Secondary metabolite Constituents, Antimicrobial Activity and Gas chromatography-Mass spectroscopy Profile of Bombax buonopozense P. Beauv. (Bombacaceae) Stem bark Extract

 

Yusuf-Babatunde A.M.1, Osuntokun, O.T.2, Ige, O.O.* 4, Solaja O.O.3

1Department of Pharmaceutical Technology, Ogun State College of Health Technology, Ilese-Ijebu.

2Department of Microbiology, Faculty of Science, Adekunle Ajasin University, Akungba Akoko, P.M.B. 001, Ondo State, Nigeria.

3Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Obafemi Awolowo University, Ile Ife,

Osun State, Nigeria.

4Department of Environmental Health Technology, Ogun State College of Health Technology, Ilese-Ijebu.

*Corresponding Author E-mail: ogbonronka@gmail.com, yusufademola4u@yahoo.com

 

ABSTRACT:

This study evaluated the phytochemical constituents, antimicrobial activity and structural characteristics using GC-MS of bioactive constituents from the stem bark extract of Bombax buonopozense. Secondary metabolite constituents was assayed using standard procedures. The antimicrobial activity was carried out using agar diffusion method to determine the susceptibility of these microorganisms: (Staphylococcus aureus (ATCC 29213), Streptococcus faecials, Bacillus subtilis (ATCC 8263), Escherichia coli (ATCC 25922), Salmonella tiphi, Pseudomonas aeruginosa (ATCC 27853), Candida albican (ATCC 90029), Aspergillusniger and Phytophera megakarya. EtOAc fraction was chromatographed on silica column to afford oil isolate and its chemical structures were proven with the employment of GC-MS. Phytochemical screening revealed the presence of saponins, flavonoids, reducing sugars, steroids, terpenoids, anthraquinone, phenols and alkaloids. The antimicrobial sensitivity test showed higher activity on Candida albican, Bacillus subtilis, Pseudomonas aeruginosa and Staphylococcus aureus, moderate to little activity on the rest organisms. The GC-MS of the stem bark oil extract revealed the main constituents as: 1, 2 Benzenedicarboxylic acid 4.10%, Diisobutyl phthalate 1.18%, Dibutyl phthalate 0.72%, Lupenone 0.62%, Lupeol 4.93%, Lupenyl acetate 86.28%, Testosterone decanoate 0.87% and 3-O-Acetyl-6-methylcycloartenol 0.67%. The findings indicate stem bark extract has bioactive compounds which may be effective in the treatment of infection diseases.

 

KEYWORDS: Bombax buonopozense, secondary metabolite, antimicrobial, gas chromatography, extract.

 

 


 

 

 

1. INTRODUCTION:

Gentiana, a cosmopolitan and important genus of the Many rare and useful herbs occur in nature, from which important drugs could be prepared or agents which may serve as starting materials for the partial synthesis of some useful drugs. The usefulness of these plant materials medicinally is due to the presence of bioactive constituents such as phenols, flavonoids, tannins and alkaloids[1].

They may also have complementary and overlapping mechanisms of action in the body, including antioxidant effects, modulation of detoxification enzymes, stimulation of the metabolism, antibacterial and antiviral. Also, related studies revealed that flavonoids apart from their antioxidant protective effects, inhibits the initiation, promotion and progression of tumors. Tannins are known to inhibit pathogenic fungi. Alkaloids play some metabolic role and control development in living system. Saponins prevent disease invasion of plants by parasitic fungi, hence having antifungal properties [1].

 

The search for newer sources of antimicrobial agents is a global challenge preoccupying research institutions, pharmaceutical companies and academia, since many infections agents are becoming resistant to synthetic drugs[2]. Infection diseases are the world’s major threat to human health and account for almost 50, 000 deaths every day[3]. Natural products (secondary metabolites) have been the most successful source of potential drug leads[4, 5, 6, 7, 8]. Natural plants have been seen as a valuable source of medicinal agents with proven potential of treating infection diseases and with lesser side effects compared to the synthetic drug agents. Currently, there is a growing interest in searching for new antimicrobial agents from natural sources such as bacteria, fungi, and plants[9, 10]. Natural products, especially microbial and plant products, constitute the major sources of new drug molecules[11]. Researchers are increasingly becoming involved in the screening of such plants with the aim of establishing their potential antimicrobial effects and identifying the compounds responsible for their antimicrobial properties[12].

 

Bombax buonopozense P. beauv (Bombacacea) is a large tropical tree that grows to 40 metres in height with large buttress roots that can spread 6 metres. The bark is covered in large, conical spines, especially when young, but shedding them with age to some degree. The branches are arranged in whorls; the leaves are compound and have 5-9 leaflets and 5-25 secondary veins. The individual leaflets have entire margins and are also large. The undersides of the leaflet may be glabrous or puberulous. It is found widely distributed in Africa from Ghana to Sierra Leone, Uganda and Gabon[13]. Common vernacular names include: Vabga (Dagbani language in Ghana), Kurya (Hausa language in Northern Nigeria), Ponpola (Yoruba language in South west, Nigeria). Many parts of the plant are used for medicinal purposes, as food, as a source of clothing fiber, as a building material, as cotton wool and as dye. The fruits are eaten by animals such as Water Chevrotain. A decoction of the leaves is used for feverish conditions, diarrhea, pains and muscle aches. Root decoction is used as antimicrobial and stomach aches[13].

 

The aim of the present study was to investigate the phytochemical and antimicrobial properties and structural identification of bioactive component using GC-MS of the stem bark extracts of B. buonopozense which have been claimed to possess some ethno-medicinal uses in infection diseases.

 

2. MATERIAL AND METHODS:

2.1. Plant collection:

The stem bark and leaves of B. buonopozense were collected in January 2015 in Ilese-Ijebu at an undeveloped land opposite Ogun State College of Health Technology campus located with latitude (030 East) and longitude (060 North). It was authenticated by Mr. Ogunowo I. I. of Herbarium unit of Department of Pharmacognosy, Faculty of Pharmacy Obafemi Awolowo University, Ile-Ife and voucher specimen (number FPI-2073) deposited at the Herbarium.

 

2.2. Preparation of plant extract and isolation:

The stem bark of B. buonopozense was air dried at room temperature and milled. The powdered stem bark (1.50 kg) was macerated with 50% aqueous ethanol at room temperature for 72 hours with constant stirring. The extract was filtered using whatman (No 1) filter paper and filtrate concentrated to dryness in vacuo on a rotary evaporator to get (120.75 g) of the ethanolic extract.100 g was liquefy in distilled water and partitioned with ethyl acetate (3 x 500 millilitres), n-butanol (3 x 500 millilitres) successively and fractions processed to dryness using rotary evaporator yielding ethyl acetate fraction (15.77 g) and butanol fraction (13.45 g). A 10 g of EtOAc was separated on silica gel column using a gradient of n-hexane (Hex), EtOAc and methanol (MeOH) yielding five fractions BSE1-BSE5. BSE1 (3.20 g) was further fractionated on silica gel using a gradient of Hex, EtOAc and MeOH which afforded the oil compound (BSE1a, 291.5 mg) and two other oil fractions BSE1b and BSE1c.

 

The ethanolic crude extract was subjected to qualitative phytochemical screening using the method of [14].

 

GC-MS analysis of the oil was performed on (Shimadzu QP-2010 Ultra Japan) with a DB-5MS capillary column of length 30m (0.25 µm internal diameter and 0.25 µm film thickness).

 

2.4. Antimicrobial Susceptibility Testing:

The susceptibility testing was carried out using the agar well diffusion method [15]. 1 ml of the standardized 24 hour broth culture of the isolate, adjusted to 0.5 McFarland scale, was mixed with 19 ml of molten Mueller Hinton agar in sterile universal bottle, poured into sterile petri dish and was left to solidify. Using a sterile corkborer of 8mm diameter, equidistant wells were made in the agar. 0.1mL of the 100mg/ml extract was dispensed into the well. 0.025mg/ml of Rifampicin and Ofloxacin were dispensed into two other wells to serve as control.

 

The plates were incubated at 37°C for 24hours. After 24 hours of incubation, the zones of inhibition were observed and measured to the nearest millimeter (mm) using a standard transparent meter rule.

 

2.5. Determination of Minimum Inhibitory Concentration (MIC):

Minimal inhibitory concentration (MIC) was carried out using the agar dilution method according to the Clinical Laboratory Standard Institute (CLSI, 2012). Various concentrations (100mg/ml, 90mg/ml, 80mg/ml, 70mg/ml, 60mg/ml, 50mg/ml, 40mg/ml, 30mg/ml, 20mg/ml and 10mg/ml) of the crude extracts were prepared. 1ml of each concentration is incorporated into 19ml of Mueller-Hinton agar and poured into sterile petri dish. A 24-hour broth culture of the test organisms, diluted to 0.5 McFarland standard was streaked on the agar plate and incubated at 37°C for 24 hours. The plates were then examined for the presence or absence of growth. The lowest concentration that inhibited growth was taken as the minimum inhibitory concentration of the respective extracts.

 

3. RESULTS:

3.1. Secondary metabolite Screening:

Secondary metabolite screening of the ethanolic crude extract was subjected to qualitative phytochemical screening using the method of [14] and the result shows the presence of saponins, flavonoids, terpenoids, anthraquinone, steroids, reducing sugar, phenols and alkaloids, while cardiac glycosides and resins are absent(Table 1).


 

Table 1: secondary metabolite profile of ethanolic extract of Bombax buonopozense

Phytochemical Tests

Alkaloid

Phenol

Steroids

Reducing sugars

Resin

Terpenoids

Saponnins

Flavonoid

Anthraquinone

Cardiac glycosides

Result

++

+++

+++

+++

-

++

++

+++

++

-

Key: +++ = highly present, ++ = moderately present, + = slightly present, - = absent.

 

Table 2: Antimicrobial activities of stem barkoil extract of B.buonopozense

Organism

Crude ethanolic extract (mg/mL) zone of inhibition (mm)*

Rifampicin (5µg) (bacterial positive control)

Flucosazole(5µg) (fungi positive control)

DMSO (negative control)

                     

100

50

25

12.5

 

Staphylococcus aureus (ATCC 29213)

16

7

7

4

23

NA

NA

Streptococcus faecalis

12

8

6

3

20

NA

NA

Bacillus subtilis ATCC 8263

13

10

6

5

28

NA

NA

Escherchia coli (ATCC 25922)

13

10

9

5

21

NA

NA

Salmonella typhi

14

10

9

4

21

NA

NA

Pseudomonas aeruginosa ATCC 27853

17

8

5

5

22

NA

NA

Candida albican(ATCC90029)

10

6

8

2

NA

21

NA

Aspergillus niger

12

8

5

2

NA

20

NA

Phytophera megakarya

10

11

8

3

NA

19

NA

Keys:

DMSO- Dimethyl sulphur oxide, NA- No activity

The zones of inhibition recorded are less the diameter of the cup, which is 8 mm.

* Represents the mean of three determinations.

 

Table 3: Minimum Inhibitory concentration (MIC) of stem barkoil extract of B. buonopozense.

Organisms

Staphy. aureus

(ATCC29213)

Strep. faecalis

B. Subtilis

(ATCC8263)

E. coli

(ATCC25922)

Salmonella typhi

P. aeruginosa

(ATCC27853)

Candida albicans (90029)

Aspergillusniger

Phytopheramegakarya

MIC (mg/mL)

50.0

60.0

50.0

60.0

50.0

60.0

60.0

50.0

40.0

 


3.2. Antimicrobial activity of the stem bark oil extract:

The table 2 shows the antimicrobial results of the extracts against the test organisms. The zone of inhibition of growth of the isolates are a function of the relative concentration of the extract. The extract shown strong antibacterial potency against P. aeruginosa (17 mm), Staphy.aureus (16 mm), and Salmonella typhi (14 mm), in comparison to standard drugs Rifampicin with (22 mm), Ofloxacin with (20 mm), antifungi activity against Aspergillus niger (12 mm), Phytophera megakarya (10 mm) and Candida albican (10 mm) in comparison toFluconazole (22 mm) respectively.

 

 

The table 3 shows the result of MIC determination on the test organisms. The lowest MIC of 40 mg/mL was demonstrated against Phytophera megakarya, 50 mg/mL was demonstrated against Staphy. aureus, Bacillus subtilis, Salmonella typhi and Aspergillus niger, while the values of60.0 mg/mL was demonstrated against Candida albicans, Escherichia coli, Pseudomonas aeruginosa, and Staphy. aureus. DMSO used as respective controls was inactive against the organisms.

 

 

 

 

 


Table 4: Chemical composition of Bombax buonopozense stem bark oil extract

S/N

NAME

Molecular Weight

Structural Formula

Ret. index

Retention time

Area %

Compound Structure

1.                         

1, 2-Benzenedicarboxylic acid,

278

C16H22O4

1908

16.043

4.10

 

2.                         

Dibutyl phthalate

278

C16H22O4

2037

16.525

1.80

 

3.                         

Diisobutyl phthalate

278

C16H22O4

1908

17.006

0.72

 

4.                         

Lupenone

424

C30H48O

2831

24.323

0.62

 

5.                         

Lupeol

426

C30H50O

2848

24.850

4.93

 

6.                         

Lupenyl acetate

468

C32H52O2

2987

25.037

86.28

 

7.                         

Testosterone Decanoate

 

 

 

442

C29H46O3

3074

25.628

0.87

 

8.                         

3-O-Acetyl-6-methoxy-cycloartenol

498

C33H54O3

3093

25.810

0.67

 

 


Table 4; The oil constituents are hydrocarbons characterized with high percentage of Lupenyl acetate (86.28%), the other components were:                                     1,2Benzenedicarboxylic acid (4.10%), Diisobutyl phthalate (1.80%), Dibutyl phthalate (0.72%), Lupenone (0.62%), Lupeol (4.93%), Testosterone decanoate (0.87%) and 3-O-Acetyl-6-methylcycloartenol (0.67%).

 


 

Fig. 1. Spectra of gas chromatography-mass spectrometry analysis of stem bark oil extract of Bombax buonopozense

 


4. DISCUSSION:

The qualitative secondary metabolite analysis of Bombax buonopozense ethanolic stem bark extract revealed the presence of saponins, flavonoids, reducing sugars, steroids, terpenoids, anthraquinone, phenols and alkaloids which are plant secondary metabolites have been established to be responsible for antimicrobial properties of most medicinal plants [16, 17, 18, 19].

 

The antimicrobial activity tests of the oil extract of Bombax buonopozense showed activity against both Gram-positive and Gram-negative bacteria and fungi. The antibacterial activity of the oil was more effective on gram negative bacteria P. aeruginosa, zone of inhibition (17 mm), gram positive bacteria Staphylococcus aureus, zone of inhibition (16 mm) and fungi Aspergillus niger, zone of inhibition (12 mm) at higher concentration of 100 mg/ml. The sensitivity of the oil extract is concentration dependent as it increases the sensitivity also increases. The broad spectrum antimicrobial activity of Bombax buonopozense extracts could be as a result of plant secondary metabolities (alkaloids, anthraquinone, flavonoid, steroids, tannin, terpenoids, phlobatannins and reducing sugars), present in the plant. The demonstration of activity against both gram-negative, gram- positive bacteria and fungi are an indication that the plant can be a source of bioactive substances that could be of broad spectrum of activity. The fact that the plant was active against both clinical and laboratory isolates is also an indication that it can be used against drug resistant microorganisms prevalent in hospital environment. The results obtained in the present study justify the use of the extracts of Bombax buonopozense leaves, stem bark and roots in traditional medicine for the treatment of microbial disease, especially those caused by S. aureus, B. subtilis, K. pneumonae, Proteus spp., P. aeruginosa and E. coli[20].

 

Gas chromatography-mass spectrometry analysis of oil the extract of Bombax buonopozense revealed eight (8) compounds with different isomers. The percentage composition of chemical compounds present in the oil are Lupenylacetate which constitute the major component (86.28%), followed by Lupeol (4.93%),               1,2Benzenedicarboxylic acid (4.10%), Diisobutyl phthalate (1.80%), Testosterone decanoate (0.87%), Dibutyl phthalate (0.72%), 3-O-Acetyl-6-methylcycloartenol (0.67%) and Lupenone (0.62%). These identified compounds fall in the category of fatty acids, triperpenes, and phytosterol. Identify fatty acid are1, 2 Benzenedicarboxylic acid, Diisobutyl phthalate, Dibutyl phthalate, triterpenoids are Lupenyl acetate, lupeol, and Lupenone, phytosterols are Testosterone decanoate and 3-O-Acetyl-6-methylcycloartenol. Triterpenoids including lupeol is highly multifunctional measured by their ability to block kappa B activation (nuclear factor), inhibit signal transducer, apoptosis, transcription, and angiogenesis [21]. Phytosterols are known to have various biological properties including anticancer activity. It is indicated that phytosterol rich diet may reduce cancer risk by 20%[22]. Phytosterols enabling anticancer responses by enhancing immune responses recognition of cancer and also have properties including cell cycle progression, apoptosis induction and tumor metastasis inhibition[23]. Hence, phytosterols could be incorporated in diet potentially prevent cancer development. Many fatty acids such as omega-3-fatty acids (α-linolenic acid) were shown to induce apoptosis in a variety of human cancer cell lines and hence is important nutritional adjuvant therapeutics in the prevention of various human cancer diseases [24]. Recently, triterpenes are possibly an alternative method for curing cancer [25].

 

5. CONCLUSIONS:

The oil extract from B. buonopozense stem-bark contains a mixture of fatty acids, triterpenesphytosterol isomers and possessed moderate antibacterial and antifungi activities, therefore justifying some of its uses in ethnomedicine.

 

6. ACKNOWLEDGEMENTS:

The authors wish to express their gratitude to the Tertiary Education Trust Fund (TETFUND) and National Research Fund (NRF) Intervention Grant (TETF/DESS/NRF/OAU/STI/ Vol 1/BI.13.10), Nigeria, and ObafemiAwolowo University for provision of financial support for this research project.

 

7. DECLARATION OF INTEREST:

The authors assert that no opposing interest exists. The authors alone are answerable for the content and writing of the paper.

 

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Received on 12.04.2019         Modified on 22.04.2019

Accepted on 01.05.2019       ©A&V Publications All right reserved

Res.  J. Pharmacognosy and Phytochem. 2019; 11(2):87-92.

DOI: 10.5958/0975-4385.2019.00016.5