Research Article

A comparative study of the phytochemical, antibacterial and scavenging effects of methanolic leaves extract of Blumea lacera (Burm. F.) DC and Blumea aurita (Linn F.) DC

Abubakar Salisu1*, Etim Veronica1, Nweke Ogechi1, Asemota Uwem, Fatokun Olakunle2

1Sheda Science and Technology Complex (SHESTCO), Biotechnology and Genetic Engineering Laboratory, P.M.B. 186, Garki Abuja 2Chemistry Advanced Laboratory Sheda Science and Technology Complex (SHESTCO). P.M.B 186, Garki Abuja

 

*For correspondence

Dr. Abubakar Salisu,

Sheda Science and Technology Complex (SHESTCO), Biotechnology and Genetic Engineering Laboratory, P.M.B. 186, Garki Abuja, Nigeria.

Email:salisuabubakar99@yahoo.com

Received: 30 October 2015

Accepted: 15 November 2015

ABSTRACT

Objective: The comparative study of phytochemical, antibacterial and antioxidant properties of Blumea lacera and Blumea aurita family (Asteraceae).

Methods: The fresh leaves were collected, washed, dried and grounded. It was extracted with 95% ethanol using cold maceration.

Results: The phytochemical screening of the ethanol extracts of Blumea lacera and Blumea aurita revealed the presence of tannins, steroids, phenols, glycoside, alkaloids and flavonoids while saponnins were present in B. aurita but absent in B. lacera, carbohydrate was present in B. lacera but absent in B. aurita. The preliminary antimicrobial screening of the two extracts against three pathogens; Escherichia coli, Salmonella typhi and Staphylococcus aureus revealed the zone of inhibition of S. lacera leaf extracts ranging between (9.00 ± 00 – 21.00 ± 00 mm) with highest zone showed on E. coli at 60 µg/disc while the leaves of B. aurita revealed (9.00 ± 00 – 22.00±00 mm) with the highest zone observed on E. coli at 60 µg/disc. The antioxidant activity of the B. lacera and B. aurita extracts were low compared to the standard Vitamin C; the scavenging effect of B. lacera is greater than that of B. aurita the IC50 was shown in increasing order, 0.12 < 0.82 < 0.86 for Vitamin C, B. lacera and B. aurita respectively.

Conclusions: The preliminary study justified the ethno-medicinal as well as pharmaceutical potentials of the plant extracts family Asteraceae.

Keywords: Antibacterial, Antioxidant, Phytochemical, Blumea lacera, Blumea aurita

Introduction

Medicinal plants have a great history in the treatment and prevention of human infectious diseases.1 Traditional medicinal herbs are widely used as home remedies in viral and bacterial infections due to their low toxicity effect in the human system.2 Selection of medicinal plants for drug discovery research is based on the knowledge of indigenous community and on the basis of their therapeutic uses.3

Phytochemical/secondary metabolites have being the subject for many research studies, because these compounds exhibit many pharmacological and biological activities. These include antibacterial, antifungal, antivirus, anticancer, anti-inflammatory and antioxidant among others.

Anti-oxidants are substances capable of mopping up free radicals and preventing them from causing cell damage. Free radicals are responsible for causing a wide number of health problems which include cancer, aging, heart diseases and gastric problems.4 Antioxidants cause protective effect by neutralizing free radicals, which are toxic by -products of natural cell metabolism. The human body naturally produces antioxidants but the process is not 100 percent effective, the production of free radicals and that of its effectiveness also declines with age5 increasing the antioxidant intake can prevent diseases and lower health problems. Fruits, vegetables and medicinal herbs are the richest source of antioxidant compounds.6 Blumea aurita (L.f) is an aromatic, annual, erect and glutinous herbaceous plant, up to 30100 cm tall and densely branched. The leaves are alternate and the base is decurrent on the stem and also possesses glandulous points. The inflorescence shows several involucres per head, formed with many white, yellow or mauve coloured small flowers with narrow bracts. The fruit is a campanulate capitule up to 1 cm large. Locally, it is used as an insect repellent. The leaves are said to have cicatrizing properties. The powdered plant or infusions are given for dyspepsia or indigestion, anodynes, painkiller and anesthetics.7 The plant is reportedly used in the treatment of constipation, inflammation, dyspepsia and aiding of wound healing. It is also used for the treatment of dysentery, rheumatic pain.8 It is also an anti hyperalgesic9, anti-inflammatory, anti-pyretic, analgesic and membrane stabilizing effects.10

B. lacera (Burm .f. ) DC. is an annual herb and grows up to 45 to 60cm long. The stem of this plant is hairy. The whole plant has a pungent smell, especially when squeezed. Flowers are bright yellow in colour. After blooming for several days prominently, the yellow spike like portion of the flower just turns into grey/white in colour which gives the flower a ball shape. Yellow achenes are nearly tetragonous and not ribbed. The plant is described in Ayurveda as bitter, astringent, anti-inflammatory, styptic, ophthalmic, digestive, anthelminthic, liver tonic, expectorant, febrifuge, antipyretic, diuretic, deobstruant, and stimulant.11 The plant extract possess anti-diarrhea, antimicrobial, anxiolytic, anti-atherothrombosis, membrane stabilizing and alpha-amylase inhibitory activities.12 The plant is also used in folk medicine for the treatment of cough, bronchitis, dysentery, wound healing.13 The plant also possesses anticancer activities.14 However, based on literature the ethno-medicinal potential of these plants are well pronounced but no report has been made about the comparative study between them despite the fact that they are from the same family. Therefore, the aim of this work is to determine the qualitative phytochemical, antibacterial and antioxidant potentials of B. aurita and B. Lacera with the intention of educating as well as providing the scientific justification of these herbs used as folk medicine by the local community.

Materials and Methods

Phytochemical analysis of the plant extracts

The extracts were screened for phytochemical using standard procedure.15

Clinical bacterial isolates

Three clinical bacterial isolates used in this research was obtained from Microbiology unit, University of Abuja Teaching Hospital, Gwagwalada. They include Escherichia coli, Salmonella typhi and Staphylococcus aureus.

Confirmation of the isolates

The isolates were sub cultured on Eosine Methylene Blue (EMB) agar for 24 to 48 hours. Colonies with green metallic sheen was observed which indicated a positive result for E. coli. The colonies were further subjected to citrate and urease test which showed a negative result for E. coli.16 Methyl red test was also positive.

The organisms were confirmed by subculture on Deocycholate Citrate Agar (DCA). A hydrogen sulphide production by Salmonella spp. It was further cultured on Simmon's Citrate Agar slant for 24 hours. A deep blue colour was observed which indicated a positive result for Salmonella spp.17 Methyl red test was also positive

The colonies that showed coagulase positive and catalase positive, in addition, yellow colonies andcleared yellow zone on mannitol salt agar confirmed the colonies are S. aureus.18

Disc preparation for antimicrobial susceptibility test

Using a perforator machine, a Whatman No.1 filter paper was punched to size standard disc of 6mm.The punched discs were sterilized using a pressure steam sterilizer (PSS YX-241D, England) at 1210C for 15 minutes. The concentrations of the extracts in each disc were obtained using serial dilution from the prepared stock solution (0.12 g). The stock solution of extracts was sterilized by filtration through 0.22 μm sterilizing Millipore express filter. Three set of discs with different concentrations were obtained (60, 30 and 15nµg/disc). Dimethylsulphoxide (DMSO) and Ciprofloxacin was used as negative and positive control respectively.

Antimicrobial assay

The In vitro antibacterial susceptibility of the extracts was carried out using the disc diffusion method by Clinical and Laboratory Standards Institute [19].The surface of Mueller Hinton agar plate was uniformly inoculated on Petri dishes with overnight stock culture of each of the bacterial species prepared in nutrient broth. The turbidity of the used clinical isolates were adjusted to match 0.5 McFarland standard 108 cfu/ml with sterile saline and inoculated onto the agar medium. Sterile Whatman No 1 filter paper discs (Whatman International Ltd., UK), 6 mm in diameter was used. The filter paper discs was soaked in the different extract concentrations, and dried at room temperature, they were carefully and aseptically placed into the Petri dishes seeded with inoculants. Ciprofloxacin (Oxoid ltd, UK) was used on the inoculated plates as positive control. Plates were kept for 30min to provide sufficient time for the test extract to diffuse into the medium and finally incubated at 37oC for 24 hours. The diameter of the zone of inhibition produced around the discs were measured with transparent centimeter ruler and converted to the nearest millimeter. All experiments were performed in duplicates and the zone of inhibitions were mean standard deviation of the duplicate.

Determination of minimum inhibitory concentration (MIC)

The MIC of isolates was carried out using tube dilution technique as described by20 McFarland turbidity standard of 0.5 which is approximately equivalent to106cfu/ml was used to standardize the suspension of test organisms. Each tube containing 2 ml of nutrient broth with addition of 20µl of different concentrations of each extract and loopful of test organisms. The tubes were prepared in duplicates and another set of tubes without extract was seeded with a loopful of the test organism to serve as the positive control while a tube containing 2 ml of nutrient broth that was not inoculated served as the negative control. After incubation for 24 hours at 37oC, the tubes were then examined for microbial growth by observing the turbidity using spectrophotometer UV/VIS (Model-ST-UV-7558).

Determination of minimum bactericidal concentration (MBC)

To determine the MBC, for each set of test tubes from the MIC determination, a loopful was streaked on a prepared sterile Mueller Hinton agar. The plates were then incubated at 37oC for 24 hours, the plates that has no visible growth was recorded as the minimum bactericidal concentration. Another Muller Hinton agar plate was streaked with the test organisms from non MIC determination tubes to serve as control.

Antioxidant activity

The ability of each extract to scavenge DPPH radicals was measured according to standard.21,22 3 ml of each plant extracts at a concentration of 100 μg/ml was mixed well with 1 ml of (0.1 mM of 2, 2-diphenyl-1-picrylhydrazyl; DPPH) in methanol. The mixture was shaken and left for 30 min in the dark at room temperature. Absorbance was measured at 517 nm against the blank using UV/VIS spectrophotometer (Model-ST-UV-7558). Ascorbic acid was used as a standard antioxidant. Controls contain only solvent and DPPH without any extract. The experiments were carried out in triplicate.

Results and Discussion

Table 1: The qualitative phytochemical screening of B. aurita and B. lacera.

Test

B. aurita

B. lacera

Tannins

+

+

Steroids

+

+

Triterpenoids

-

-

Saponins

+

-

Phenols

+

+

Glycosides

+

+

Alkaloids

+

+

Terpenoids

-

-

Carbohydrate

-

-

Flavonoids

+

+

The preliminary phytochemicals screening (Table 1) revealed presence of alkaloids, flavonoids, phenols, tannins, saponins, steroids, terpenoids, triterpenoids carbohydrates and glycosides. These phytochemicals immensely contributed to their antibacterial and pharmacological potential.23 Alkaloids, steroids, phenols, flavonoids and tannins are present in both the plants extract and this may be likely linked with the fact that the plants are from the same family (Asteraceae).

Table 2: Average zone of inhibition (mm) of B. lacera against clinical isolate.

Organisms

15

30

60

CPL

DMSO

E. coli

13.50±0.5

16.00±00

21.00±00

26.00±00

NA

S. typhi

13.00±00

16.50±0.5

20.00±00

26.00±00

NA

S. aureus

9.00 ±00

11.00±00

16.00±00

24.00 ±00

NA

NA: No Activity

Table 3: Average zone of inhibition (mm) of B. aurita against clinical isolate.

Organisms

15

30

60

CPL

DMSO

E. coli

15.00±00

18.00±0.5

22.00±00

26.00±00

NA

S. typhi

14.50±0.5

17.50±00

20.50±05

26.00±00

NA

S. aureus

10.00±0.5

14.00±00

18.00±00

24.00 ±00

NA

NA: No Activity

Table 4: Minimum inhibitory concentration (µg/ml) and minimum bactericidal concentrations of B. lacera and B. aurita.

Test Organisms

Concentrationsv(µg/ml)

B. lacera

B. aurita.

E. coli

3.5

++

++

7.5

+

+

15

MIC

MIC

30

MBC

MBC

S. typhi

3.5

++

++

7.5

+

+

15

MIC

MIC

30

MBC

MBC

S. aureus

3.5

++

++

7.5

++

++

15

+

+

30

MIC

MIC

highly turbid - ++; slightly turbid - +

The antibacterial properties B. lacera and B. aurita extracts (Table 2& 3) were promising on two Gram negative test organisms namely E. coli (21.00±00 – 22.00±00 mm) and S. typhi (20.00±00 mm) at 60 µg/disc each for B. lacera and B. aurita respectively. The extracts also showed activity on Gram positive bacteria namely S. aureus (16.00±00 -18.00±00 mm) at 60µg/disc each for B. lacera and B. aurita respectively. The antibacterial results activities agreed with12 on the activity of methanolic extract of B. lacera on eight (8) clinical isolates of bacteria both Gram negative and Gram positive. A research work was carried out by24 reported the antimicrobial activity of aqueous ethanol extract of B aurita on certain Gram negative and Gram positive bacteria. The MIC and MBC of both extracts where determined within 15 µg/ml to 30µg/ml on two tested Gram negative bacteria, but for S. aureus the MIC was determined at 30 µg/ml while the MBC was not determined within the prepared concentration range see Table 4.

Vit. C: Vitamin C., B. a: Blumea aurita, B. l: Blumea lacera

Figure 1: Antioxidant curve of B. aurita and B. lacera in comparisons with standard antioxidant Vit. C.

The antioxidant potential of the two extracts are low compared with standard antioxidant ascorbic acid (Vitamin C) while the B. lacera showed more promising antioxidant activity than B. aurita (Figure 1). Both extracts showed concentration dependency. The free radical scavenging activity of each extract was expressed as the percentage of DPPH decrease. It was expressed as IC50 which is defined as the concentration of each extract required for scavenging of 50% of DPPH radicals compared to that of ascorbic acid. The lower IC50 value is also an indication of higher scavenging activity or higher antioxidant activity of plant extracts as represented in increasing order 0.12 < 0.82 < 0.86, for Vitamin C, B. lacera and B. aurita respectively.

Medicinal plants are important sources of antioxidants.25 The secondary metabolites like phenolic and flavonoids from plants have been reported to be potent free radical scavengers. They are found in all parts of plants such as leaves, fruits, seeds, roots and bark.26 To avoid certain effects caused by synthetic antioxidant such as ageing, diabetes, skin lesions ,immune depression, pancreatitis, infertility, heart disease, hypertension, shock and trauma, memory loss and depression, asthma, pulmonary disease, hemorrhage, rheumatoid arthritis, uremia, peptic ulcer, inflammatory bowel disease, tumors and cancer, chronic renal failure and liver damage4 there is a need for more effective, less toxic and cost effective antioxidants. Medicinal plants appear to have these desired comparative advantages; hence, there is growing interest in natural antioxidants from plants.27-29

Conclusions

The current research work justifies some of the local medicinal uses of the two plants, and the findings showed that the antibacterial activity of B.aurita is more potent than that of B. lacera, while the antioxidant potentiality of B. lacera was more pronounced than that of B. aurita. The research confirms that B. aurita and B. lacera are good candidates for antibacterial and antioxidant uses. Thus, this may explain the traditional basis of using these plants in the treatment of various bacterial infections and other disease conditions.

Acknowledgements

The authors would like to express their sincere thanks to all staff of Biotechnology Advanced Laboratory for their enormous contribution to the success of this research.

Funding: No funding sources

Conflict of interest: None declared

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