Comparative analysis of four different solvent extracts of Annona muricata bark based on bioactive compounds and antibacterial effectiveness

Abstract

Traditional treatment of diseases has a great impact in every era due to the non-synthetic and various beneficial effects of medicinal plants. The miraculous medicinal plant Annona muricata L. (Magnoliales: Annonaceae) contains multiple pharmacologically active compounds. From the ethnic period, the plant has been used in the formulation of traditional medicines in the South-Asian region without knowing its actual therapeutic values. In this study, four different solvent extracts of the A. muricata bark were analyzed by GCMS, and their antibacterial efficacy was evaluated against multidrug-resistant (MDR) pathogenic bacteria. The GC-MS analysis detected the highest number of active compounds (thirty-two) in chloroform extract, while, n-hexane, ethanol, and ethyl acetate extract also contain seven, six, and three bioactive compounds respectively. The most prominent compounds present in the bark extracts having antibacterial activity are heneicosane, 13-docosenamide, hexadecane, hexadecanoic acid esters, benzyl benzoate,1,2-benzene dicarboxylic acid esters, 2,5-di-tert-butyl-1,4-benzoquinone (DTBBQ), eicosane, 9-octadecanamide etc. The in vitro antibacterial activity of the extracts was monitored against two Gram-positive and two Gram-negative multi-drug resistance (MDR) bacterial strains. The chloroform bark extract showed the lowest MIC values (75–120 μg/mL) and the highest inhibition against four bacteria (two Gram-positive and two Gram-negative), whereas, moderate activity was observed in other three extract. The presence of various bioactive compounds in the extracts certainly has potential therapeutic values and can prevent MDR pathogenic bacteria. Thus, the bark of A. muricata can be used as a potent natural antimicrobial agent and also reduce the chance of secondary infection caused by opportunistic pathogens.

Graphical abstract

Highlights

• •Annona muricata plant used in this study was grown in Bangladesh.
• •GCMS profiling of the bark extracts of A. muricata with four different polar to non-polar solvents.
• •Highest number of bioactive compounds were found in chloroform extract.
• •Chloroform extract showed potential antibacterial activity against Multidrug Resistance (MDR) bacterial strains.

1. Introduction

The modern world is highly reliant on synthetic drugs to treat various kinds of infections and diseases. Treatment with synthetic drug induces the human pathogens into a deadly multidrug resistance (MDR) form which leads our medical science to a dead end. Therefore, researchers are now fostering medicinal plants that have been applied in ethnomedical treatment for many years. Annona muricata L. a member of the Annonaceae family is being considered as a valuable medicinal plant in the herbal sciences from ancestor period. This tree is native to the tropical and subtropical regions of South and North America and is known as soursop [1,2]. Now this tree is also cultivated in several regions of Southeast Asia including Bangladesh. In Bangladesh, the fruit is more popular with common people than the tree and locally known as Corossol. Furthermore, researchers are more concerned about the health benefits and therapeutic potential of the A. muricata tree [3]. Various parts of the tree, particularly the leaves have been widely studied and different phytochemical compounds like alkaloids [4], flavonoids and antioxidants [5,6], protein, glycosides, terpenoids, saponins, tannins, anthraquinones, phenolics [7], cyclopeptides, and essential oil [8,9] are also found in the studies. However, scientists are highly focused on the annonaceous acetogenin compound [10] due to its special structure and extensive biological activities [11,12] and so far 120 acetogenins have been identified in A. muricata tree [13]. Unlike the leaf part of A. muricata tree, the bark extract is less vigorously studied in the existing literature. As far the compounds listed in the bark and stem bark portion of A. muricata included alkaloids, epoxymurin, gallic acid, lichexanthone, and β-sitosterol-3-O-β-D-glucopyranoside [3,4,[14], [15], [16]]. Moreover, another compound annonacin was detected and quantified in ethyl acetate bark extract using HPLC-MS [17].

Annona muricata tree has been used as a part of herbal medicine since ancient times and contains many potential therapeutic constituents. Leaves of this tree showed anti-diabetic, anti-cancer, hypoglycemic, and antiplasmodial activity, fruits showed anti-diarrheal and anti-inflammation activity, stem bark showed antimicrobial, sedative, and febrifuge activity and seed showed insecticidal and anticonvulsant activities [2,6,14,18,19]. A recent study in Bangladesh also found that methanol crude extract of A. muricata fruit showed notable antidiarrheal activity but do not have any antibacterial potential [20]. There is much evidence of the antibacterial activity from the leaf, stem bark, root, and seed extract of A. muricata against different microorganisms [21,22]. The study of Bento et al., 2013 [23] isolated some pharmacological natural compounds from A. muricata and they suggested the natural compounds have a possible use in antimicrobial therapy against MDR bacterial strains. The study on the antimicrobial activity of the bark extract of the A. muricata tree is limited but still there are few research works have been done and the methanolic bark extract showed antibacterial activity against Escherichia coli [19]. Recent computational and experimental studies have been done on the bark extract of the tree and the study suggested that the methanolic, ethyl acetate and n-hexane bark extract of A. muricata has an antibacterial activity which can compete favorably with streptomycin and also has more potent antifungal activity when compared with amphotercin B [14].

Annona muricata tree has a long history of being effectively used in the formulation of herbal medicine in different regions of Southeast Asia. From the perspective of Bangladesh, people are now cultivating A. muricata without knowing about the phytochemical compounds and medicinal values of the tree. Besides, few research works have been performed, especially on the bark of A. muricata grown in Bangladesh. So, this current research work comprehensively investigates the phytoconstituents present in A. muricata bark using four distinct solvents (n-hexane, chloroform, ethyl acetate, and ethanol). The aim of this study is to explore the therapeutic values of the detected bioactive compounds and the future perspective of A. muricata bark as a new drug candidate to treat multiple infectious diseases. Thus, the current research work was pursued to observe the possible in vitro antibacterial activity of the crude bark extract of A. muricata against various human pathogenic MDR bacterial strains.

2. Method and materials

2.1. Sample collection

The bark sample of the A. muricata tree was collected from Station Road, Rajshahi (Latitude 24.3744°N; Longitude 86.6134°E) in September 2019. The collected voucher specimen was deposited at the Department of Botany, Jahangirnagar University, Bangladesh; accession no. JUH 10097. Fresh bark samples were collected and kept in a labeled sterile bag which was brought directly to the laboratory for further processing.

2.2. Sample processing and extraction

Freshly collected bark of A. muricata was washed with tap water to remove unwanted dirt. Then the bark was cut into small pieces (∼6–8 mm) and sun-dried in a shaded area for two weeks. The properly dried bark pieces were ground into a fine powder using a grinder machine (Fritsch, Germany).

The powder was extracted with four different nonpolar to polar organic solvents (n-hexane, chloroform, ethyl acetate, and ethanol) using the soxhlet apparatus (Glassco, UK). Ten grams of bark powder was taken in a filter paper cone and placed into the soxhlet apparatus. A 150 mL of solvent was taken in the round bottom flask and attached to the previously set soxhlet apparatus. The soxhlet apparatus was placed on the heating mantle and the temperature was set at 65–80 °C. At this temperature, the organic solvent was vaporized and reached the condenser chamber where it condensed back into the liquid and returned into the cone containing the sample. Compounds that were solubilized in that solvent eventually come into the round bottom flask. This process has been repeated several times to ensure the maximum amount of extraction of soluble compounds with the organic solvent. The extracts were then separated from the solvent using a rotary evaporator (H50-500, Lab Tech, Italy) at 25 °C. The bark sample was sequentially extracted using hexane, chloroform, ethyl acetate, and ethanol as solvent. For simplification of further data analysis extract has been given short names like BH: n-hexane bark extract, BC: chloroform bark extract; BE: ethyl acetate bark extract, and BEt: ethanol bark extract. The whole research work was performed between September 2019 to March 2020 and during this period the sample was stored at 4 °C.

2.3. Gas chromatography-mass spectroscopy analysis

2.3.1. Sample preparation

The dried sample of each extract was diluted with methanol (GC grade, Sigma Aldrich, USA) and after passing through a syringe filter (0.22 μm) was taken into a 4 mL GC vial.

2.3.2. Gas chromatography-mass spectroscopy condition

The gas chromatographic analysis of the four different solvent extracts of the bark sample was performed by GCMS-QP2020 (SHIMADZU, Japan) equipped with an auto-sampler (AOC- 20s) and auto-injector (AOC-20i). The chromatography method was carried out by a capillary column prepared with diphenyl (5 %) and dimethyl poly-siloxane (95 %). Detailed GC-MS conditions are given in Table 1. The mass spectrum of given chromatogram in GC-MS was matched with the database of the National Institute of Standards and Technology (NIST) library 2008 and 2014 editions.

Table 1.

GC-MS condition for phytochemical characterization of A. muricata bark.

GCMS Condition
Capillary column	Length	30 m
	Inner diameter	0.25 mm
Injection temperature		220 °C
Initial oven temperature & rate		80 °C & 5 °C/min
Ion source temperature		280 °C
Carrier gas		Helium
Flow rate		1.72 ml/min
Sample injection volume		5 μL
Solvent cut time		3.20 min
Total running time		50 min

2.4. Bacterial strains collection

Bacterial specimens were collected from the outside area of hospitals in Rajshahi city and aseptically transferred to our laboratory. Firstly, the strains were identified by cultural and biochemical tests and then the pure culture of bacterial strains was finally identified by the MicroStation ID system (BIOLOG, USA; ELx808BLG).

2.5. Bacterial culture preparation for MicroStation ID system

The MicroStation ID system is a semi-automated rapid microbial identification system that gives species-level identification based on 71 carbon source utilization and 23 chemical sensitivity assays in GENIII microplate test panels. MicroStation ID system is a computerized rapid detection system that can identify more than 2900 species of microorganisms by comparing them with their database. Cell suspensions were tested with a panel of pre-selected carbon sources and compared against more than 2900 identification profiles of environment and fastidious organisms from diverse fields of microbiology. Bacterial samples are sub-cultured on nutrient agar (Difco™, USA) and incubated at 37 °C for 18 h to gain pure culture. A single colony from cultured nutrient agar plates was transferred to Inoculating Fluid-A (IF-A) purchased from BIOLOG and transmittance (95–98 %) was adjusted with a turbidity meter (BIOLOG, USA) for protocol-A. The liquid media was transferred to a 96-well GEN III microplate with an eight-channel electronic pipettor (BIOLOG, USA) and incubated at 33 °C. After 16–22 h of incubation, the microplates were read on a plate reader (BIOLOG, USA). The result was analyzed using the GENIII database 2.8.0 database using the software program Mirolog™6.2 (BIOLOG, USA).

2.6. Antibiogram

The Antibiotic susceptibility test of the identified strains was conducted by agar disc diffusion method using commercial discs (Himedia, India). The strains were subcultured on a nutrient agar plate at 37 °C for 24 h and one to three colonies of each strain were dissolved in sterile normal saline (0.85 % NaCl) to achieve 0.5 McFarland standard (O.D. 600 = 0.01 to 0.1). Each bacterial culture (100 μL) was spread in a MHA plate with a sterile cotton bar and antibiotic discs were placed on the plate and incubated at 37 °C for 16–22 h. The antibiotic discs tetracycline (TE) 30 μg, chloramphenicol (C) 30 μg, ciprofloxacin (CIP) 5 μg, kanamycin (K) 30 μg, neomycin (N) 30 μg, oxytetracycline (O) 30 μg, gentamicin (GEN) 10 μg, ampicillin (AMP) 25 μg, ceftriaxone (CTR) 30 μg, nalidixic acid (NA) 30 μg, imipenem (IMP) 10 μg, cefotaxime (CTX) 30 μg, co-trimoxazole (COT) 25 μg, erythromycin (E) 15 μg were used in this study according to the standard guidelines of Clinical and Laboratory Standards Institute (CLSI, 2020) [24].

2.7. Antimicrobial activity

The antimicrobial activity of the bark extract of A. muricata was tested by broth microdilution assay and the minimum inhibitory concentration (MIC) value was also determined [[25], [26], [27]]. Four different solvent extracts of bark (described above) were assayed for antimicrobial activity against four different organisms (listed in Table 3). Firstly, the bark extracts were prepared in three different concentrations (100, 150, and 200 μg/mL) to observe whether the bark extract exhibited any reduction in microbial population by monitoring the absorbance and comparing with positive and negative control data and then the MIC value was also determined ranging from 1024 μg/mL to 75 μg/mL. Chloramphenicol was used as the positive control and DMSO (10 %) as the negative control.

Table 3.

List of the bio-active compounds with their biological activity detected in four different crude bark extracts of A. muricata by GCMS.

Sl. No.	Name	Extract Name	Conc. (%)	Activity	Reference
1.	Hexadecanoic acid methyl ester	BH	12.53 ± 0.02	Hypocholesterolemic, hemolytic, antiandrogenic	[28]
2.	Heneicosane, (pheromone)	BH	8.49 ± 0.02	Pheromone, control Aedes aegypti, antimicrobial	[29]
		BC	2.70 ± 0.20
3.	1,2-Benzenedicarboxylic acid	BH	19.32 ± 0.03	Antibacterial, in vitro anti-inflammatory	[30,31]
4.	13-Docosenamide	BH	30.32 ± 0.07	Antimicrobial, anti-inflammatory	[32,33]
		BC	0.75 ± 0.01
		BE	14.18 ± 0.05
5.	Methyl stearate	BC	0.19 ± 0.01	Anti-inflammatory, antinociceptive, antioxidant, antifungal	[32,33]
6.	1,2-Benzenedicarboxylic acid, bis(2-methyl propyl) ester	BC	5.64 ± 0.02	Antimicrobial, hypoglycemic, cytotoxic activity, cell apoptosis, and potential chemotherapeutic agent	[34]
		BE	38.90 ± 0.04
7.	1,2-Benzenedicarboxylic acid, butyl 2-methyl propyl ester	BC	59.12 ± 0.05	α-Glucosidase, Hypoglycemic	[35,36]
8.	1,2-Benzenedicarboxylic acid, butyl 2-ethylhexyl ester	BC	1.92 ± 0.04	Anti-inflammatory, cytotoxic, antifungal, and mycolytic activity	[37,38]
9.	2,5-di-tert-butyl-1,4-benzoquinone	BC	0.11 ± 0.01	Antibacterial component	[39]
10.	Eicosane	BC	3.54 ± 0.04	Antifungal, antibacterial	[40,41]
11.	9-Octadecanamide	BEt	1.97 ± 0.05	Hypolipidemic, anti-inflammatory,	[42]
12.	Stigmast-4-en-3-one	BEt	2.36 ± 0.06	Steroids, antimicrobial	[43]

2.7.1. Microbial inoculum preparation

A pure culture of microbial cells from a nutrient agar plate was inoculated in sterile normal saline and then adjusted to 0.5 Macfarland standard (O.D. 600 = 0.01 to 0.1, ∼10⁸ CFU/mL).

In a 96-well microtiter plate, 100 μL of each concentration of plant extracts was dispensed in the specified well containing 100 μL of sterile Luria broth (LB) and then 15 μL of microbial inoculums was added to each well of the microtiter plate. After mixing the inoculums with plant extract by shaking the microtiter plate with hand gently, the plates were incubated at 37 °C for 24 h. The absorbance was measured at 600 nm in a microtiter plate reader (Erba Lisa Scan II, Germany), and for determining the MIC value the 96 well plate was observed for visible turbidity.

2.8. Statistical analysis

All the experiments were carried out in triplicate and the data were expressed as mean ± standard deviation (SD, n = 3) for reproducibility. Data was analyzed by one-way ANOVA using IBM SPSS Statistics 22 and p values were considered significant at p > 0.05.

3. Results

3.1. Gas chromatography-mass spectroscopy analysis

The GCMS analysis of four crude extracts has been given a mass spectrum of unknown components and after matching with the known component of the library (NIST library 2008 and 2014 editions), seven components have been found in BH (Fig. 1a), thirty-two components in BC (Fig. 1b), three components in BE (Fig. 1c) and six components in BEt (Fig. 1 d). The identified compounds are listed in Table 2.

Fig. 1.

GCMS chromatogram of bark extract using four different solvents (a) BH (b) BC (c) BE and (d) BEt.

Table 2.

Bio-active compounds in four different solvent extracts of A. muricata bark analyzed by GCMS.

Sl No.	Compound Name	Retention Time	Relative Concentration				Molecular Formula
			BE	BC	BE	BEt
1.	Hexadecanoic acid, methyl ester	28.182	12.53 ± 0.02	1.07 ± 0.05	–	–	C17H34O2
2.	Heneicosane	29.870	8.49 ± 0.02	2.70 ± 0.20	–	–	C21H44
3.	9,12-Octadecadienoic acid, methyl ester, (E,E)-	31.757	2.87 ± 0.03	–	–	–	C19H34O2
4.	11-Octadecenoic acid, methyl ester	31.913	20.37 ± 0.07	–	–	–	C19H36O2
5.	1,2-Benzenedicarboxylic acid, diisooctyl ester	39.618	19.32 ± 0.03	–	–	21.00 ± 0.07	C24H38O4
6.	13-Docosenamide, (Z)-	43.441	30.32 ± 0.07	0.75 ± 0.01	14.18 ± 0.05	–	C22H43NO
7.	17-α-21-β-28,30-Bisnorpane	47.732	6.04 ± 0.05			–	C28H48
8.	Eicosane	13.527	–	3.54 ± 0.04	–	–	C20H42
9.	Naphthalene, 2,3-dimethyl-	13.989	–	0.16 ± 0.01	–	–	C12H12
10.	Naphthalene, 1,6-dimethyl-	14.096	–	0.10 ± 0.01	–	–	C12H12
11.	Dimethyl phthalate	14.257	–	0.39 ± 0.01	–	–	C10H10O4
12.	Benzoic acid, 2-(1-oxopropyl)-, methyl ester	14.791	–	3.75 ± 0.04	–	–	C11H12O3
13.	2,5-di-tert-butyl-1,4-benzoquinone (DTBBQ)	15.060	–	0.11 ± 0.01	–	–	C14H20O2
14.	Ethanone, 1-[4-(1-hydroxy-1-methyl ethyl)phenyl]	15.460	–	0.26 ± 0.01	–	–	C11H14O2
15.	Hexadecane	16.051	–	0.18 ± 0.01	–	–	C16H34
16.	Pentadecane, 3-methyl-	18.073	–	0.32 ± 0.01	–	–	C16H34
17.	Octadecane	19.208	–	8.08 ± 0.07	–	–	C18H38
18.	Tetradecane, 5-methyl-	23.795	–	0.18 ± 0.01	–	–	C15H32
19.	Benzyl benzoate	24.180	–	0.35 ± 0.01	–	–	C14H12O2
20.	Heptadecane, 3-methyl-	24.358	–	0.36 ± 0.01	–	–	C14H12O2
21.	Nonadecane	25.215	–	5.18 ± 0.03	–	–	C19H40
22.	Cyclotetradecane	25.366	–	0.12 ± 0.01	–	–	C14H28
23.	1,2-Benzenedicarboxylic acid, bis(2-methyl propyl) ester	26.066	–	5.64 ± 0.02	38.90 ± 0.04	–	C16H22O4
24.	1,2-Benzenedicarboxylic acid, butyl 2-methyl propyl ester	27.060	–	59.12 ± 0.05	–	–	C16H22O4
25.	1,2-Benzenedicarboxylic acid, butyl 2-ethylhexyl ester	27.797	–	1.92 ± 0.04	–	–	C20H30O4
26.	Heptadecane	28.567	–	0.09 ± 0.01	–	–	C17H36
27.	1,2-Benzene dicarboxylic acid, butyl octyl ester	28.896	–	4.04 ± 0.03	–	67.66 ± 1.01	C20H30O4
28.	1,1′-Bicyclopropane, 2,2,2′,2′-tetrachloro-1,1′-dimethyl-	29.664	–	0.11 ± 0.01	–	–	C8H10Cl4
29.	Butane, 1-(2,2-dichloro-3-ethyl cyclopropyl)-	31.413	–	0.05 ± 0.01	–	–	C9H16Cl2
30.	9-Octadecenoic acid (Z)-, methyl ester	31.942	–	0.18 ± 0.01	–	–	C19H36O2
31.	4-Methyldocosane	32.224	–	0.04 ± 0.01	–	–	C23H48
32.	Methyl stearate	32.488	–	0.19 ± 0.01	–	–	C19H38O2
33.	Hexacosane	32.709	–	0.12 ± 0.01	–	–	C26H54
34.	Cyclopropaneoctanoic acid, 2-[[2-[(2-ethyl cyclopropyl) methyl]cyclopropyl]methyl], methyl ester	35.194	–	0.11 ± 0.01	–	–	C22H38O2
35.	Methyl-9,10- epoxy-12,15-octadecadienoate	35.597	–	0.13 ± 0.01	–	–	C19H32O3
36.	Docosane	37.487	–	0.64 ± 0.01	–	–	C22H46
37.	Bis(2-ethylhexyl) phthalate	39.620	–	–	46.90 ± 0.08	–	C24H38O4
38.	Cyclohexanamine, N, N-dimethyl-	3.879	–	–	–	4.48 ± 0.05	C8H17N
39.	Hexadecanoic acid, ethyl ester	29.704	–	–	–	2.46 ± 0.04	C18H36O2
40.	9-Octadecenamide	43.431	–	–	–	1.97 ± 0.05	C18H35NO
41.	Stigmast-4-en-3-one	47.019	–	–	–	2.36 ± 0.06	C29H48O

Values were recorded in three replicates (n = 3) and expressed as mean ± SD.

Here, BH: n-hexane bark extract, BC: chloroform bark extract; BE: ethyl acetate bark extract, and BEt: ethanol bark extract.

The BH contained 13-docosenamide (30.32 %); 11-octadecenoic acid, methyl ester (20.38 %); 1,2-benzenedicarboxylic acid, di-isooctyl ester (19.32 %) and hexadecanoic acid, methyl ester (12.54 %) most prominently.

According to the GCMS spectrum of BC, the most prominent constituents were 1,2-benzenedicarboxylic acid, butyl 2-ethylhexyl ester (59.15 %); hexadecane (8.09 %); 1,2-benzenedicarboxylic acid, bis (2-methyl propyl) ester (5.64 %); nonadecane (5.18 %); 1,2-benzene dicarboxylic acid, butyl octyl ester (4.04 %) and benzoic acid, 2-(1-oxopropyl)-, methyl ester (3.76 %). The other significant compounds were 1,2-benzenedicarboxylic acid, butyl 2-ethylhexyl ester (1.91 %); hexadecanoic acid, methyl ester (1.08 %); 13-docosenamide, (Z)- (0.75 %); heneicosane (0.64 %); benzyl benzoate (0.35 %); ethanone, 1-[4-(1-hydroxy-1-methylethyl)phenyl]- (0.26 %); tetradecane, 5-methyl (0.18 %); cyclotetradecane (0.12 %),1,1′-bicyclopropane, 2,2,2′,2′-tetrachloro-1,1′-dimethyl- (0.11 %) etc.

In BE bis(2-ethylhexyl) phthalate (46.90 %) is highly abundant and the other two detected compounds are 1,2-benzenedicarboxylic acid, bis (2-methylpropyl) ester (38.91 %) and 13-docosenamide, (Z)- (14.18 %).

On the contrary, 1,2-benzene dicarboxylic acid, butyl octyl ester (67.65 %) is the most significant compound present in BEt, and 1,2-benzenedicarboxylic acid, di-isooctyl ester (21.07 %) is also present in considerable amount in this respective extract. Other detected compounds were cyclohexylamine, N, N-dimethyl- (4.48 %); hexadecanoic acid, ethyl ester (2.46 %); stigmast-4-en-3-one (2.36 %) and 9-octadecenamide (1.97 %).

Few detected compounds like erucamide; heneicosane; hexadecane; benzyl benzoate; bis (2-ethylhexyl) phthalate; oleamide; 13-docosenamide; 1,2-benzene dicarboxylic acid esters; 2,5-di-tert-butyl-1,4-benzoquinone (DTBBQ); ecosane; 9-octadecanamide have different biological activities in literature (Table 3).

3.2. Identification of microbial strains by semi-automated MicroStation ID system BIOLOG

The clinical bacterial strains were identified by a semi-automated MicroStation system using the software program Mirolog™ 6.2, the software gives strain level identification of microbial strain by comparing with the GENIII database 2.8.0 for protocol A. The results are listed in Table 4.

Table 4.

Microbial strain identification by MicroStation ID system BIOLOG.

Sl No.	Species	Organism Type	DIST	ΔDIST/Probability	SIM
1.	Pseudomonas aeruginosa	G(−)	5.001	1.674	0.602
2.	Escherichia coli	G(−)	6.066	1.944	0.582
3.	Bacillus ruris	G(+), Rod	5.574	0.360	0.617
4.	Bacillus subtilis	G(+), Rod	4.037	1.08	0.563

Note: G (+) = Gram Positive; G (−) = Gram Negative; SB = Single bacilli.

3.3. MDR pattern of identified microbial strains

Fourteen antibiotics with different modes of action were tested against the four identified bacterial strains. Pseudomonas aeruginosa showed resistance against 8 antibiotics, while E. coli showed resistance against 6 and B. subtilis & B. ruris showed resistance against 7 antibiotics. The results are presented in Fig. 2.

Fig. 2.

MDR pattern of four isolated nosocomial bacterial strains.

3.4. Antimicrobial activity of bark extract

The antibacterial activity of four different solvent extracts BH, BC, BE and BEt was tested against four MDR bacterial strains (mentioned in the 3.2 section). Primarily, the three different concentrations of extracts (200, 150, and 100 μg/mL) were added to the microbial culture, and absorbance was recorded. In comparison with positive and negative control data, all extracts were able to reduce the microbial population to some extent. Both the BC and BE showed more significant activity against both Gram-positive and Gram-negative bacteria (Table 5). The MIC values of all four extracts were between 75 and 1200 μg/mL and the BC showed the lowest MIC values of 75 μg/mL for E. coli, P. aeruginosa, and B. subtilis and 120 μg/mL for B. ruris. However, the BH showed the highest MIC value (1200 μg/mL) against B. subtilis and BEt against both B. subtilis and B. ruris.

Table 5.

Antibacterial activity of A. muricata bark extract against pathogens.

Sample name	Concentration (μg/mL)	Absorbance against MDR bacteria
		E. coli	P. aeruginosa	B. subtilis	B. ruris
BH	200	0.54 ± 0.07a	0.55 ± .07a	0.79 ± 0.08cd	0.59 ± 0.06ab
	150	0.78 ± 0.04cd	0.75 ± 0.02c	0.93 ± 0.05de	0.74 ± 0.05bc
	100	0.95 ± 0.04e	0.77 ± 0.04c	1.22 ± 0.05f	0.98 ± 0.03e
BC	200	0.17 ± 0.03abc	0.16 ± 0.05ab	0.12 ± 0.15a	0.15 ± 0.04ab
	150	0.28 ± 0.05bcd	0.25 ± 0.04abcd	0.15 ± 0.04ab	0.22 ± 0.05abc
	100	0.38 ± 0.04de	0.30 ± 0.03cde	0 0.24 ± 0.06abc	0.42 ± 0.08e
BE	200	0.25 ± 0.04a	0.42 ± 0.06abc	0.32 ± 0.07ab	0.36 ± 0.08ab
	150	0.26 ± 0.07a	0.56 ± 0.03cde	0.49 ± 0.05bcd	0.71 ± 0.04ef
	100	0.38 ± 0.04ab	0.62 ± 0.03def	0.74 ± 0.10ef	0.77 ± 0.09f
BEt	200	0.87 ± 0.03abc	0.66 ± 0.10a	0.84 ± 0.09abc	0.72 ± 0.07ab
	150	1.04 ± 0.06cd	0.84 ± 0.05abc	1.05 ± 0.06cd	0.89 ± 0.15bc
	100	1.19 ± 0.13de	1.02 ± 0.11cd	1.33 ± 0.07e	1.22 ± 0.06de
Chloramphenicol	30	0.41 ± 0.08	0.56 ± 0.04	0.44 ± 0.04	0.55 ± 0.04
DMSO	10 %	1.21 ± 0.09	1.38 ± 0.04	1.45 ± 0.09	1.28 ± 0.05

Note: values were recorded in three replicates (n = 3) and expressed as mean ± SD where ANOVA was performed to test the concentration of bioactive compounds significantly different at the level of 5 % (p < 0.05). Identical uppercase letters of average values in the same column do not differ significantly.

4. Discussion

Folk medicine is imperative in developing countries where people with lower income have a great value towards this treatment but sometimes without having any scientific proof, this treatment can also have an adverse health impact. The traditional use of A. muricata as a part of folk medicinal treatment has been reported since 1956 in different parts of the world. In Indian ayurvedic medicine, A. muricata tree is used to treat kidney trouble, and high blood pressure, as a respiratory stimulant, febrifuge, abortifacient, for scorpion stings, etc [44,45]. Different parts of the tree, especially the bark, are used as a tonic in Indian folk medicine [18]. The uses of this plant as a part of herbal medicine are increasing day by day, but no detailed study was done on the bark of the A. muricata tree, especially in the Southeast Asian region. In the current study, we focused on the phytochemical constituents of the bark extract of the A. muricata tree grown in Bangladesh using four different non-polar to polar solvents. Also, we revealed the antimicrobial activity of the plant extract on MDR bacterial strains.

In phytoscience, the identification of bioactive compounds helps to interpret the toxicity and the adverse effects, along with determining the appropriate dosages and finding the most suitable method to extract them. In this study, the phytochemical analysis of A. muricata bark extract revealed some crucial and valuable data through GC-MS analysis. Unlike the other parts of the A. muricata tree, the bark extract is less vigorously studied in the existing literature. The current study revealed the predominant presence of fatty acids along with different esters, alkanes, aromatic hydrocarbons, ketenes, amides, steroids, etc. in the bark extracts using different solvents (Table 2). The highest number of compounds (thirty-two) were presented in the BC part and of them, the most prominent compounds are 1,2-benzenedicarboxylic acid, and butyl 2-methylpropyl ester (59.13 %), which have hypoglycemic activity in literature [35,36]. Other detected compounds 1,2-benzenedicarboxylic acid, butyl 2-ethylhexyl ester, and its other active derivatives (1.92 %) have anti-inflammatory, cytotoxic, antifungal, and mycolytic activity [37,38]. Heneicosane which acts as a pheromone and also has antimicrobial activity [29], was detected both in BH (8.5 %) and BC (2.7 %). Another compound 13-docosenamide found in BH (30.32 %), BC (0.748 %), and BE (14.18 %) has anti-inflammatory, antioxidant, and antimicrobial activity [32]. The compound 9-octadecanamide found in BEt (1.97 %) has hypolipidemic, anti-inflammatory, and antibacterial activity [42]. The bark extract was enriched with different derivatives of 1,2-benzenedicarboxylic acid esters which were considered the main constituents of the Elaeagnaceae plant and had anti-tumor, anti-inflammatory, and antibacterial functions [46]. Compounds detected in A. muricata bark extract with extensive and important biological activity are listed in Table 3.

In this study, MDR bacterial strains were isolated directly from the hospital area of Rajshahi, Bangladesh, and also identified in the laboratory and assayed for antibiogram (Table 4, Fig. 2). All four bacteria were resistant to the antibiotics that are commonly used to treat infectious diseases. The pathogens, P. aeruginosa, E. coli, B. ruris, and B. subtilis have a history of causing nosocomial secondary infection and a potential hazard to human health. Therefore, natural antimicrobial agents that significantly inhibit MDR pathogens are appreciable in the medical sector. The noteworthy antibacterial activity exerted by BC and BEt (Table 5, Table 6) in microdilution assay against four MDR bacterial strains. The bark extract of A. muricata contains the bioactive compound 1,2-benzenedicarboxylic acid ester derivatives and 13-docosenamide which have the potential as an antimicrobial agent [32,33,46]. A study by Oyebamji et al., 2019 [14] also found the antibacterial and antifungal activity of ethyl acetate bark extract of A. muricata against a few strains. Besides that, the BC contained two other derivatives of 1,2-benzene dicarboxylic acid esters, DTBBQ, eicosane, heneicosane, methyl stearate, etc. which, are also listed as a potent antimicrobial compound in literature [29,[39], [40], [41]].

Table 6.

MIC value (μg/mL) of BH, BC, BE, and BEt extract of A. muricata.

Microorganisms	BH	BC	BE	BEt	Controla
B. subtilis	1200	75	250	1200	64
B. ruris	550	120	250	750	72
E. coli	550	75	120	1200	40
P. aeruginosa	500	75	500	850	64

^aChloramphenicol was used as a positive control.

The plant is widely used as a source of chemically active metabolites and its different curative properties [2,47]. The extensive therapeutic potential and widespread application of A. muricata in traditional medicines manifest that, if properly investigated, the bioactive components present in the tree could give affordable and accessible treatment with great health benefits. However, there are few studies on the antimicrobial activity of A. muricata bark extract and no study has been performed to consider A. muricata bark extract as a potent antimicrobial agent against MDR bacterial strains. This study reveals that the bark extracts of A. muricata (BH, BC, BE, and BEt) significantly inhibit bacterial growth at 200 μg/L concentration by lowering the absorption. The absorption data also coincide with the MIC value of the extracts. Thereby, the bark of A. muricata contains bioactive compounds with bacteriostatic activity that can inhibit the pathogens successfully. A recent study also showed the molecular docking pattern of some valuable phytochemicals and revealed that phytochemicals like annonaine and coclaurine acted as good antifungal and antibacterial agents and predicted that they could also be used against the MDR pathogenic bacteria and fungi [14]. However, due to the crude extract, the bacterial inhibitory concentration is much higher in our study. So, a detailed study should be needed to know the effectiveness and the pathway of the bioactive compounds that inhibited the bacteria. As the use of synthetic drugs makes the microorganisms surrounding us resistant to them, leveraging natural sources as an alternative for killing them is mandatory now. The findings of this study indicate that the soursop tree grown in Bangladesh have essential biological active compounds and could be used as a promising source for the development of new antibacterial drugs. The bark of the tree has been shown significant antibacterial activity against MDR bacteria and thus, making the tree more endearing for further research.

5. Conclusion

The phytoconstituents of a plant certainly change with geographical and topographical variation and the bio-active compounds of A. muricata bark were not studied previously in the perspective of Bangladesh. This study reveals that the different solvents bark extract of the A. muricata tree grown in Bangladesh contains compounds like heneicosane, 13-docosenamide, hexadecane, hexadecanoic acid esters, benzyl benzoate, dibutyl phthalate, 1,2-benzenedicarboxylic acid esters, DTBBQ, eicosane, 9-octadecanamide which contains antimicrobial potential in literature. In this current study, the crude extracts also showed broad-spectrum antimicrobial activity against four MDR bacterial strains (P. aeruginosa, E. coli, B. subtilis & B. ruris) which were almost resistant to traditional antibiotics. The results obtained in this research will help to broaden the alley to use the bark of A. muricata tree in different ayurvedic and herbal medicine formulations to reduce the efficacy of pathogenic bacteria. However, in the future, for proper use of the tree in medication against various types of illness, isolation of the bio-active compounds and a more detailed study on their mechanism of action is required. The findings of this study validate that the bark extract of the tree is a natural antimicrobial agent and therefore can be used in medication to inhibit bacterial infection.

CRediT authorship contribution statement

Subarna Sandhani Dey: Writing – original draft, Methodology, Investigation, Funding acquisition, Formal analysis. Mahci Al Bashera: Writing – original draft, Methodology, Investigation, Funding acquisition, Formal analysis. Shyama Prosad Moulick: Investigation, Formal analysis. Firoz Ahmed: Writing – review & editing, Conceptualization. Md Zafar As Sadiq: Visualization, Data curation. Mst. Sarmina Yeasmin: Investigation, Formal analysis, Data curation. G.M. Masud Rana: Formal analysis, Data curation. Trissa Saha: Visualization, Data curation. Musharrat Jahan Prima: Writing – review & editing. Md Murshed Hasan Sarkar: Validation, Supervision, Resources.

Ethical statement

Ethical statement is not applicable for this current study.

Data availability statement

Data to support the findings of the current study are available within the article and its supplementary files.

Funding statement

The whole investigation was performed as a part of an R & D project that was supported by Bangladesh Council of Scientific and Industrial Research (BCSIR), Dhaka −1205, Bangladesh.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.