aqueous, acetone and ethanolic extract of nerium indicum l

International Journal of Applied Environmental Sciences

ISSN 0973-6077 Volume 12, Number 9 (2017), pp. 1721-1732

© Research India Publications

http://www.ripublication.com

Aqueous, Acetone and Ethanolic extract of Nerium

indicum L. as potential antibacterial agent against

Pseudomonosa aeruginosa.

Kavita Chetwani*, Dr. R.K. Agnihotri and Dr. Purti Chaturvedi

Khandari Campus, Department of Botany, Dr. B.R. Ambedkar University Agra, India

Abstract

Bacterial diseases are widespread all over the world. The problem of bacterial

infections of Pseudomonas aeruginosa which effect environment as well as

human causes nosocomial infections hence a result of it is ubiquitous nature,

ability to survive in moist environments and resistance to many antibiotics and

antiseptics. Nerium indicum which act as antibacterial potential, parts of plant

are accounted as a curative agent and have been used in wisdom in a variety of

ailments. The leaves are the important part of this plant which is rich in

bioactive compounds such as glycoside, Oleandrin, flavonoid, tannin, neriin,

phytosterin and l-strophnathin, rosaginin and nerlin, volatile oil, fixed oil,

neriodorin and neriodorein, which exhibited antimicrobial activities. In present

study, N. indicum extracts were analysed for various bio-molecules and their

antibacterial affectivity was studied. N. indicum extracts viz, acetone, ethanol

and aqueous at different concentrations (200, 100, 50, 25, 12.5, 6.25 and 3.125

mg/ml) were prepared and their effectiveness as an antibacterial were studied

against P. aeruginosa by using disc diffusion method and MIC. Observations

of study showed the effectiveness in all the extracts tested. It was also

observed that acetone extract was most effective in comparison to ethanol and

aqueous extracts.

Keywords: Antibacterial activity, Bioactive compounds, MIC.

1722 Kavita Chetwani, Dr. R.K. Agnihotri and Dr. Purti Chaturvedi

INTRODUCTION

N. indicum Linn.commonly known as “kaner”, belongs to family Apocynaceae. It is

widely distributed in Mediterranean region and Subtropical Asia but is native of

Indian subcontinent and cultivated elsewhere. The plant originates from the

Mediterranean region and is indigenous to Indo-Pakistan subcontinent (Patel, 2010).

All parts of the plant are utilizing as curative agent and have been used in folklore in a

variety of ailments. It is also an ornamental plant with leathery evergreen leaves and

with clusters of red or pink or white flowers. The plant leaves and bark are used as

heart tonic, diuretic, expectorant, diaphoretic and emetic (Patel et al.,2010). Leaf juice

in very small doses is given in snake and other venomous bites. Juice of young leaves

are effective in ophthalmic with lacrimation infusion of the leaves is abortive. Root

past is helpful in haemorrhoids, in various kinds of cancer like Ulceration and leprosy

(Vinayagam et al.,2011, Ahmed, 2006, Chauhan et al., 2013, Sikkarwar et al., 2003).

The selected plant is also used in ayurveda for the treatment of various diseases.

Various phytochemical can be obtained from the plants which are very beneficial for

mankind and medicinal plants have become the richest biological resource for such

chemicals which are utilize in manufacturing of traditional drugs as well as in modern

nutraceuticals, food supplements, medicines, folk medicines, raw material and

pharmaceutical intermediates for synthetic drugs (Tumwine, 2011). The presence or

absence of such compounds totally depends on the extent of accumulation, the

amount of plant material used and the analytical method selected (Harborne, 1973).

Harmful bacteria that cause bacterial infections and diseases are called pathogenic

bacteria. Bacterial diseases occur when pathogenic bacteria get into the body and

begin to reproduce and cloud out healthy bacteria, or grow in tissues that are normally

sterile. Bacteria may also give out toxins that damage the body. The control of

bacterial infection has been remarkably effective so the discovery of antibacterial

drugs is necessary (Rios and Recio, 2005). Infectious diseases remain the leading

cause of death and account for one-quarter of all death in the world (WHO, 1998).

Antibiotics are one of our most important weapons in fighting bacterial infections and

much benefited to the health-related quality of human life since their introduction.

Hence, over the past few decades, these health profits are under the threat and many

commonly used antibiotics have become less and less effective against certain

illnesses, because many of them produce toxic reactions. This situation has forced

researchers to search for new antimicrobial substances from various sources as novel

antimicrobial chemotherapeutic agents, but the cost production of antibiotics are high

and they produce adverse effects compared to plant derived drugs (Abiramasundari et al., 2011). The existence of effective antibiotic resistant or multi-resistant strains are

gently appearing, imposing the necessary need to search and development of new

drugs (Silver, 1993).

Aqueous, Acetone and Ethanolic extract of Nerium indicum L. as potential… 1723

Consequently, step must be taken to overcome this problem, for example, to control

the excess use of antibiotic, investigation should be done to better understand the

genetic mechanisms of resistance, and to continue studies to develop new drugs,

either synthetic or natural. The ultimate aim is to offer appropriate and efficient

antimicrobial drugs. Hence the current investigation aim to screen and evaluate

certain antibacterial activity of N. indicum.

MATERIALS AND METHODS

Collection and storage of plant material: The leaves of plant were collected from

Department of Botany, S.L.S., Khandari campus, Dr. B.R. Ambedkar University,

Agra. The stored leaves were thoroughly washed with tap water and followed by

distilled water. After cleaning the leaves were dried in shade and grinded into

powdered form with the help of grinder.

Test Organisms used: Clinical pure culture of Pseudomonas aeruginosa were used

in this study. This was obtained from the Department of Botany, Microbiology.

S.L.S., Khandari campus, Dr. B.R. Ambedkar University, Agra. A 24 hour broth

culture of the selected species was used for antibacterial screening.

Extraction of Plant Materials:

Aqueous extract: About 50gm of leaf powder was added to distilled water and boiled

on slow heat for 4-5 hrs. It was then filtered through muslin cloth. The filtrate thus

obtained was further stained through Whatman No. 1 filter paper. The extraction was

carried out at room temperature.

Organic extract: Soxhlet apparatus was used (Okeke et al. 2001). About 50gm of dry

powder material was uniformly packed into a thimble with 300ml solvent and runs

into a soxhlet apparatus. Extract is store in airtight bottle at 4ºC in a refrigerator for

further use.

Preliminary phytochemical analysis (Debela 2002): The plant extracts (acetone,

ethanol and aqueous) were subjected to phytochemical studies to qualitatively analyze

the active components present in them. The compounds screened are alkaloids, tannin

compounds, glycosides, flavonoids, steroids and triterpenoids, carbohydrates, proteins

and amino acids.

Antibacterial activity: Disc diffusion method (Mukherjee et al.1995)

Minimum Inhibitory Concentration (MIC): Different concentrations of the

N.indicum leaves were prepared. 25µl overnight broth culture of the test organism

were inoculated into the dilutions with different concentrations and incubated at 37°C

for 24 hours. The lowest concentration of the extract that inhibited the growth of the


test organism was recorded as the MIC.

Minimum Bactericidal Concentration (MBC): This was described by (Usman et al. 2007), the inhibited test tubes resulted from MIC was streaked using a sterile wire

loop on to the agar plate free of bacteria and incubated at 37ºC for 24 hours. The

lowest concentration of extract which showed no bacterial growth was noted and

recorded as the MBC.

RESULTS

Preliminary phytochemical analysis: Phytochemical constituents are secondary

metabolites of plants and several other aromatic compounds are secondary

metabolites of plants that act as a defence mechanism against the microorganisms,

insects and other herbivores (Bonjar et al., 2004) The antimicrobial activity may due

to a wide variety of secondary metabolites, such as tannins, terpenoids, alkaloids, and

flavonoids that have antimicrobial activities (Crown, 1999). This may therefore

explain the demonstration of antibacterial activity of N. indicum.

Table-1: Preliminary Phytochemical analysis of different leaf extracts of Nerium indicum leaves:

S.No. Phytochemical

constituents Name of the Test Ethanol Acetone Aqueous

1. Alkaloids Mayer’s Test + + +

2. Tannin compounds Ferric chloride

Test + + +

3. Glycosides Borntrager’s Test - - -

4. Flavonoids Alkaline Reagent

Test + + +

5. Steroids and

Triterpenoids Salkowski Test + - -

6. Carbohydrates Benedict’s Test + + +

7. Proteins Ninhydrin Test - - -

8. Amino Acids Buiret Test - - -

+ = Present; - = Absent

Antibacterial activity of different leaf extracts of Nerium indicum:

Different concentrations of N. indicum leaves extract was prepared and was evaluated

by agar disc diffusion method. It was observed that all leaves extract of N. indicum which were prepared were effective against Gram negative bacteria and displayed


broad spectrum of activity. The disc diffusion method was mainly used for the

determination of antibacterial action. Among the different methods, disc diffusion has

been used more frequently to examine the antimicrobial activity of natural

antimicrobials (Kim & Kim, 2007; Mayachiew et al., 2010).

The results obtained are encouraging as the ethanol, acetone and aqueous extracts

showed considerable effectivity. It was observed that most active result was exhibited

in acetone extract and ethanol leaves extract had shown moderate result.

Comparatively in case of aqueous leaves extract starting three concentrations showed

moderate result in comparison to both two extracts and rest four concentrations

showed least activity with respective zone of inhibitions against the P. aeruginosa,

given in table-3.

Table-3: The Antibacterial activity of different leaf extracts of Nerium indicum

against P. aeruginosa:

Bacterial

pathogen

Different

concentration

(mg/ml)

Acetone Ethanol Aqueous

Mean S.D. Mean S.D. Mean S.D.

P. aeruginosa

200 mg/ml 16.83 1.21 10.33 2.68 12.33 2.74

100 mg/ml 14.16 0.94 9.16 1.46 10.66 1.49

50 mg/ml 11.83 0.68 8.83 1.21 9.16 0.9

25 mg/ml 9.83 0.37 8.83 1.21 7.33 3.4

12.5 mg/ml 9.16 0.69 8.66 0.94 7.00 3.21

6.25 mg/ml 8.16 0.37 7.5 0.5 5.00 3.55

3.125 mg/ml 8.33 0.46 7.5 0.5 2.5 3.54

S.D.= Standard Deviation

ANNOVA TABLE:

Source of Variation SS DF MS F P-value at 1%

Between Column 44.95 2 22.47 10.07 6.01

Between Raw 110.46 6 18.41 8.25 4.46

Within Groups 26.87 12 2.23

Total 182.28 20 43.11

CD = 1.946 at 1%; **Significant (P = 0.01)


Figure 1: Graphical representation of antibacterial activity of different leaves extracts

of N. indicum against P. aeruginosa:

MIC and MBC of N. indicum leaf extracts against P. aeruginosa: Micro dilution

method was used to determine the lowest concentration of the plant extracts that

inhibiting the growth of the bacteria and found to be effective in the assessment of

MIC.

The MIC value of acetone and ethanol leaves extract of N. indicum was found to be

25mg/ml against P. aeruginosa. The MIC value of aqueous leaves extract of N. indicum was found to be less potent 100mg/ml.

Table 4: The MIC and MBC of Nerium indicum leaf extracts against Staphylococcus aureus and Pseudomonas aeruginosa.

S.No. Bacterial strains Plant extracts MIC

(mg/ml) MBC

2. P. aeruginosa

Acetone 25

No microbial

death Ethanol 25

Aqueous 100

DISCUSSION

Many naturally occurring compounds present in plants have been shown to possess

antimicrobial properties and perform as a source of antimicrobial agents against

pathogens (Deans and Ritchie 1987; Kumar et al., 2006). Higher plants are a very

Zo

ne

of

inh

ibit

ion

s (m

m)

Different Concentrations (mg/ml)


good source of medicines that made significant contribution for human health

(Anesini and Perez, 1993, Adoum, 2009). The presence of antibacterial substances

used for the treatment of many diseases in the higher plants is well originated.

(Fridous et al., 1990, Didry et al., 1998).

The main aim of the present study was to evaluate the ability of the plant extract to

inhibit the growth of pathogenic bacteria. Study conducted by Zaheer et al., 2010, in

vitro antibacterial activity of P.alba against Gram positive bacteria Staphylococcus aureus, Bacillus subtilis, and Gram negative bacteria Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi was carried out and fruitful result was observed against

Staphylococcus aureus and Bacillus subtilis.

Phytochemical analysis of the plant extracts:

Phytochemical compounds such as alkaloids, tannins, flavonoids, saponins, and

several other aromatic compounds are secondary metabolites of plants that act as a

defence mechanism against the microorganisms, insects and other herbivores (Bonjar

et al., 2004). The present study investigation was carried out on the leaves extracts

which revealed the presence of the medicinally active constituents.

The phytochemical analysis of N. indicum summarized in Tables before and the

bioactive compounds of N. indicum may differ in their solubility depending on the

extractive solvents used. This is supported from the research of De-Boer et al., 2005

that successful prediction of botanical components from plant material is largely

dependent on the type of solvent used in the extraction procedure. Earlier study of

Suganya et al., 2012 elaborated that the ethanol extract of N.oleander showed positive

for carbohydrates, proteins, alkaloids, cardiac glycosidase, tannins and for acetone

extract showed positive for alkaloids, cardiac glycosidase, and tannins, and these

findings are similar to Santhi R et al., 2011 whereas Malik et al., 2015 exhibited that

the highest activity of the N. oleander might be due the presence of terpenoids,

saponins andamino acids.

Antibacterial Activity of the Plant Extracts:

In present study, all the extracts of N. indicum leaves were found to be active against

the bacterial strain for their antibacterial activity. It was noted that acetone extract has

greater effect in the inhibition from aqueous extract, which may be due to the fact that

acetone is the best solvent for the active compounds extracted from the plant when

compared with distilled water used in the case of aqueous extracts. (Jouda 2013)

It was observed that the acetone leaves extract of N. indicum was more active and

showing inhibition effect up to dilution of 3.125mg/ml whereas Ates et al (2003),

reported that the acetone extracts of Cinnamomum cassia bark extracts showed no


antibacterial activity against Y. enterocolitica

The ethanolic leaves extract of N. indicum was found to be moderate and showing

inhibition effect up to dilution of 3.125mg/ml whereas Malik et al., 2015 ethanolic

extract of Nerium oleander showed biggest zone of inhibition 28mm at highest

concentration (900mg/ml), whereas at lowest concentration (500mg/ml) of extract had

the minimum zone of inhibition 18mm was observed against P.aeruginosa also Malik

et al., 2015, showed that the ethanolic extract of N. tabacum showed the maximum

zone of inhibition (26mm) against Staphylococcus aureus whereas there was a less

difference in zones were 20mm and 21mm against Escherichia coli and P. aeruginosa respectively.

The Aqueous leaves extract of N. indicum was found to be least. Bakhiel et al.(2008)

showed that the aqueous extract of C. sativum was active against S. aureus, P. aeruginosa and S. typhi. Whereas, in result of Seddik et al., 2010 and Mohamed et al., 2010, which demonstrated that A. herba-alba aqueous extracts had a weak

antibacterial activity against E.coli, but in the present study leaves aqueous extract of

N. indicum showed efficacy for both the bacterial strains.

MIC of the plant extracts:

The MIC value of acetone and ethanol leaves extract of N. indicum against P. aeruginosa was found to be 25 mg/ml and these extracts of N. indicum gave the best

antibacterial activity. Jouda 2013, reported that the MIC value of the methanol leaves

extract of F. sycomorus was efficiently active and exhibiting the highest potency with

MIC from 6.25-3.125 mg/mL against S.aureus and same for aqueous extract also,

whereas the MIC values obtained showed that ethanol extract of E. camaldulensis has

the most potent effect against P. aeruginosa.

The MIC value of aqueous leaves extract of N. indicum was found to be less potent

100mg/ml against P. aeruginosa. Present study was supported by the Britto et al., 2011, investigated that the methanol extracts for plants of Apocynaceae family had

shown 12.5mg/ml concentration was found to be the minimum inhibitory

concentration against most of the pathogens.

CONCLUSION

Many natural compounds, especially plants have been used for the treatment of

several diseases and an impressive number of new medicines have been developed

from them. Thus the plant kingdom is really a potential source of medicinal

properties. Plant have their own chemical compounds, some of those chemical

compounds also have curative values for us. In recent years, ethno-botanical and

folkfore uses of natural compounds, especially of plant origin received much attention


as they are well tested for their efficacy and generally believed to be safe for human to

use. Therefore, our results indicate that this plant extracts have inhibitory effect for

growth of microorganism against the bacteria under studies, due to presence of some

active compounds in this plant which act as natural antibiotics that can be use instead

of antibiotics after experiments have performed on it. Hence, in addition to numerous

other medicinal properties, the antibacterial activities of N. indicum are also coming to

light; effective steps are to be taken for its propagation so that this valuable tree does

not face the danger of extinction.

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