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Chemical Compositions and Antioxidant Activity of Volatile Oils from Morinda citrifolia and Beta vulgaris Leaves from Nigeria

Morinda citrifolia L. and Beta Vulgaris L leaves are both ethnomedicinal use for the treatment of arthritis, indigestion and skin infections with no reports on their essential oils compositions. The colourless volatile oils with a percentage yield of 0.6 and 0.4 (w/w) for Morinda citrifolia L. and Beta Vulgaris L respectively were obtained. Forty-five compounds representing 94.31 % of the total percentage compositions were identified in the leaf essential oil of M. citrifolia with the most abundant compound as 14-beta-H-pregna- (33.13%). Forty-eight compounds representing 74.18% of the total oil composition were identified in the leaf oil of B. Vulgaris with phytol (24.20%) as the dominant compound. The essential oils showed good free radical scavenging activity when compared to ascorbic acid used as control, with % inhibition varying from 88.74 ± 0.010 to 96.61 ± 0.004 as compared to 95.68 ± 0.010 to 97.31±0.003 of the ascorbic acid at (100 to 6.25 mg/ml) concentrations. The leaves essential oils of Morinda citrifolia L. and Beta Vulgaris L contains chemical compounds that might be responsible for their antioxidant activity. This result


INTRODUCTION
The Morinda citrifolia L (figure 1), also known as Noni in Nigeria, belongs to the Rubiaceae family (Arunachalam, 2018). It is a plant native to Southeast Asia and Australia that has long been used medicinally. According to reports, M. citrifolia can treat a wide range of medical conditions, including arthritis, heartburn, headaches, wounds, and skin infections. Additionally, there are some reports on the antitumor and anticancer activity of this plant and biological activities such as antihypoglycemia have been reported (Algenstaedt et al., 2018).
Beta Vulgaris L. (figure 1), popularly known as beetroot, is a member of the Chenopodiaceae family (Mello et al., 2008). It is a biennial herbaceous plant. It is eaten in Nigeria as a vegetable. It has traditionally been believed to treat or prevent conditions like arthritis, colon, prostate, dyspepsia, and skin infections. Beta Vulgaris leaf consumption lowers the risk of diabetes mellitus, obesity, and cardiovascular illnesses. It is also recognized for its potential to treat cancer (Kumar et al., 2016). It is known to have a wide range of biological activities, including properties that are antibacterial, antioxidant, anticancer, antiviral, and anti-diabetic (Kavitha et al., 2020). Consequently, in continuation of our search for biologically active compounds from plants with ethnomedicinal uses (Okpala et al., 2021;Okpala et al., 2022;Onanuga and Oloyede, 2021), we present chemical compositions of the volatile oils and antioxidant activities of leaves of Beta Vulgaris L. and Morinda citrifolia L. The chemical compositions of the essential oils (EOs) of the leaves of both plants have not been reported in the literature to the best of our knowledge.

Beta Vulgaris Leaves
Morinda citrifolia Leaves

Plant Material
The

Extraction of the Essential Oil
The air dried and pulverized leaves of M. citrifolia (200 g) and B. Vulgaris (200 g) were weighed and separately subjected to extraction using hydro-distillation method with Clevenger type apparatus for four hours following British Pharmacopoeia specifications with modifications (British Pharmacpoeiae, 1980). The samples were added into a 2 L round-bottomed flask containing 1.0 L distilled water and heated to boiling. There was the evaporation of the essential oils together with water vapour and these were collected in a condenser. The upper phase that contained the EOs was separated from the lower one and anhydrous sodium sulphate was used for drying the oils isolated. Oils extracted were preserved in a sealed amber glass vial at 4 o C until analyses. The percentage yields (w/w) were determined.

Gas Chromatograph-Mass Spectrometry (GC-MS) of the Oils
GC-MS analysis of the oils was performed using Gas chromatography 7890 coupled with mass spectrometer 5975 Agilent technology. The chemical components were identified by matching their mass spectra with those recorded in the mass spectra library (W11N17 main). The stationary phase was the column of model Agilent technologies HP-5 MS of length 30 m, the internal diameter of 0.320 mm and thickness of 0.25 µm while the mobile phase was helium gas. The oven temperature was at 80 o C held for 2 mins at 12 degrees per minute to the final temperature of 240 o C held for 6 mins. The ion source was set at 240 o C and electron ionization at 70 eV. The scan ranges were from 50 to 550 amu and the interface temperature between GC and MS was 250 o C. A sample of (1.0 µL) of diluted oils in hexane was manually injected into the GC-MS.

Antioxidant Assay
The antioxidant activity of the essential oils was evaluated using the DPPH (2, 2-diphenyl-1picrylhydrazyl) free radical scavenging ability method. The concentrations (100 mg/mL, 50 mg/mL, 25 mg/mL, 12.5 mg/mL and 6.25 µg/mL) of the essential oils were mixed with 100 µM methanol-DPPH solution (2.0 mL) prepared by dissolving 3.94 mg of DPPH in 100 ml of methanol to give a purple solution. The mixture was shaken vigorously and left to incubate for 30 minutes in the dark at room temperature and the absorbance was then measured at 517 nm and recorded as A (sample) using a GS UV-12, UV-VIS Spectrophotometer. In its radical form, DPPH absorbs, but upon reduction by antioxidant species, its absorption reduces. A blank experiment was carried out applying the same procedure without essential oil (DPPH + Methanol) and the absorbance was recorded as A (control). Ascorbic acid was used as a standard antioxidant for comparison. The free radical scavenging activities of each essential oil were then calculated as percentage inhibition according to the following equation:
Some similarities of the ethnomedicinal uses of the two plants could be related to the presence of compounds such as phytol, neophytadiene, mesitylene, 3-hexanol, decane, Docosane, tetracosane, eicosane and heneicosane which were identified in the essential oils of both plants. The GC-MS chromatograms of the essential oils are given in Figures 2 and 3. The antioxidant activity of the essential oils of the leaves of M. citrifolia and B. Vulgaris are presented in Tables 4 and 5. The DPPH scavenging ability of the essential oils was compared with ascorbic acid a known standard antioxidant.      (Pino et al., 2010). 14-b-H-Pregna, a steroid considered to be a sex pheromone specific to males, and also a defensive chemical with diabetic retinopathy prevention and treatment effects. The presence of 14-b-H-pregna has been reported in the essential oils from different parts of some plants, including Scutellaria plants, Urginea indica Kunth, Allium rotundum, Gundelia tournefortii L (Farhang et al., 2016). Citrus limon (Akhila et al., 2015). Dehpour et al.2012 reported that 14-b-H-pregna was the major compound in the essential oil of lower Allium rotundum which displayed antibacterial activity. Neophytadiene is a good analgesic, antimicrobial, antipyretic, antioxidant and anti-inflammatory compound (Venkata et al., 2012). Phytol, known to exhibit antioxidant and antinociceptive effects is a precursor of synthetic vitamins E and K and is cytotoxic against breast cancer cell lines (MCF7) (Casuga et al., 2016;Sermakkani and Thangapandian, 2012).
The measured absorbance values and calculated percentage inhibition show that the antioxidant activity of the two essential oils and standard (Ascorbic acid) is concentration dependant. From the results, the % inhibition of the essential oils of both plants exhibited good scavenging ability on DPPH radical which were comparable to ascorbic acid at all concentrations (100-6.25 mg/ml) investigated.

CONCLUSIONS
Analysis of the essential oils of M. citrifolia and B. Vulgaris leaves showed that they contained different major constituents. The major constituent of M. citrifolia leaves essential oil were steroids: 14-beta-H-pregna-(33.13%) and 1-Hexacosane (11.11%), while the dominant compounds identified in B. Vulgaris were phytol (24.20 %) and 1,3-dimethylbenzene (14.84%. These major constituents of both oils have been reported to possess similar properties such as antibacterial, antioxidant and antinociceptive. Most of the chemical compounds identified from the essential oils of M. citrifolia and B. Vulgaris were biologically active compounds and the essential oils exhibited good scavenging ability at all concentrations investigated. The plants' parts could be a source of drug development for oxidative diseases.