CHEMICAL COMPOSITION AND ANTIBACTERIAL ACTIVITY OF MEDICINALLY USEFUL ESSENTIAL OIL FROM THE INFLORESCENCE OF EUPHORBIA HELIOSCOPIA L. GROWN IN EGYPT

CHEMICAL COMPOSITION AND ANTIBACTERIAL ACTIVITY OF MEDICINALLY USEFUL ESSENTIAL OIL FROM THE INFLORESCENCE OF EUPHORBIA HELIOSCOPIA L. GROWN IN EGYPT

Amira Mohammed Beltagy

Lecturer of Pharmacognosy and Phytochemistry, Department of Pharmacognosy, Faculty of Pharmacy, Damanhour University, Damanhour, Egypt.

ABSTRACT: The genus Euphorbia has about 2000 members making it one of the largest ones among the flowering plants. Euphorbia helioscopia L. As the chemical composition of the essential oil from Euphorbia helioscopia developed in Egypt has not yet been explored, along these lines the present to extricate the essential oil and investigate the oil chemical composition and antimicrobial activity. Essential oil from the whole inflorescence of the flowering plant, Euphorbia helioscopia Linn. was extracted by hydrodistillation method. The chemical composition of the obtained volatile oil was investigated by GC-MS. The major components include thymol (48.36%), Caryophyllene (23.57%), carvacrol (6.70%), caffeic acid (6.48%) and caryophyllene oxide (5.58). Minor components as translongipinocarveol (2.77%) and DL-malic acid (1.9%). Screening antimicrobial activity of the isolated volatile oil against four bacteria showed that it was active only against Bacillus subtilis with an inhibition zone of 11 mm ± 0.25 and MIC value 17 mg /ml. Gentamycin was utilized as a positive antibacterial control. The data of this study might be considered as the first identification of the chemical composition of the essential oil of the plant species grown in Egypt. Essential oils may have a moderate potential as a natural medicine for microbial infections against Bacillus subtilis.

Euphorbia helioscopia, Essential oil, GC/MS, Antibacterial activity

INTRODUCTION: Euphorbiaceae is a vast plant family with 300 genera and 5000 species. The genus Euphorbia is the biggest genus in F. Euphorbiaceae. The genus has around 2,000 members making it one of the biggest genera of flowering plants. All govern latex and have unique flower structures. The plant is local to calm districts however has likewise adjusted to sub-tropical regions everywhere throughout the world.

In Egypt, the plant is basic in the Nile Delta and Upper Egypt ¹. The plant, Euphorbia helioscopia Linn. subsp. helioscopia, is known as sun spurge or wolf’s milk as an English name.  The plant has great therapeutic significance, often used to treat ascites, edema, pulmonary tuberculosis, tinea and cervical tuberculous lymphaden ². Leaves and stems are used as febrifuge and vermifuge. The oil from the seeds has purgative properties while the roots are used as anthelmintic ³.

Uzair M et al., (2009) reported that on mixing the seeds with roasted pepper, the mixture could be used in the treatment of cholera ⁴. The plant contains diterpenes, flavonoids, tannins, polyphenols, glycosides, lipids and volatile oils ³. Ethanolic extract of E. helioscopia amongst medicinal plants, collected from Islamabad and the Murree region of Pakistan was studied for insulin secretagogue activity and was found to be active at 10 μg/mL (p<0.05) ³. Other plant biological activities as vasodepressor ⁵, anti-allergic ⁶ and were reported. Also, the plant extract was found to have considerable antiviral activity ⁷.

Three extracts of E. helioscopia namely petroleum ether, dichloromethane, and methanol, were tested for their antifungal activity against Cladosporium cucumerinum and found to be devoid of antifungal activity ⁸. Dichloromethane and methanol extracts of the aerial parts of E. helioscopia were also tested against Trichophyton longifusus, Candida albicans, Aspergillus flavus, Microsporum canis, Fusarium solani, and Candida glabrata. Among them, dichloromethane extract showed 90% Inhibition against Fusarium solani. Antifungal activity of the dichloromethane extract of the aerial parts of the plant was also confirmed by Uzair ⁴.

Different plant extracts of the Iranian species was active against Bacillus anthracis and inactive against Klebsiella pneumoniae, Proteus vulgaris, Shigella sonei, Vilorio cholera, Escherichia coli, Staphylococcus aureus and Salmonella paratyphi A.

Assessment of the growth inhibitory effects of Euphorbia helioscopia L. extracts and fractions of the alcoholic extract on different five human cancer cell lines were performed and found to possess anticancer activity and could be a chemopreventive agent against human cancer ⁹. Allelopathic impact of the water extracts of various plant organs and infested soil of E. helioscopia L. on the seed germination and seedling development of wheat and lentil was reported ¹⁰.

A study on E. helioscopia species grown in Saudi Arabia amongst other local plants investigated the plant nutritional value and explored the lipoidal plant profile. While the main components of essential oil of Saudi Arabia species were reported as elemol and β-eudesmol ¹¹.

Analysis of volatile steam oil obtained from the inflorescence of E. helioscopia grown in Greece resulted in the identification and quantification of 40 constituents (94.3%). The major compounds were phytol (21.2%), trans-Caryophyllene (10.0%) and docosanoic acid methyl ester (8.1%) ¹². The chemical composition of the essential oil of the whole flowering herb of Euphorbia helioscopia grown in Egypt has not been identified yet. Thus this work aimed at identifying the plant essential oil composition and at investigating the oil antimicrobial activity.

MATERIALS AND METHODS:

Plant Material: The whole flowering and fruiting plant of Euphorbia helioscopia Linn. was collected from its natural habitats from wheat farms in El- Gharbia governorate in Egypt in April 2017. The plant was identified by Prof. Dr. Kamal Shaltout, Department of Botany, Faculty of Science, Tanta University, Tanta, Egypt. A voucher specimen was deposited (with accession no. EH 202) in Herbarium of Faculty of Science, Tanta University, Tanta, Egypt.

Essential oil Extraction: The aerial parts of the plant were air dried with high care to keep the seeds. The air-dried powder (200 gm) whole inflorescence was communicated and at once subjected to hydrodistillation using Clevenger -type distillation apparatus for 3 h. The resulted extracted oil was then protected from light and stored at - 4 ºC till GC/MS analysis.

Antimicrobial Activity: Antimicrobial activity assay using a cut plug method as described previously ¹³. Two Gram-negative bacteria (Pseudomonas aeruginosa ATCC 11921 and Salmonella typhimurium isolates), two Gram-positive bacteria Staphylococcus aureus ATCC 6538) and Bacillus subtilis ATCC were selected for antibacterial activity assay. Gentamycin was used as a positive antibacterial control.

Cut plug method recorded by Pridham et al., ¹⁴ was employed to determine the antimicrobial activity of the essential oil. Three replicates were made for each test, and all plates were incubated at 32 °C for 24 h. Then the average diameters of the inhibition zones were recorded in millimeters (mm) and compared for all plates.

MIC Determination of the Essential Oil against the Most Susceptible Microorganism: Half-fold serial dilutions were made for an obtained essential oil to prepare concentrations of 6.25, 12.5, 25, 50 and 100 mg/mL in distilled water. Distilled water was considered as a negative control. A previously prepared (0.5 mL of about 106 cells/mL) pure spore suspension of the most susceptible microorganism was mixed with 9.5 mL of each concentration in sterile test tubes, incubated at 32 °C for 24 h; then the optical density of growth was measured spectrophotometry at 620 nm for each incubated mixture. Quantification of the growth-inhibiting effect was made by determining the percentage of the surviving cells that represented percent optical density ¹⁵.

Antibacterial activity was repeated three times. Inhibition zone was expressed as mean ± standard deviation (SD). MIC against the susceptible organism was determined.

RESULTS: The yield of the essential oil obtained was 0.18v/w. The composition of the essential oil was identified and quantified by GC/MS.  Eighteen constituents from E. helioscopia were identified and quantified. These compounds and their corresponding mass spectra, as well as their retention times, are given in Table 1. The major component is the monohydric phenol, thymol with concentration 48.36% followed by the sesquiterpene, Carophyllene with a concentration of 23.57%. Other major constituents included carvacrol (6.70%) and caffeic acid (6.48%). In general, the volatile oil composition can be classified as phenols (thymol and carvacrol) with total percentage composition of 55.06%. Sesquiterpenes represented by caryophyllene was identified in a concentration of 23.57%. Oxygenated sesquiterpenes as caryophyllene oxide, isoaromadendrene epoxide, and translongipino carveol, were represented in the volatile oil by a total concentration of 9.03%. Organic acids (caffeic acid and DL-malic acid) add up to 8.39% to the total constituents.

Alcohols (linalool and endoborneol) represented only 0.82 % of the total oil composition. The remaining detected compounds are fatty acids namely transglutaconic acid, palmitic acid and oleic acid with a total percentage of 0.84%. Methylated Fatty acids namely 2-pentadecane 6, 10, 14-trimethyl and methyl-linolenate were represented by 0.30 percent concentration. The biological amine; 2-adamantanamine is represented by a percent concentration of 1.08.

TABLE 1: GC/ MS RESULTS OF VOLATILE OIL OF EUPHORBIA HELIOSCOPIA L.

S. no.	Retention time (min)	Percent of total	Compound  & Molecular Formula	Structure & Mass Spectrum
1	3.159	0.1	3-Heptanol, 5-methyl- C8H18O
2	3.709	0.07	2-Mercaptoethanol HOCH2CH2SH
3	4.319	0.18	1,6-Octadien-3-ol, 3,7-dimethyl- C10 H18 O ( Linalol)
4	5.670	0.64	Endoborneol C10 H18 O
5	8.846	6.70	Phenol, 2-methyl-5-(1-methylethyl)- | C10H14O Carvacrol
6	9.917	48.36	Thymol C10H14O
7	11.802	1.08	2-Adamantylamine C10H17N
8	11.827	6.476	Caffeic Acid C9H8O4
9	12.463	1.912	DL-Malic acid C4H6O5
10	12.768	23.57	Caryophyllene C15H24
11	17.625	5.583	Caryophyllene oxide C15H24O
12	19.415	0.675	Isoaromadendrene epoxide   C15H24O
13	19.801	2.766	Longipinocarveol, trans- C15H24O
14	22.517	0.291	trans-Glutaconic acid C5H6O4
15	23.602	0.182	2-Pentadecanone, 6,10,14-trimethyl- C18H36O
16	26.588	0.398	n-Hexadecanoic acid C16H32O2 (palmitic acid)
17	29.084	0.119	Methyl linolenate   C19H32O2
18	30.015	0.148	Oleic Acid C18H34O2

TABLE 2: MIC OF VOLATILE OIL ISOLATED FROM E. HELIOSCOPIA AGAINST THE SENSITIVE MICROORGANISM BACILLUS SUBTILIS

Essential oil isolated from E. helioscopia showed antibacterial activity against the Gram-positive bacteria, Bacillus subtilis with MIC value 17 mg/ ml. Inhibition zone of the volatile oil was 11 mm ± 25 while as inhibition zone of gentamycin against Bacillus subtilis was 10 mm. The other tested microbes were resistant against this volatile oil.

FIG. 1: GROWTH INHIBITION OF THE ESSENTIAL OF EUPHORBIA HELIOSCOPIA L. AGAINST BACILLUS SUBTILIS

DISCUSSION: Essential oil composition of Euphorbia helioscopia herb changes with various geographical sources. Looking at the oil composition collected from the Nile Delta of Egypt with those from Saudi Arabia and Iran brought about distinguishing proof of various constituents. This study recognized the significant constituents of the oil extricated from Egyptian plant as thymol and caryophyllene. While as the major compounds essential oil of Saudi Arabia species was represented as elemol and β-eudesmol ¹¹. Major oil constituents of the plant gathered from Greece were phytol, trans-Caryophyllene and docosanoic corrosive methyl ester ¹².

Carvacrol that constitutes 6.70% of the essential oil was reported to possess antioxidant activity ¹⁶. Caffeic acid is an organic compound found in the plant as a key intermediate in the biosynthesis of lignin which composes biomass plant component. The other organic bicarboxylic acid in the oil, malic acid is used as a food additive.

The antibacterial activity of the essential oil against Bacillus subtilis may be related to its composition of phenols especially thymol that comprises 48.36%. This study can presume that some obtrusive field weeds, for example, Euphorbia Helioscopia that is normally developed in Egypt on wheat and pea might be advantageous plants to be proposed for culture as a therapeutic plant.

CONCLUSION: The present work was designed to identify the chemical composition of the essential oil from the inflorescence of the plant Euphorbia helioscopia grown in Egypt. Also, this work was aimed at testing for possible antimicrobial activity of the isolated oil. GC–MS analysis of the isolated oil resulted in the identification of thymol and caryophyllene as the major components in the oil. The oil showed antimicrobial activity against Bacillus subtilis. This may be due to its high percentage of phenolic content. Further investigation of the plant is recommended to clarify its phytochemical constituents and also for possible pharmacological effects as the Egyptian plant is rarely worked on.

ACKNOWLEDGEMENT: The author is thankful to Prof. Dr. Kamal Shaltout, Department of Botany, Faculty of Science, Tanta University, Tanta, Egypt for authentication of the plant. The author is also thankful for the members of “The Central Lab. of Tanta University”, Tanta, Egypt for providing the necessary facilities to carry out the study.GC/MS analysis.

CONFLICT OF INTEREST: The author declares no conflict of interest.

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    How to cite this article:

    Beltagy AM: Chemical composition and antibacterial activity of medicinally useful essential oil from the inflourescence of Euphorbia helioscopia L. grown in Egypt. Int J Pharm Sci & Res 2019; 10(8): 3660-67. doi: 10.13040/IJPSR.0975-8232.10(8).3660-67.

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