemical compounds are responsible for these properties.^[3,4] EsO are vital components of the agricultural industry and have been used in the food products, drinks, pharmaceuticals, perfumes and cosmetics industries.^[5,6] EsO added to edible products, either by direct mixing or in edible coatings and active packaging, may serve as a valid alternative to prolong shelf life and prevent autoxidation.^[7] For this purpose, the food industry requires large amounts of EsO.^[1] The medicinal properties of EsO and its key component, terpenes, have been newly acknowledged for their roles as antipyretics, sedatives, anxiolytics and expectorants, as well as inhibitors of bacterial and fungal growth.^[8] Along with all-natural constituents, EsO has biological properties and is considered to be specific due to its radical scavenging activities.^[9] Volatile oils have many remedial uses in human medicine due to their antiviral, antiphlogistic, antinociceptive, antioxidant, anticancer, antibacterial and fumigant properties.^[10–12] Volatile compounds might be utilized by massage therapy or inhalation. EsO can be helpful in treating dementia patients and may protect the human body from food poisoning.^[13] EsO plays an imperative role in preventing respiration as well as ion circulation and thus the demolition of bacterial cells.^[14] Moreover, plants have negligible EsO; they degrade easily in their surroundings, and some of them have insufficient negative effects.^[15] The family Boraginaceae includes the genus Cordia, which contains approximately 300 species. One of them has the botanical name Cordia dichotoma Forst., a small to medium-sized deciduous tree with a short, crooked trunk, a short bole, and a spreading crown, also known as Lasura.^[16,17] The flowers are bisexual, white to pinkish in color, and appear as loose corymbose cymes. The leaves are simple and dentate, and the bark and stem are brownish, smooth and wrinkled.^[18] Fruits are edible and have a mass of gooey flesh that turns black as they ripen and become viscid.^[17] Tropical and subtropical regions of the world are where these trees grow.

For many years, the therapeutic properties of C. dichotoma were known. Bark and stem water are used to treat burning sensations, leprosy, gonorrhea, dyspepsia, fever, and diarrhea.^[19] The medicinal properties of the leaves include anthelmintic, astringent, diuretic, demulcent, purgative, expectorant, tonic, ulcer, and cough.^[17] Fruit is primarily used as an astringent, expectorant, purgative, anti-helminthic, or diuretic.^[20] Antioxidants and anti-inflammatory agents are known to exist in seeds.^[21,22] For phytochemical screening, several compounds, including amyrins, betulin, octacosanol, lupeol-3- rhamnoside, sitosterol, sitosterol-3-glucoside, hentricontanol, hentricontane, taxifolin-3,5-dirhmnoside, and hesperetin-7-rhamnoside, are abundant in C. dichotoma.^[19]

A literature survey revealed that no work has been conducted on the chemical composition and antibacterial activity of essential oils from the aerial parts of C. dichotoma, i.e., fruits, stems, seeds and leaves. Therefore, the purpose of the current study was to investigate the chemical composition of C. dichotoma essential oils and their antibacterial effects.

Materials and methods

Collection and identification of plant materials

C. dichotoma fruits, stems, seeds and leaves (800 g each) were collected from the small village Rano Bughio, which is located in the District of Matiari, Sindh Province, Pakistan. The plant authentication procedure was confirmed by Prof. Tahir Rajpoot, a taxonomist at the Botany Institute, Sindh Varsity Jamshoro/Pakistan.

Exploration of essential oils composition

The pulverized powders of C. dichotoma fruits, stems, seeds and leaves (100 g each) were hydrodistilled with deionized water (1000 mL) for 4 h by using a Clevenger-type apparatus. The obtained aqueous extract (800 mL) was further mixed with 100 mL of n-hexane (analytical grade) to obtain oils by using a separating funnel. After completion of 20 min, the upper layer of n-hexane was separated, treated with anhydrous magnesium sulfate and instantly analyzed by GC-MS.

Identification of chemical constituents

GC-MS verified the presence of EsO in C. dichotoma fruits, stems, seeds, and leaves. A GC instrument (Agilent 6890 N) fixed with an MS detector was used for the GC-MS analysis (MS-5975). The capillary column used was an HP-5 MS with dimensions of 30 m in length, a 0.25 mm internal diameter, and a 0.25 m film thickness. Additionally, the 90°C oven temperature was set for 1 min, increased to 200°C at 5°C/min (hold time kept at 6 min), and then decreased to 290°C at 20°C (hold time kept 10 min). Additionally, a 1 L sample of HPLC-grade n-hexane was injected using splitless mode, and electron ionization (EI) mode at 70 eV was used for MS detection. At a 1.5 mL/min flow rate and MS temperature, helium (carrier gas) was used to maintain the injector and transfer line at 220 and 290 degrees, respectively.

Identification of constituents

The chemical constituents of C. dichotoma fruits, stems, seeds and leaves were examined by matching their RIs and HP-5 MS data with those in the literature. The retention indices of all the constituents were studied by using standard (C8–C32) n-alkanes and the National Institute of Standards and Technology (NIST) mass spectral library with the help of the Kovats method; the values were above 90%. The oil percentage composition was calculated from the peak areas of gas chromatography.

Evaluation of in vitro antibacterial activity

With the assistance of the American Type Culture Collection (ATCC), antibacterial effects of C. dichotoma fruit, stem, and seed and of leaves against S. aureus and E. coli were tested on Mueller Hinton agar (MHA), a medium for the growth of microorganism species. To investigate the antibacterial activity of the four different fractions, a modified disc diffusion method was chosen.^[3,17] To ensure the antibacterial action of the fruit, stem, seed, and leaf essential oils, five different concentrations, 1000, 750, 500, 250, and 125 µg/mL, were prepared using 100% DMSO (dimethylsulfoxide) as the solvent. DMSO was also used as a negative control.^[14] With the aid of a sterile cotton swab, the bacterial suspensions were stretched onto solid Petri plates and adjusted to 106 CFU/mL. The surface of the microbial Petri plates was then covered with a 5 mm disc (Whatman No. 1 filter paper) that had been moistened with 15 L of EsO at various concentrations (diluted in 100% DMSO). The plants were kept in the incubator for 24 h at 37°C. By measuring the inhibition diameter around the discs in millimeters (mm)^[23] and calculating the MIC and MBC values after the incubation period, the antibacterial action of each microbial strain was recorded. All the results were verified in triplicate and are presented as the mean ± standard deviation (SD).

Results and discussion

Chemical composition of essential oils (EsO)

One of the well-known and economically advantageous hydro-distillation techniques was used in this study to isolate EsO from the fruits, stems, seeds, and leaves of C. dichotoma.^[21] Fruit, stem, seed, and leaf hydro-distillation occurred in 0.09, 0.04, 0.07, and 0.06% (dry weight) respectively, of the EsO. By using GC-MS, a total of 25 compounds (covering 57.12% of the total volatiles) were discovered in the seeds, and 25 compounds (covering 52.48%) were discovered in the leaves. In total, nearly 27 and 23 compounds (comprising 64.83% and 31.55%, respectively, of the total volatiles) were detected in the fruits and stems. Table 1 and Figures 1, 2 , 3 , and 4 show that the products were eluted on the HP-5 MS column.

Figure 1. GC‒MS chromatogram of C. dichotoma fruit.

Figure 2. GC‒MS chromatogram of C. dichotoma stem.

Figure 3. GC‒MS chromatogram of C. dichotoma seeds.

Figure 4. GC‒MS chromatogram of C. dichotoma leaves.

Table 1. Chemical composition of essential oil (EsO) from C. dichotoma fruit, stem seed and leaves.

Compounds	CC^a	Rt^b	RI^c	Current work				Reported work ^[Citation24]
				Percent composition (%)
				Fruit %	Stem %	Seed %	Leave %	C. myxa Leave %
Sabinene	MH	6.01	961	3.31	2.11	–	0.23	–
3-Methylnonane	HC	6.10	976	1.98	2.10	–	4.73	–
a-Pinene	MH	6.31	1028	0.88	0.54	0.75	1.08	–
Nonanal	OC	6.42	1089	5.77	6.52	12.14	3.16	2.9
Linalool	OM	6.55	1092	–	1.17	2.19	2.29	8.4
2,3-Octanedione	KC	7.15	1115	2.78	2.16	2.22	1.11	–
a-Terpineol	OM	7.53	1195	–	1.02	1.23	1.43	–
Safranal	MH	8.71	1197	2.22	0.13	1.17	1.15	1.5
Vitispirane	Oth	10.33	1271	3.11	–	2.55	–	–
2-Undecanol	OC	11.90	1277	1.50	1.34	2.42	4.64	–
Nonanoic acid	AC	12.12	1297	2.32	0.23	1.27	1.32	–
2-Methoxy-4-vinylphenol	PC	14.39	1315	3.05	–	3.18	–	–
β-Damascenone	KC	15.11	1388	4.12	–	–	–	0.7
Tetradecane	HC	16.15	1400	–	0.15	0.52	0.64	–
Trans-Geranylacetone	KC	17.34	1454	2.17	–	–	–	3.9
3,4-Dehydro-β-ionone	KC	18.16	1485	4.06	–	1.55	3.52	–
2,4-di-t-Butylphenol	PC	18.19	1513	2.13	1.03	1.38	10.25	–
Dihydroactindiolide	KC	19.11	1525	2.86	1.33	3.35	1.13	–
Trans Nerolidol	OS	19.88	1564	–	2.09	2.12	0.27	–
Dodecanoic acid	AC	20.33	1570	–	–	3.16	–	–
Hexadecane	HC	22.45	1600	–	0.20	0.25	0.39	–
Guaiol	OS	22.79	1602	1.12	–	0.11	1.20	–
γ-Eudesmol	OS	24.07	1630	1.03	–	2.10	2.14	–
Methyl jasmonate	EC	25.10	1655	3.32	1.65	3.11	2.10	–
Heptadecane	HC	25.72	1700	0.33	0.53	1.66	0.96	–
Germacrene D	SH	26.33	1710	1.34	1.22	2.31	2.11	–
Hexahydrofarnesyl acetone	KC	26.56	1838	2.23	–	–	2.21	7.00
Nonadecane	HC	26.99	1900	0.22	3.22	–	2.13	–
Farnesyl acetone	KC	29.22	1918	2.20	0.57	–	0.08	3.7
E-15-Heptadecenal	OC	30.65	2084	2.04	0.84	–	–	–
Phytol	DT	35.53	2106	3.22	0.15	0.26	2.21	19.2
Linoleic acid	AC	36.43	2134	1.22	1.25	0.99	–	–
Oleic acid	AC	37.77	2140	4.30	–	5.13	–	–
Total				64.83	31.55	57.12	52.48
Monoterpene hydrocarbons	MH			6.41	2.78	1.92	2.46
Oxygenated monoterpenes	OM			ND	2.19	3.42	3.72
Oxygenated sesquiterpenes	OS			2.15	2.09	4.33	3.61
Sesquiterpene hydrocarbons	SH			1.34	1.22	2.31	2.11
Diterpenes	DT			3.22	0.15	0.26	2.21
Oxygenated compounds	OC			9.31	8.70	14.56	7.80
Hydrocarbons	HC			2.53	6.20	2.43	8.85
Phenolic compound	PC			5.18	1.03	4.56	10.25
Ketonic compounds	KC			20.42	4.06	7.12	8.05
Acidic compounds	AC			7.84	1.48	10.55	1.32
Ester compound	EC			3.32	1.65	3.11	2.10
Others	Oth			3.11	–	2.55	–

^a= classes of compounds, ^b = Retention time, ^c = Retention Indices

The main components of the fruit oil were Nonanal (5.77%), β-Damascenone (4.12%), Oleic acid (4.30%), Methyl jasmonate (3.32%), Sabinene (3.31%), Phytol (3.22%, 3, 4-Dehydro-β-ionone (4.06%), and Vitispirane (3.11%). While the major compounds of the stem oils are Nonanal (6.52%), 2, 3-Octanedione (2.16%) and Nonadecane (3.22%). In addition the major compounds of the seed oils are Nonanal (12.14%), Dihydroactindiolide (3.35%), 2-Methoxy-4-vinylphenol (3.18%), Oleic acid (5.13%), Dodecanoic acid (3.16%) and Methyl jasmonate (3.11%). Similarly, the major components of the leave oils are 2, 4-di-t-Butylphenol (10.25%), 3-Methylnonane (4.73%), 2-Undecanol (4.64%), 3, 4-Dehydro-β-ionone (3.52%) and Nonanal (3.16%). The chemical compounds in C. dichotoma fruit stem seeds and leaves, which are complex mixtures of diverse substances, are explained in Table 1. The different classes of compounds (CC) were classified as follows: monoterpene hydrocarbons (MH), Oxygenated monoterpenes (OM), Oxygenated sesquiterpenes (OS), Sesquiterpene hydrocarbons (SH), Diterpenes (DT), Oxygenated compounds (OC), Hydrocarbons (HC), Phenolic compound (PC), Ketonic compounds (KC), Acidic compounds (AC), Ester compound (EC) and Others (Oth). It is examined that the prime chemical compounds are Ketonic compounds (KC) in fruit (20.42%) but slight concentrations in stem (4.06%), seed (7.12%) and leave (8.05%) have been observed. Whereas the major constituents are Oxygenated compounds (OC) in stem (8.70%), fruit (9.31%) and seed (14.56%). on the contrary, low concentrations in leave (7.80%) has been found. Similarly, the main constituents are Phenolic compounds (PC) in leave (10.25%), however, little concentrations in fruit (5.18%), stem (1.03%) and seed (4.56%) have been examined. Table 1 shows that there are several similarities between the compositions of the fruits, stems, seeds and leaves of C. dichotoma. The results revealed that a-pinene, Nonanal, 2,3-Octanedione, Safranal, 2-Undecanol, Nonanoic acid, 2,4-di-t-Butylphenol, Dihydroactindiolide, Methyl jasmonate, Heptadecane, Germacrene D and Phytol are identical components of fruit, stem, seed and leaf essential oils.

Previous investigations of C. myxa leaf EsO revealed that a total of forty compounds were present. Phytol (19.2%) and linalool (8.4%) were found to be the major components in the left part.^[24] A comparison of the data obtained from C. dichotoma (Table 1) with those formerly reported for C. myxa demonstrated that nonanal, linalool, safranal, β-damascenone, trans-geranyl acetone, hexahydro farnesyl acetone, farnesyl acetone and phytol are common EsO constituents.^[24] In another study, the EsO of Cordia curassavica leaf shows only α-humulene through hydrodistillation^[25] which is not found in current study, while in 2020 Cordia africana Lam. specie shows the four different EsO compounds namely β-caryophyllene, germacrene D, δ-cadinene and phytol. These all merely were studies in leaf part of Cordia Africana, while in current study germacrene D and phytol were analyzed in all parts (leaves, Seed, flower and stem) of C. dichotoma plant.^[26] In another study, various EsO compounds were identified namely: α-pinene, sabinene, β-caryophyllene, ar-curcumene, β-sesquiphellandrene, 4-cyclodecen-1-one, ar-turmerone in leaves of Cordia curassavica by hydrodistillation method. While in stem and leaves of Cordia curassavica, α-humulene and β-caryophyllene EsO compounds were obsereved.^[27]

Antibacterial activity of essential oils (EsO)

Table 2 shows that C. dichotoma fruit, stem, seed and leaf EsO had patchy antimicrobial effects on two strains of bacteria, namely, E. coli (gram-negative) and S. aureus (gram-positive). In addition to E. coli, the EsO fruit exhibited a maximum zone of inhibition of 12, 9, 6, 4 and 2 mm at concentrations of 1000, 750, 500, 250 and 125 μg/mL, respectively, while the EsO fruit exhibited 10, 8, 5 and 2 mm zones at concentrations of 1000, 750, 500 and 250 μg/mL, respectively; however, there was no zone at 125 μg/mL. Similarly, stem EsO had a maximum inhibitory effect on E. coli strains of 9, 4, and 3 mm in diameter at concentrations of 1000, 750 and 500 μg/mL, respectively, but had no effect on any of the other zones at 250 and 125 μg/mL, while next to S. aureus, stem EsO had an inhibitory effect on 6, 4, and 2 mm diameter groups at concentrations of 1000, 750, and 500 μg/mL, respectively; however, no zone was formed at 250 and 125 μg/mL. Furthermore, seed essential oils against E. coli exhibited 13, 11, 8, 5 and 3 mm zone diameters at 1000, 750, 500, 250 and 125 μg/mL, respectively, while against S. aureus, seed EsO showed zones of inhibition of 9, 6 and 3 mm, respectively, at 1000, 750 and 500 μg/mL, but no inhibition zones were observed at 250 and 125 μg/mL. Similarly, in addition to E. coli, the leaf EsO had zone diameters of 10, 7, 4 and 2 mm at 1000, 750, 500 and 250 μg/mL, respectively, but no zone was observed at 125 μg/mL. Hence, leaves of S. aureus with EsO had zone diameters of 10, 7, 3 and 1 mm at 1000, 750, 500 and 250 μg/mL, respectively; however, no zone was detected at 125 μg/mL.

Table 2. Antimicrobial activity of C. dichotoma fruits, stems, seeds and leaves.

Concentrations (μg/mL)	Zone of inhibition diameter in mm ± SD
	E. coli				S. aureus
	Fruit	Stem	Seed	Leave	Fruit	Stem	Seed	Leave
1000	12 ± 0.04	9 ± 0.03	13 ± 0.04	10 ± 0.03	10 ± 0.03	6 ± 0.02	9 ±0.04	10 ± 0.04
750	9 ± 0.02	4 ± 0.02	11 ± 0.04	7 ± 0.02	8 ± 0.03	3 ± 0.00	6 ±0.02	7 ± 0.02
500	6 ± 0.02	3 ± 0.01	8 ± 0.03	4 ± 0.01	5 ±0.02	2 ± 0.00	3 ±0.01	3 ± 0.01
250	4 ± 0.01	-	5 ± 0.02	2 ± 0.01	2 ± 0.01	-	-	1 ± 0.00
125	2 ± 0.00	-	3 ± 0.01	-	-	-	-	-
MIC (μg/mL) MBC (μg/mL)	125 (1000)	500 (1000)	125 (1000)	250 (1000)	250 (1000)	500 (1000)	500 (1000)	250 (1000)

The minimum inhibitory concentration is abbreviated as the MIC, known as the lowest concentration of oil; this concentration is used to control bacterial expansion.^[28,29] The in vitro data in Table 2 indicate that, compared with S. aureus, EsO was very efficient next to E. coli (gram-negative), with an MIC of 125 μg/mL (gram-positive), with an MIC of 250 μg/mL. Likewise, EsO from seeds was also observed to be massively potent against E. coli, with an MIC of 125 μg/mL, compared to S. aureus, which has an MIC of 500 μg/mL. Similarly, stem EsO was perceived to be slightly less effective against E. coli (MIC of 500 μg/mL) and S. aureus (MIC of 500 μg/mL). Hence, leaf EsO was found to be slightly more effective against E. coli (MIC of 250 μg/mL) and S. aureus (MIC of 250 μg/mL) than was stem EsO but less effective against fruit and seed EsO. The control, DMSO, exhibited no antibacterial effects on either E. coli or S. aureus. Thus, C. dichotoma fruit, stem seed and leaf essential oils were shown to have better bactericidal effects against E. coli and S. aureus. After the MIC was calculated, the minimum bactericidal concentration (MBC) was also measured at 1000 μg/mL against both bacteria, which showed that the MBC for each bacterium was identical to the variation in MIC.

Conclusion

This study reports essential oil results of the fruit, stem, seed and leaf of the C. dichotoma plant. Overall, the results showed that the total amount of compounds in fruit was higher than in stem, seed, and leaf. Further examination of the fruit and seed revealed that they were primarily made of phenolic (14.56%) and ketonic (20.42%) compounds, respectively. Additionally, current research indicates that the use of C. dichotoma as a source of the antibacterial agent, which may be the presence of EsO is crucial. Additionally, research should be done on both the isolation of compounds and their in vivo examinations.

Acknowledgments

The authors would like to extend their sincere appreciation to the Researchers Supporting Project Number (RSP2024R301), King Saud University, Riyadh, Saudi Arabia. The complete address of Mohsin Kazi’s affiliation is as follows: Department of Pharmaceutics, College of Pharmacy, PO Box 2457, King Saud University, Riyadh 11451, Kingdom of Saudi Arabia.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Correction Statement

This article has been republished with minor changes. These changes do not impact the academic content of the article.