Evaluation of Antimicrobial and Phytochemical Properties of some Indigenous Indian plants

Robinka Khajuria, Loveleen Kaur*, Aditi Kaushik and Gurpreet Saredia

Department of Biotechnology, Lovely Professional University, Phagwara-144402

*Corresponding Author E-mail: loveleen.16406@lpu.co.in

ABSTRACT:

Methanolic, ethanolic and aqueous extracts of ten indigenous Indian plants Alstonia scholaris, Anthocepalus cadamba, Elaeocarpus ganitrus, Madhuca indica, Butea frundosa, Berberis aristata, Abutilon indicum, Nyctanthes arbor tristis, Aegle marmelos and Urginea indica were investigated for antimicrobial activity against E. coli, B. subtilis and S. aureus followed by determination of Minimum inhibitory concentration of the plant extracts. A primary phytochemical screening was done to determine the presence of Terpenoids, Tannins, Anthraquinone, Steroids, Saponins, Alkaloids and Flavonoids. Methanolic extract of B. aristata was found to have potent antibacterial activity against three bacterial cultures followed by A. indicum. The largest zone of inhibition (24mm) was observed against B. subtilis in the presence of crude Methanolic extract of B. aristata. Minimal Inhibitory concentration B.aristata was found to vary from 40µg/ml to 73µg/ml for the three bacterial cultures. All the tested phytochemicals were present in B.aristata and A. cadamba indicating that these plants could be further exploited for production of bioformulations for treating infectious diseases.

KEYWORDS: Antimicrobial property, Berberis aristata, Indigenous plants, Minimum inhibitory concentration, Phytochemicals

INTRODUCTION:

Since the past few decades, emerging and re-emerging multidrug resistant pathogenic strains of microorganisms have become a chief cause of concern for the medical practitioners worldwide. Screening of several medicinal plants for their potential antimicrobial activity is being noticed due to the increasing failure of conventional chemotherapeutics and antibiotics (Colombo et al.1996, Rojas et al 2003, Bandow et al 2003, Zinn et al 2004). In recent years, secondary plant metabolites (phytochemicals), formerly having unknown pharmacological activities, have been extensively investigated as a source of medicinal agents (Joshi et al 2011). The beneficial products from plants result from combinations of different secondary metabolites like alkaloids, saponins, tannins, anthraquinone, etc. Exploitation of these phytochemicals produced by certain plants can serve as a solution to the ever increasing problem of drug resistant pathogens.

India is one of the World’s top 12 mega diversity countries with 10 biogeographical regions. The assemblage of climatic and altitudinal variations coupled with variations in ecological habitats has led to the development of unique diversity in medicinal plants which serve as source of raw material for traditional medicine systems as well as for pharmaceutical industries.(Pushpangadan 2002). India has more than 1/4^th of World’s known medicinal plants with over 8000 plants species. Over one and a half million traditional healers use a wide range of medicinal plants for treating ailments of both humans and livestock across the length and breadth of the country. Different parts of ethnomedical plants or their extracts are used for the treatment of various diseases. This study was undertaken in order to carry out preliminary investigative studies on the antimicrobial properties of ten indigenous Indian plants against three test organisms. Moreover, phytochemical analysis was carried out in order to detect the presence of different phytochemicals known for their therapeutic effects.

Table1: List of plants used in the study and their traditional importance

Family	Botanical Name	Local Name	Plant part Tested	Major Traditional Uses (References)
Apocynaceae	Alstonia scholaris	Sataparni (Devil Tree)	Leaves	Treatment of Epilepsy, Asthma, Malarial Fever, Urticaria, Chronic Dysentery, Diarrhoea (Lee et al, 2012)
Rubiaceae	Anthocepalus cadamba	Kadam	Leaves	Antidiuretic, Anthelmintic, Blood purifier, Analgesic
Elaeocar-paceae	Elaeocarpus ganitrus	Rudraksh	Leaves	Treatment of mental diseases, epilepsy, asthma, hypertension, arthritis and liver diseases (Hule and Juvekar, 2009)
Sapotaceae	Madhuca indica	Mahua	Leaves	Pain killer, Anthelmintic, Antidiabetic, Analgesic, Antiarthritis agent (Shekhawat and Vijayvergiya, 2010)
Fabaceae	Butea frundosa	Palash	Leaves	Astringent, Diuretic, Depuretive, Aphrodisiac and Tonic properties. Effective in leprosy, leucorrhoea and gout. (Das et al, 2012)
Berberidaceae	Berberis aristata	Indian Barberry or Daru Haridra	Leaves	Antipyretic, antibacterial, antihepatotoxic, anticancer antihyperglycaemic, antioxidant and antilipidemic agent. (Potdar et al, 2012)
Malvaceae	Abutilon indicum	Atibala	Leaves	Aphrodisiac, laxative, diuretic, sedative, astringent,tonic, anti-inflammatory, anthelmintic, and analgesic agent. (Rajakaruna et al, 2002)
Oleaceae	Nyctanthes arbortristis	Parijat or Harsingar	Leaves	Antibacterial, Anthelmintic, Antiinflammatory, Hepatoprotective, Immunopotential, Antipyretic, Antioxidant and Anti fungal. (Saxena et al, 2002)
Rutaceae	Aegle marmelos	Bael or Bilvapatra	Leaves	Antibacterial, Treatment of constipation and other gastrointestinal problems. (Pattnaik et al,1996)
Asparagaceae	Urginea indica	Jangli Kanda	Leaves	Expectorant, cardiac, stimulant, diuretic, bronchitis, chronic asthma, deobstruent (Shiva-Kameshwari et al, 2012)

MATERIAL AND METHODS:

Collection of plant material:

Leaves of all the ten indigenous Indian plants, Alstonia scholaris, Anthocepalus cadamba, Elaeocarpus ganitrus, Madhuca indica, Berberis aristata, Abutilon indicum, Nyctanthes arbor-tristis, Aegle marmelos, Urginea indica and Butea frondosa were collected from Botanical garden of Panjab University, Chandigarh and Herbal Garden, Lovely Professional University, Phagwara (Table 1)

Preparation of plant sample:

Fresh Green leaves were washed under running tap water, dried in hot air oven at 60⁰C for 24 hours and ground to fine powder.

Maintenance of bacterial culture:

24 hour old broth cultures of Escherichia coli, Bacillus subtilis and Staphylococcus aureus were maintained in actively growing state by regular subculturing.

Preparation of Plant Extracts:

15 gm of leaf powder was extracted with methanol by continuous hot extraction for 8 hours. The extract obtained was concentrated in vacuum rotary evaporator and was used for determination of antibacterial activity as described by Joshi et al (2011). The crude samples were subjected to phytochemical screening for the presence of Terpenoids, Tannins, Anthraquinone, Steroids, Saponins, Alkaloids and Flavonoids. Similarly extraction was carried out using ethanol as extractant. 15 gm of leaf powder was also used for preparation of hot water extract and extraction was carried out according to Parekh and Chanda (2007).

Table2: Determination of zone of inhibition of indigenous Indian plant extracts against different test organisms.

	*Zone of Inhibition(diameter in mm)
Test Organism Plants	*E. coli*			*S. aureus*			*B. subtilis*
Test Organism Plants	M	E	A	M	E	A	M	E	A
Alstonia scholaris	12.3	12.0	12.3	6.0	6.0	6.0	5.7	ND	5.6
Anthocepalus cadamba	ND	ND	ND	6.3	ND	6.3	8.3	7.5	8.3
Elaeocarpus ganitrus	8.3	6.5	8.3	8.3	8.0	8.3	6.0	6.0	6.0
Madhuca indica	ND	ND	ND	ND	ND	ND	8.3	7.0	8.3
Butea frundosa	6.3	6.0	6.3	6.0	6.0	6.0	ND	ND	ND
Berberis aristata	22.0	20.0	19.0	20.0	20.0	18.0	24.0	20.0	20.0
Abutilon indicum	14.0	18.0	12.0	20.0	16.0	13.0	18.0	14.0	14.0
Nyctanthes arbor – tristis	18.0	16.0	16.0	14.0	14.0	13.0	14.0	16.0	12.0
Aegle marmelos	16.0	11.0	12.0	16.0	16.0	13.0	9.0	12.0	8.0
Urginea indica	12.0	11.0	11.0	11.0	9.0	9.0	9.0	9.0	8.0
CD@5%	3.29	2.37	1.47	1.48	1.77	1.49	1.74	1.57	1.42

M- Methanolic Extract, E- Ethanolic Extract, A- Aqueous Extract, ND- Not Detected

* Average of three replicates

Antimicrobial assay:

Lawn culture of the three bacterial cultures were prepared on nutrient agar plates using sterile swabs. Wells were prepared using 4mm cork borer and 100µl of methanolic extract, ethanolic extract and hot aqueous extract was added in their respective wells. 1mg/ml of Ampicillin was used as Positive control and respective solvent was used as a negative control. Plates were than incubated at 37⁰C for 24 hours. Thereafter diameter of zone of inhibition was measured and recorded. Zone less than 5mm was considered to be non-detectable.

Antimicrobial assay:

Minimal Inhibitory Concentration (MIC):

Since methnolic extracts showed better inhibition of microorganisms, so the residues of plant extracts were dissolved to 25 mg/ml using methanol. All extracts were serially diluted two-fold to1.95 μg/ml in a 96-multi- well microplate. 100 μl (1 × 10⁶ CFU/ml) of actively growing bacterial culture was added to each well. Ampicillin was added as reference antibiotic in each assay. Extract-free solution was used as the negative control. Pre-incubation absorbance values were read at 630nm. The microplates were then incubated overnight at 37°C and absorbance values were read after 24h. MIC values were recorded as the lowest concentration of the extract that completely inhibited bacterial growth (Eloff 1998).

Phytochemical screening:

Phytochemical screening was done to detect the presence of various secondary metabolites (Tiwari et al 2011, Joshi et al 2011)

Tannins: 0.5gm plant powder dissolved in 10ml distilled water and filtered. To 2ml filtrate, few drops of 1% FeCl₃was added and observed for the occurrence of blue black, green or blue green precipitate.

Steroids: 0.2gm powder was dissolved in 2ml of acetic acid. Conc. sulphuric acid was added to the ice cold sample and checked for violet to blue or bluish green coloration.

Terpenoids: 0.5gm of powder was dissolved in 2ml chloroform. To this 3ml conc. sulphuric acid was added and observed for the occurrence of reddish brown layer at the interface.

Saponins: 1gm powder was dissolved in 5ml distilled water and filtered. To the filtrate, 3ml of distilled water was added and shaken for 5 min and was observed for persistence of frothing after boiling.

Flavonoids: 0.5 gm powder was dissolved in distilled water and filtered. To this 2ml of 10% NaOH was added to give yellow colour. Change from yellow to colourless on keeping was observed.

Alkaloids: 1 gm powder was dissolved in 5ml of 1% aqueous HCl, stirred and filtered. 1ml of Mayer’s reagent was added to 1ml of the filtrate and observed for formation of buff coloured precipitates.

RESULTS AND DISCUSSIONS:

Determination of antimicrobial activity of plant extracts:

When methanolic, ethanolic and aqueous extracts of ten plants were compared, it was found that methanolic extract was the most effective against all the three bacterial cultures. E.coli was found to be most susceptible to all the plant extracts followed by S. aureus and B.subtilis though the maximum zone of inhibition (24mm) was exhibited by methanolic extract of B.aristata against B.subtilis followed by E.coli (22mm). Extracts of A. indicum and N. arbor-tristis also proved to be effective inhibitors against all the three bacterial cultures (Table 2). It is not surprising that there are differences in the antimicrobial effects of plant species as this could be due to differences in the phytochemical properties amongst the species. It is quite possible that some of the plants eg Madhuca indica that were ineffective in this study do not possess antibiotic properties, or the plant extracts may have contained antibacterial constituents, just not in sufficient concentrations so as to be effective. It is also possible that the active chemical constituents might not be soluble in the solvents used. Moreover, the drying process may have caused conformational changes to occur in some of the chemical constituents found in these plants.

Minimum inhibitory concentration

Two-fold serial dilutions of methanolic extracts of plants were used to determine MIC against E. coli, S. aureus and B. subtilis. MIC for B. aristata was evaluated as 40µg/ml, 69 µg/ml, and 73 µg/ml, for B. subtilis, S. aureus and E. coli respectively showing that it was potent even at low concentrations. When compared with all plant extracts, M. indica was found to be effective at very high concentration (402-525 µg/ml) indicating its poor antimicrobial activity (Table 3).

Table3: Minimum inhibitory Concentration of plant extracts against E.coli, S.aureus and B.subtilis

Plants Microorganism	Minimum Inhibitory Concentration (µg/ml)
Plants Microorganism	E.coli	S.aureus	B.subtilis
Alstonia scholaris	87	135	102
Anthocepalus cadamba	175	289	253
Elaeocarpus ganitrus	137	149	102
Madhuca indica	402	525	438
Butea frundosa	149	188	203
Berberis aristata	73	69	40
Abutilon indicum	103	107	125
Nyctanthes arbortristis	140	158	98
Aegle marmelos	156	238	222
Urginea indica	292	250	188

Incubation Temperature: 37^oC

Table4: Phytochemical analysis of Plant leaf powder.

Phytochemicals Plants	Terpenoids	Tannins	Anthraquinone	Steroids	Saponins	Alkaloids	Flavonoids
Alstonia scholaris	+	+	+	+	--	--	+
Anthocepalus cadamba	+	+	+	+	+	+	+
Elaeocarpus ganitrus	--	+	+	+	+	+	+
Madhuca indica	--	+	+	+	+	+	+
Butea frundosa	+	+			--	+	--
Berberis aristata	+	+			+	+	+
Abutilon indicum	+	--			+	-	+
Nyctanthes arbortristis	+	+			--	+	+
Aegle marmelos	+	+			--	+	+
Urginea indica	+	+			--	+	--

+- Present -- Absent

The MIC can be very crucial for development of bioformulations from this plant. Similar antimicrobial activity of B. aristata against some human pathogenic bacteria has also been demonstrated by Wagh and Vidhale (2010) against Staphylococcus and Pseudomonas spp.

Phytochemical analysis

Preliminary phytochemical analysis of extract in powder forms revealed the presence of Terpenoids, Tannins, Anthraquinone, Steroids, Saponins, Alkaloids and Flavonoids. Flavanoids were present in the leaf powder of all the plants while tannins were detected from all plant except A. indicum. Presence of other phytochemicals viz. Terpenoids, saponins and alkaloids varied from plant to plant. B. aristata and A. cadamba were found to be rich in all the tested phytochemicals (Table 4). Phytochemical analysis demonstrated valuable phytoconstituents which can be very useful for therapeutic purposes. Plant extracts exhibiting potent antimicrobial activity were found to contain most of the tested phytochemicals.

CONCLUSION:

All the ten plant species used in this study exhibited antimicrobial activities. Most effective results were exhibited by the methanolic extract of Berberis aristata with a MIC of 40µg/ml, against B. subtilis. Primary phytochemical analysis revealed variations in the phytochemical profiles of plants which explains the differences in their antimicrobial actions.

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Received on 13.11.2013 Modified on 10.12.2013

Res. J. Pharmacognosy & Phytochem. 6(1): Jan.-Mar. 2014; Page 05-08