INTRODUCTION:

Plants having medicinal values are natural resources, which are oftenly used in the treatment of various microbial diseases. ⁽¹⁾ Over the last few decades, medicinal plants are highly rich source of effective and safe medicines. In current times there has been much focus on plants with antimicrobial activity. There are various reports on the effectiveness of wild plants against broad spectrum microorganisms and as a result, plants are still recognized as the backbone for modern medicine to treat various infectious diseases.⁽²⁾ Antimicrobial properties of medicinal plants are being progressively reported from different parts of the world. ^(3’4’5)Some food substances such as garlic and onion containing allicin and allin (cinnamic aldehyde) respectively can be use for protection against pathogenic microorganisms.⁽⁶⁾ Many published reports shown that the higher plants have could be a potential source for the new antimicrobial agents. ⁽⁷⁾

The antimicrobial compounds from plants may inhibit bacterial growth by different mechanisms than those presently used. Antimicrobials therefore, may have a significant clinical value in treatment of resistant microbial strains.⁽⁸⁾ Besides, the antimicrobial activity of plant oils and extracts have formed the basis of many applications including raw and processed food preservation, pharmaceuticals, alternative medicine, and natural therapies.⁽⁹⁾ Although M. Sudha Devi and M. Thenmozhi (2011)⁽¹⁰⁾ investigated about antibacterial activity of Plumbago zeylanica leaf extracts pharmaceutical studies on these plants are inadequate. This study was therefore carried out in order to investigate the antimicrobial activity.

Natural products can be selected for biological screening based on ethnomedical use of plants, because many infectious diseases are known to have been treated with herbal remedies throughout the history of mankind. . Even today, plant materials continue to play a major role in primary health care as therapeutic remedies in many developing countries ^(11’12).

In this study, Plumbago zeylanica leaf extracts, having traditional claims for several diseases were investigated for the common pathogenic microorganisms that show highest degrees of resistance against many antimicrobial agents. The ethnobotanical data on the traditional uses of this plant and selection of the plant parts to be tested was complemented with a literature review and information gathered from traditional healers.

MATERIALS AND METHODS:

Plant materials:

Fresh plant materials Plumbago zeylanica was collected from different sites of their occurrence. Leaves of plants were used to assay.

Preparation of crude extracts:

Extraction of plant material was done by commercially available kit (behr Labor – Technik GmbH) with some modifications.

The plant materials were dried in shade at room temperature and ground separately in grinder _{before filling them in thimbles for extraction.}

Figure : Crude P. zeylanic

Figure : Soxhlet extraction

The ground weighed material (fine coarse powder), was extracted by different solvents viz petroleum ether, chloroform, acetone, methanol, ethanol and distilled water in a specific sequence based on increasing polarity. The soxhlet hot extraction procedure for each of the above solvents was run for about 6 hours, until a colorless solvent was seen in the siphon tube, which indicated complete extraction. The solvents were removed under reduced pressure and controlled temperature by rotary evaporator. The extracts were dried and stored in a clean glass bottle and kept at 4-6⁰C for further _{antimicrobial screening.}

Microorganisms and condition for cultivation:

In this study, the test organisms were obtained from Microbiology department of JN Medical College, Belgaum (KLES University)

Antimicrobial assays:

The antimicrobial activities of the Soxhlet extracts of plants were analyzed by the disc diffusion method^{. (13’14)}

The inoculums size of each group of bacteria and yeast were prepared by using a no. 0.5 McFarland standard to give a concentration of 1 x 10⁸ bacteria and 1 x 10⁶ yeast per milliliter. In order to test the antimicrobial activity of plants, 15 ml of Mueller Hinton Agar (MHA) were poured in Petri dishes which were then inoculated with strains of bacteria by taking 0.1 ml from cell culture media. It was kept to solidify at room temperature for a kindly specifies the time range. Whatman filter paper no. 1 was used to prepare discs approximately 6 mm in diameter, sterilized filter paper disks were socked in liquefied crude extracts and were subjected to air drying , placed on inoculated culture media. Plant extracted using each solvent was tested against the three microorganisms. Disks that were only impregnated with extractant solvents were used as a control.

RESULT:

A plant used in our study was selected for its importance in ethnobotanic. The antimicrobial activity of Soxhlet extract of Plumbago zeylanica was evaluated in vitro against three microorganisms, which are known to cause hospital acquired infections in humans. Results of antimicrobial activity of Soxhlet extract of used plants against the test microorganisms are shown in Table 1.

Plant name	Solvent	Concentration	Result
Plant name	Solvent	Concentration	E.coli	S.aures	C.albicans
Plumbago zeylanica	P.ether	1 g/ml	+++	+++	---
	Chloroform	1 g/ml	---	---	---
	Acetone	1 g/ml	+++	+++	---
	Methanol	1 g/ml	---	---	---
	Ethanol	1 g/ml	---	+	+
	Water	1 g/ml	---	---	---

Table 2: Secondary antimicrobial screening of P. zeylanica using selected solvents at 100mg/ml. NT = Not tested, “=” No zone of inhibition

Plant name	Solvent	Concentration	Result/Zone of inhibition (mm)
Plant name	Solvent	Concentration	E.coli	S.aures	C.albicans
Plumbago zeylanica	P.ether	100 mg/ml	8	34	=
	Acetone	100 mg/ml	14	36	=
	Ethanol	100 mg/ml	=	14	13
Control	Vancomycin	10 µg/disk	NT	28	NT
	Gentamycin	10 µg/disk	25	37	NT
	Ciprofloxacin	10 µg/disk	34	NT	NT

Table 3: Minimum inhibition concentration of Plumbago zeylanica using petroleum ether, acetone and ethanol. NT= Not tested, “=” Zone of inhibition.

Plant name

MIC Zone of inhibition (mm)

E. coli

S. aureus

C. albican

Plumbago zeylanica

100 mg/ml

50 mg/ml

30 mg/ml

25 mg/ml

10 mg/ml

100 mg/ml

50 mg/ml

30 mg/ml

25 mg/ml

10 mg/ml

100 mg/ml

50 mg/ml

30 mg/ml

25 mg/ml

10 mg/ml

Petroleum ether

Acetone

Ethanol

Table 4: Secondary antimicrobial screening of medicinal plants using selected solvents at 50mg/ml. NT = Not tested, “=” No zone of inhibition

Plant name	Solvent	Concentration	Result/Zone of inhibition (mm)
Plant name	Solvent	Concentration	E. coli	S. aureus	C. *albicans*
Plumbago zeylanica	P.ether	50 mg/ml	=	21	=
	Acetone	50 mg/ml	13	31	=
	Ethanol	50 mg/ml	=	10	12
Control	Vancomycin	10 µg/disk	NT	28	NT
	Gentamycin	10 µg/disk	25	37	NT
	Ciprofloxacin	10 µg/disk	34	NT	NT

After getting results from primary screening, shown in (Table 1), secondary screening was done for the initiation to determine minimum inhibitory concentration (MIC).

The second screening was done in concentrations of 100 mg/ml and 50 mg/ml. Firstly the liquefied plant extract was again dried to get the initial weight, and then it was dissolved in its corresponding solvents where they originally extracted from, to obtain the respective concentration. The disks were then aseptically impregnated with 10 µL per disk of the 50 mg/ml using micropipette and left to dry at room temperature. The same impregnation was also performed to the 100 mg/ml.

The antibiotic control disks used were selective for the gram positive microorganism (Staphylococci aureus), Viz Vancomycin of 10µg/disk and Gentamycin, Ciprofloxacin of 10 µg/disk each for gram negative organisms (Escherichia coli).

According to the satisfactory result found (Table 2), P. zeylanica was further investigated for determining the minimum inhibition concentration (MIC).

Minimum inhibitory concentration

Determination of MIC was on the basis of measurement obtained from the zones of inhibition by the concentration dilution method. Dilution of extracts was done on the basis of result found from secondary screening. Plumbago zeylanica was randomly diluted to obtain 100 mg/ml, 50 mg/ml, 30 mg/ml, 25 mg/ml, 10 mg/ml.

The result illustrated that antimicrobial activity of non-polar and polar extract of Plumbago zeylanica showed high activity profile against the three strains using petroleum ether, acetone and ethanol. This result supports the fact that the active compounds are more concentrated in this extracts. The results were supported by Nurdan Sarac and Aysel Ugur (2007)⁽¹⁵⁾

DISCUSSION:

In general, among the tested microbial strains, bacteria were found to be more sensitive to Plumbago zeylanica than fungi. The antibacterial activity was more pronounced on the gram-positive bacteria (S. aureus) than the gram-negative bacteria (E. coli). The reason for the difference in sensitivity between gram-positive and gram-negative bacteria might be described to the differences in morphological constitutions between these microorganisms, gram-negative bacteria having an outer phospholipidic membrane carrying the structural lipopolysaccharide components. This makes the cell wall impermeable to antimicrobial chemical substances. The gram-positive bacteria on the other hand are more susceptible having only an outer peptidoglycan layer which is not an effective permeability barrier. Therefore, the cell walls of gram negative organisms are more complex in lay out than the gram positive ones acting as a diffusional barrier and making them less susceptible to the antimicrobial agents than are gram positive bacteria.^(16’17)

Staphylococci and E. coli are among the most commonly encountered pathogens in clinical practice. S. aureus is a major cause of hospital acquired infections, food poisoning, osteomyelitis, pyoarthritis, endocarditis, toxic shock syndrome, and a broad spectrum of other disorders ^{(18’19’20).} In recent years, there has been an alarming increase in hospital acquired staphylococcal and E. coli infections by strains with multiple drug resistance ^{(21’22’23)}. At present, this situation is leading to the evaluation of staphylococcal pathogens potentially resistant to any available antibiotic ^{(24’25’20)}

When the antimicrobial activities of Plumbago zeylanica was compared to that of the positive controls, Plumbago zeylanica (at a concentration of 100 mg/ml) was found to have almost comparable activity to the standard Vancomycin against bacteria. The negative controls which were impregnated in the solvents were only found to be devoid of any antimicrobial activity. In general, results of antimicrobial screening of the extract initiated to do the minimum inhibition concentration (MIC) of the plants which showed activity at 100 mg/ml concentration.

The point of the treatment of hospital acquired infections, it was a consequential decision. Therefore, this result may suggest that the value of ethno-botanical significance can be widely seen from the studies made on herbal traditional medications in search of new lead drug sources. From this study the plant P. zeylanica can be considered as an important source for future drug synthesis to treat the claimed illnesses in human.

ACKNOWLEDGEMENT:

Authors are thankful to the management of JN Medical College, Belgaum (KLES University) for providing necessary facilities.

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Received on 27.08.2012 Modified on 13.09.2012

Research J. Pharm. and Tech. 5(10): October 2012; Page 1297-1300