INTRODUCTION:

In general, plants cover a large variety of medicinally desirable biological activities in both conventional and modern treatments; these natural compounds are commonly used to improve the health of people with relatively few or no side effects^1-3. Fruits are among the main plant parts that have a natural dietetic significance, they are also high in nutrients and contain high-quality minerals, making them an excellent source of minerals, digestive fibers, vital amino acids, pectic materials, fatty acids, vitamins, antioxidant agents (polyphenols, sulfur compounds, Phytosterol), and other nutrients for a well-balanced diet⁴. A variety of natural and synthetic products containing herbal materials are employed for biomedical applications. These applications include antioxidants, cellular, and antimicrobials activities^5-7.

There's a great deal of evidence that reactive oxygen species (ROS) are blamed to be involved in a variety of inflammatory and other diseases, and that consuming a diet rich in antioxidants will improve heart health and avoid illness⁸. C. myxa is a type of flowering plant that belongs to the Boraginaceae family; it is known to be grown naturally in the sub-tropics in addition to many regions in the tropics of Africa, Australia, and Asia including Iraq^9,10.Several chemical compounds like alkaloids, flavonoids, terpenoids, triterpenoids, and phenols were separated and characterized from Boraginaceous plants¹¹. The tree is known in Iraq as "Bumber", (figure 1), is popularly used for respiratory tract infection as an expectorant and demulcent, in addition to its use as diuretic, and anti-diarrheal agent. Biological investigations of various extracts and compounds were isolated from the genus Cordia. Several studies have demonstrated that the C. myxa fruit is effective as an analgesic, anti-inflammatory, and in the treatment of various viral infections including COVID-19 management^12-14. As a result, the goal of this study was to use GC-MC and HPLC analysis to determine the appearance of bioactive components in an active ethanol extract of C.myxa fruit, as well as the relative antimicrobial properties of the fruit extract.

Figure 1: Illustrates a branch of the C. myxa tree (a) and the fresh fruits (b) used in the current study.

MATERIALS AND METHODS:

Plant fruits collection:

Fresh fruits of C. myxa were obtained from trees grown at farmlands in Agricultural Research Central, Baghdad, Iraq, during the period from July and August 2020. The harvested plant material was packaged in a polyethylene bag to avoid moisture loss during transport to Al-Nahrain University's Research Center laboratory, Baghdad.

Preparation of extract:

The procedure of C. myxa fruit extract was done according to a previous study reported by Ibrahim et al.¹⁵, with some modifications. A soaking technique with petroleum ether was used to defat 1000g of powdered material (40:60). The remaining material was extracted with a 70% ethanol solution. After concentration with a rotary evaporator at 42°C, the extract was lyophilized. The residual material was preserved under −20°C until use.

HPLC analysis:

Phytochemicals were measured and identified using a high-performance liquid chromatographic technique, as described by Andlauer et al.¹⁶ with minor modifications. For this purpose, the crude extract was dissolved at a concentration of 28mg/mL in 0.5mL of 100% ethanol. HPLC instrument from (Waters Corporation, Milford, USA) was utilized for the separation. The procedure was done by utilizing a Varian Star HPLC solvent supply and a control system that included an automatic sample injector and an adjustable Varian UV-vis detector. A C18 column (Sigma Aldrich, USA) with a diameter of 250mm × 4.6mm; 5µm was used for chromatographic separation. The auto sampler injected twenty microliters of samples into the column perfectly.

Estimation of total phenolic content:

The total phenolics of the plant extracts were determined according to the method described by Ainsworth et al.¹⁷. The obtained data were presented as gallic acid equivalents (GAE).

Estimation of Tannins:

The quantity of tannins was estimated by subtracting the non-tannin phenolics from total phenolics¹⁸. The analyses were done in triplicates and the obtained data were presented as tannic acid equivalents (TAE).

Estimation of flavonoids:

The flavonoid moiety of the extracts was estimated quantitatively based on the method specified by Murthy et al.¹⁹. Rutin was adopted as the standard for such quantitative estimation of flavonoids. The experimental procedure was repeated in triplicate and the obtained data were presented as rutin equivalents (RE).

GC-MS analysis:

Ethanol fruit extract of C. myxa was subjected to analysis using GC-MS (Shimadzu Co., Japan). The analysis was done by injection of one microliter of the extract at a 20:1 split ratio. Helium gas (99.9 percent) was utilized at the rate of one milliliter/minute in a gaseous state. The research was conducted with 70 eV of ionization energy in the EI (electron impact) mode. The temperature of the injector was held at 250°C (constant). The temperature of the column oven was set at 50°C (held for 3 mins), increased to 280°C (held for 3 mins) at 10°C per min, and lastly held for 10mins at 300°C. After comparing the spectral configurations acquired with the available mass spectral database, the compounds were established (NIST and WILEY libraries (multiplier voltage of 1kV.

Antimicrobial Assays:

The antimicrobial effect of ethanol extract was investigated using S. aureus (ATCC 29213), E. coli (ATCC 35218), S. enterica (ATCC 13076), B. subtilis (ATCC 6633), P. aeruginosa (ATCC 27853), A. brasiliensis (ATCC 16404), and S. cerevisiae (ATCC 9763). For this purpose, a defined amount (1mL) of a standard stock suspension of microorganisms containing10⁶ CFU/mL was diluted with 100mL of Mueller–Hinton agar medium and kept at 45°C. The Mueller–Hinton agar media was divided into aliquots (20mL) and placed in previously sanitized plates agar medium and permitted to set at room temperature and then 4 chambers of 10mm diameter were created in each of these plates using a sterilized cork borer (No. 4). Different volumes of ethanol extract (25g/mL, 50 mg/mL, and 100mg/mL) according to previous published work were put into the holes inside the agar discs and left untouched at room temperature for 2 hours to diffuse. Then, the plates were incubated at 37°C overnight. After that, the plates were checked for signs of bacterial growth, and the diameter of the inhibition zone was measured.

Statistical Analysis:

Statistical package for social scientists (SPSS) was utilized to analyze data. The mean± standard deviation of triplicate measurements, a one-way variance analysis (ANOVA) was used to conduct data analysis and the significant difference was set at p≤0.05.

RESULTS AND DISCUSSIONS:

GC-MC results:

The relative concentration of the exhibited components was estimated according to the height of the peaks. The mass spectrometer examines the compounds eluted at various periods to determine their nature and structure (Figure 1.), peaks correlating to bioactive compounds were identified by comparing their peak retention time, peak area %, and mass spectral fragmentation patterns to those of known compounds explained in the National Institute of Standards and Technology (NIST) library. GCMSS revealed the presence of mainly 19 phytochemical compounds in the ethanol extract of C. myxa fruit, (Table 1), these compounds that were identified to be present in large amounts include; Vitamin E (22.511%), 2-Furancarboxaldehyde, 5-(hydroxymethyl) (22.123%), seven compounds of esters (21.597%), Ethyl iso-allocholate (8.883%), Ethyl Oleate(7.512%), Alpha-l-rhamnopyranose (4.800%), α-D-Glucopyranoside, O-α-D-glucopyranosyl-(1.fwdarw.3)-β-D-fructofuranosyl (4.421%), 3-O-Methyl-d-glucose(3.412%),4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl (2.477%), n-Hexadecanoic acid (1.359%), Cyclotetradecane (0.187%), and Sucrose (0.717%). According to the literature, there is an antimicrobial activity that has been associated with α-D-Glucopyranoside, O-α-D-glucopyranosyl-(1.fwdarw.3)-β-D-fructofuranosyl²⁰. Olaniyan et al. investigated the beneficial effects of the flavonoid content in methanol extract of Plukenetia conophora seeds (4H-Pyran-4-One 2,3-Dihydro-3,5-Dihydroxy-6-Methyl) on male Wistar rats' reproductive function²¹, and, Teoh et al. reported the antifungal activities for this compound²². Other compounds, such as methyl esters, Vitamin E, Ethyl Oleate have been discovered to have interesting biological activity against various infections. For instance, the antioxidant, antimicrobial, and other biomedical applications^23-26.

Figure 2: GC-MS chromatogram of ethanolic extract of C.myxa fruits.

Table 1: Identity description and concentration of components GC-MS identified by GC-MS profile

P. No	RT (min)	Ident.	Formula	Prob%	CAS	% of total
1	7.901	α-D-Glucopyranoside, O-α-D-glucopyranosyl-(1.fwdarw.3)-β-D-fructofuranosyl	C18H32O16	55.30%	597-12-6	4.421
2	9.277	4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl	C6H8O4	74.00%	28564-83-2	1.448
3	12.765	Hexadecanoic acid, methyl ester	C17H34O2	58.90%	112-39-0	6.062
4	14.300	Vitamin E	C29H50O2	55.00%	59-02-9	22.511
5	15.990	9,12-Octadecadienoic acid, ethyl ester	C20H36O2	24.20%	7619-08-01	1.123
6	18.040	Dasycarpidan-1-methanol, acetate (ester)	C20H26N2O2	14.00%	55724-48-6	7.902
7	18.537	Ethyl iso-allocholate	C26H44O5	17.90%	24560-98-3	8.883
8	21.223	2-Furancarboxaldehyde, 5-(hydroxymethyl)	C6H6O3	91.70%	67-47-0	22.123
9	22.817	4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl	C6H8O4	83.10%	28564-83-2	1.029
10	23.348	Alpha-l-rhamnopyranose	C6H12O5	9.44%	35810-56-1	4.800
11	23.557	Ethyl Oleate	C20H38O2	41.50%	111-62-6	7.513
12	24.625	Hexadecanoic acid, ethyl ester	C18H36O2	90.50%	628-97-7	0.928
13	24.676	n-Hexadecanoic acid	C16H32O2	56.60%	57-10-3	1.359
14	25.275	3-O-Methyl-d-glucose	C7H14O6	34.00%	5296-62-8	3.412
15	25.323	9,12-Octadecadienoic acid (Z, Z)-, methyl ester	C19H34O2	11.60%	112-63-0	3.239
16	26.762	Sucrose	C12H22O11	42.10%	57-50-1	0.717
17	27.213	Cyclotetradecane	C14H28	5.05%	295-17-0	0.187
18	28.655	9-Octadecenoic acid (Z)-, methyl ester	C19H36O2	6.92%	112-62-9	0.785
19	35.607.	Dasycarpidan-1-methanol, acetate (ester)	C20H26N2O2	11.50%	55724-48-6	1.549

Table 2: Peaks of the HPLC test

Peak	Compounds	R. Time	Area	Height	Area%
1	Gallic acid	1.252	3110204	113010	28.434
2	unknown	2.707	3785630	174974	22.939
3	Ferulic acid	3.021	3389248	213302	25.619
4	Chlorogenic	3.243	1358875	135717	22.294
5	Caffeic acid	5.407	1948659	20158	6.960
6	Cummaric acid	6.798	884224	19727	20.737

Figure 3: HPLC chromatogram of C.myxa fruit samples

Table 3: Phytochemical screening of C. myxa fruit extract

Peak	Compounds	R. T	Area	Area%	Formula	Structure
1	Gallic acid Mw=170.12 pharmacological activities: antioxidant, anti-inflammatory	1.252	3110204	28.434	C₇H₆O₅
2	Unknown	2.707	3785630	22.939
3	Fereulic acid Mw=194.18 pharmacological activities: antioxidant,	3.021	3389248	25.619	C₁₀H₁₀O₄
4	Chlorogenic Mw=354.31 pharmacological activities: antidiabetic effect, DNA protective effect	3.243	1358875	22.294	C₁₆H₁₈O₉
5	Caffeic acid Mw=180.16 reduce inflammation, anti-cancer, anti-toxicity, prevent diabetes	5.407	1948659	6.960	C₉H₈O₄
6	Cummaric acid Mw=164.0473 antioxidant, antimicrobial, antitumor, anti-inflammatory	6.798	884224	20.737	C₉H₈O₃

Results of phytochemical screening:

C. myxa fruit extract grown in Iraq contains polyphenols, flavonoids, and tannins. The total phenolic content in fruit extract recorded 113.71 8.40mg gallic acid/g dried extract using the gallic acid calibration curve. The total flavonoids content of the extract was calculated using the quercetin calibration curve and was found to be 68.76 4.18mg quercetin/g dried extract. According to the catechin calibration curve, C. myxa fruit extract contained condensed tannins in the range of 26.64 1.80 mg catechin/g extract, which corresponded to a catechin calibration curve. Table 3 illustrated these results.

Antimicrobial activity:

The antibacterial activity of C.myxa ethanol fruit extract was determined using the disc diffusion method. The plant extract was found to possess selective antimicrobial activity, as it was ineffective at both the tested concentrations (25, 50, and 100mg/mL) against Salmonella enterica, Bacillus subtilis, Pseudomonas aeruginosa, Aspergillus brasiliensis and Saccharomyces cerevisiae, it was found to be selectively effective against the isolates of Streptococcus mutans, which was resistant to the standard drug kanmycin. Zone of inhibition at the higher concentration 500μg/mL was found to be 25±2.1mm which was comparable to the zone of inhibition shown by ampicillin for other bacterial strains. As shown in (Table 4). It is clear from the findings that specimens showed moderate antimicrobial activity against all pathogenic microbes, in a manner dependent on concentration. A better zone of inhibition was recorded for ethanol extract of C. myxa, against S. aureus (17.7mm at 100mg/ml concentration). On the other hand, the growth of E. coli was also negatively inhibited by extract, (14.5mm at 100mg/ml concentration).^30,31Moreover, the other microbial species; Salmonella enterica, Bacillus subtilis, pseudomonas aeruginosa, Aspergillus brasiliensis and saccharomyces cerevisiae exhibited significant inhibition by ethanol extract at 25, 50, and 100mg/ml concentrations with the diameter of inhibited zones as 13.4, 15.9, 14.5, 12.6 and 13.8mm respectively. These results are in agreement with the results of antimicrobial activity of S. calophyllifolium fruit extract which previously reported by Sathyanarayanan et al.¹⁶. In a similar study, the maximum antibacterial activity in an aqueous extract of the C. myxa showed that the concentration of 200mg/ml for Pseudomonas fluorescens recorded 26mm of inhibition zone diameter and for Salmmonila 25mm, for E. coli 24 mm³². These findings are consistent with the outcomes of antimicrobial activities^33-36, and this could be used to continue the hunt for the active substance in the extract of C. myxa fruit which is ineffective against all these strains. The results suggested that fruit extract could be used to reduce microbial infections caused by S. aureus, E. coli, S. enterica, B. subtilis, P. aeruginosa, A. rasiliensis, and S. cerevisiae, which are among the most common causative agents of various infections, due to their bioactive components.

Table 4: Antimicrobial activity of C. myxa fruit crude extract

Zone of Inhibition (diameter in mm)
Microbes	Ethanolic Extract (mg/mL)			Kanamycin (mg/ml)
Microbes	25	50	100	25	50	100
S. aureus	13.5 ± 1.0	15.8 ± 1.5	17.7 ± 1.0	21.3 ± 2.1	26.0 ± 1.5	33.4 ± 2.0
E. coli	12.3 ± 1.5	12.5 ± 1.5	14.8 ± 1.0	18.6 ± 2.5	28.0 ± 3.0	35.3 ± 2.5
S. enterica	11.5 ± 0.5	12.3 ± 1.0	13.1 ± 1.5	16.3 ± 1.5	25.6 ± 1.5	31.0 ± 2.0
B. subtilis	10.7 ± 0.5	14.2 ± 0.1	15.9 ± 1.0	14.0 ± 1.0	29.0 ± 1.0	32.0 ± 7.0
p. aeruginosa	12.0 ± 0.5	13.1± 1.0	13.9 ± 1.6	17.0 ± 1.2	26.0 ± 1.0	28.0 ± 6.0
A. brasiliensis	10.4 ± 1.5	12.2 ± 1.1	13.1± 1.5	14.0 ± 2.7	25.0 ± 1.0	26.0 ± 3.0
S. cerevisiae	10.8 ± 0.1	12.7 ± 1.6	13.6 ± 1.0	13.0 ± 1.3	24.0 ± 1.0	29.0 ± 1.0

CONCLUSION:

The antimicrobial activity of an ethanol extract of C. myxa fruits was confirmed in this investigation. The presence of phytochemicals was also determined using the standard technique, and the chemical content of the ethanol extract was determined by GCMSS and HPLC.

GC-MS was used to identify 19 phytochemicals in the ethanol extract of C. myxa fruits grown in the Baghdad regions of Iraq. The ethanol extract exhibited noticeable antibacterial and antifungal properties and this may be due to the presence of active compounds such as Gallic acid or its analogs. In conclusion, the antimicrobial behaviour and phytochemical analyses of the C. myxa fruits suggest expanding the use of such fruits in the food industry, particularly in new human health-oriented products. So that further studies are needed to separate and identify specific moieties from such fruit extract along with the roots and stem of the plant for anti-microbial and biomedical applications.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

This work is supported by the College of Biotechnology, Al- Nahrain University, Iraq, and Department of Medical Basic Sciences, University of Misan, Iraq.

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Received on 11.10.2021 Modified on 17.02.2022

Research J. Pharm. and Tech. 2022; 15(7):2871-2876.

DOI: 10.52711/0974-360X.2022.00479