Phytochemical analysis and Antimicrobial screening of Murayya koenigii Extracts

 

R Namitha*, J S Ampily

Department of Chemistry, RSM SNDP Yogam Arts and Science College, Koyilandy,

Kozhikode. 673305, Kerala. India

*Corresponding Author E-mail: namithasajish@gmail.com

 

ABSTRACT:

Murayya koenigii leaf was extracted by using two solvents- chloroform and ethyl alcohol. Determined the number of chief different components present in extracts of curry leaf by thin layer chromatography. The phytochemical screening revealed the presence of alkaloids, carbohydrates, Phenolic compounds, that the alcohol extract and tannins, quinines, alkaloids, carbohydrates in the chloroform extract.  The in vitro antimicrobial activities of both extracts were examined against Staphylococcus aureus (S.a), Escherichia coli (E.c), Pseudomonas Sp (P.Sp), Streptococcus pyogens (S.p), Staphylococcus aureus (S.au) and Candida albicans. Two extracts exhibited activities against bacterial and fungal stains tested. Results were favorably compared with standard antibiotics.

 

KEYWORDS: Murayya koenigii, Phytochemical, Staphylococcus aureus, terpenoids, TLC.

 

 


INTRODUCTION:

Several plants have been beneficial to mankind since ancient times1. Ayurveda, Unani, Amchi and Siddha are some of the Indian practices that use medicinal plants for treatment of several human as well as animal diseases2. Currently, in addition to antibiotics and chemically synthesized drugs, the trend to look out for alternative medicines such as natural or herbal medicines is increasing because they may have fewer side effects or toxicity owing to their natural sources3.

 

Compounds such as proteins, fats, carbohydrates, enzymes, oils, flavonoids and terpenoids are known to be present in plants, out of which several compounds are biologically active and are used in the cure and control of numerous diseases.

 

One such medicinal plant is the Murraya koenigii, known as Kari patta or Meethi neem in the local dialect. It belongs to the family Rutaceae (Citrus family) that consists of approximately 150 genera and 1500               species4-6. Murraya koenigii is found to be native mainly to India and Sri Lanka. Additionally, it can be found in some other South Asian countries as well. It is an important ingredient in Indian curries owing to its fragrance and aroma. This plant is known to be the richest source of carbazole alkaloids. Carbazole alkaloids, present in In M. koenigii (L.) Spreng, is responsible for its multiple biological properties such as its anti-tumor, anti-mutagenic, anti-oxidative and anti-inflammatory activities7-9.

 

The leaves of Murraya koenigii contain proteins, carbohydrate, fiber, minerals, carotene, nicotinic acid, Vitamin C, Vitamin A, calcium and oxalic acid. It also contains crystalline glycosides, carbazole alkaloids, koenigin, girinimbin, iso-mahanimbin, koenine, koenidine and koenimbine. Triterpenoid alkaloids cyclomahanimbine, tetrahydromahanimbine are also present in the leaves.

 

This study aims at investigating the phytochemical screening and biological activity of different extracts of curry leaves (Murraya koenigii).

 

EXPERIMENTAL:

MATERIALS AND METHOD:

Thin layer chromatography (TLC) was performed using glass plates coated with silica gel. Chloroform and ethyl acetate was used as eluant. Spots was visualized with iodine.

 

Collection of plant materials:

The experiment was conducted in the year 2018-2019 in the college laboratory. Leaves were collected from Murrya koenigii plant and it was ensured that the plant was healthy and uninfected. The leaves were washed under running tap water to eliminate dust and other foreign particles and to cleanse the leaves and dried in the shade for several days.

 

Preparation of leaf extract:

Drying process:

Fresh leaves of Murraya koenigii were shade dried in chemistry lab until constant weight was obtained. They were kept away from high temperature and direct sunlight to avoid destroying active compounds.

 

Extraction process:

Cold Solvent Extraction:

The powdered plant material was soaked in 150ml of the solvent in a conical flask. This was placed on a shaker for about 5 days. The resulting extract was passed through muslin cloth and filtered through Whatmann filter paper. The resulting extract was evaporated to dryness. The residue obtained was reconstituted back in the corresponding solvent.

 

Chromatographic identification and separation:

Thin layer chromatography:

The first practical application of thin layer chromatography was given by Stahl10. Compared to paper chromatography, the special advantage of TLC is the versatility, speedy and sensitive. TLC is an adsorption chromatography 11 where samples are separated based on the interaction between a thin layers of adsorbent attached on the plate. The technique mostly employed for the separation of low molecular weight compounds.

 

Preparation of TLC plates:

The TLC plates were prepared by using Silica gel ‘G’ as 30 gm of silica gel was weighed and made to a homogenous suspension with 60 ml distilled water for two minutes, this suspension was distributed over the plate which was air dried until the transparency of the layer disappeared. The plates were dried in hot air oven at 110°C for 30 mins and then stored in a dry atmosphere and used whenever required.

 

Method:

Samples were prepared by diluting the crude extracts with appropriate solvent and it was applied on pre-coated TLC plates by using capillary tubes and developed in a TLC chamber using suitable mobile phase. The mobile phase was prepared by mixing10%, 15%, 20%, 25%, 30% ethyl acetate in chloroform. The TLC plates were developed by dipping in it. The developed TLC plates were air dried. The movement of the analyte was expressed by its retention factor (Rf). Values were calculated for different sample.

 

                  Distance traveled by the solute

Rf = ------------------------------------------------------

           Distance traveled by the solvent  front TLC plates

 

Analyte detection:

After drying the plates, they were exposed to Iodine vapours by placing in a chamber that was saturated with iodine vapours. The Rf value of the different spots that were observed was calculated.

 

Phytochemical evaluation:

Chemical constituents of Murraya koenigii were determined by qualitative chemical analysis.

 

Identification of Constituents by Phytochemical Test:

The extracts were subjected to quantitative test for detection of phytoconstituents in it viz. alkaloids, carbohydrates, flavanoids, terpinoids, vitamins, tannins, saponnins, anthraquinones etc.

 

Test for Alkaloids:

A little fraction of the solvent free alcohol extracts were mixed individually with a small amount of drops of dilute hydrochloric acid and it is filtered. The filtrated was evaluated carefully with different alkaloidal reagent such as,

 

a. Mayer’s reagent:

To a few ml of filtrate, a drop or two of Mayer’s reagent was added by the side of the test tube.

 

b. Dragendroff’s reagent:

To a few ml of filtrate, few drops of Dragendroff’s reagent were added by the side of the test tube.  

 

c. Hager’s reagent:

To a few ml of filtrate, few drops of Hager’s reagent were added by the side of the test tube.

 

d. Wagner’s reagent:

To a few ml of filtrate, few drops of Wagner’s reagent were added by the side of the test tube.

 

 

Test for carbohydrates:

The least amount of extracts was dissolved in 5 ml distilled water and it is filtered. The filtrate was subjected to analysis for the presence of carbohydrates and glycosides.

 

a. Molisch’s Test:

The filtrate was mixed with 2-3 drops of 1% alcoholic alpha naphthol and along the sides of the test tube; 2ml of concentrated sulphuric acid was added.

 

b. Fehling’s Test:

The filtrate was heated with 1 ml of Fehling’s A and B solution.

 

Test for glycosides:

a. Legal’s Test:

The Hydrosylate was mixed with a alcohol and then alcohol layer was separated. To this identical quantity of dilute ammonia solution was added.

 

Test for Phenolic Compounds:

Small quantity of extracts were dissolved individually in water and tested for the presence of phenolic compounds and tannins with

 

a. Ferric chloride Test:

1 ml extract was dissolved in 2ml of distilled water. To this few drops of 10% ferric chloride solution was added.

 

Test for tannins:

Ferric chloride test:

1 ml solvent extract was dissolved in 10 ml distilled water and filtered. 1% aqueous Iron chloride (FeCl3) solution was added to the filtrate.

 

Test for quinines:

The extracts were treated separately with Alc. KOH solution.

 

Test for carboxylic acid:

One ml of the various extracts was separately treated with a few ml of sodium bicarbonate solution.

 

Test for flavonoids:

a. Mixed with aqueous sodium hydroxide solution.  

 

Test for Terpenes/steroid (Liebermann - Burchard’s Test)

200mg plant material in 10 ml alcohol, filtered; 2ml of filtrate is mixed with 2ml of acetic anhydride and 1ml of conc. Sulphuric acid.

 

Test for saponnins (Frothing Test)

0.5 ml of filtrate mixed with 5ml of distilled water, shaken for 30 sec.         

 

Test for Anthraquinones (Bortrager’s Test)

100 mg of powered plant in 5ml alcohol, filtered, 2ml of filtrate is mixed with 10% of Ammonium hydroxide.

 

Pharmacological activities:

Antimicrobial activity (Well diffusion method)12

Antibacterial and antifungal activities were studied. The antimicrobial activity was evaluated by measuring the diameter of inhibition zone.

 

Antibacterial activities:

Antibacterial activities of extract was tested  against the Staphylococcus aureus (S.a), Escherichia coli (E.c), Pseudomonas Sp (P.Sp), Streptococcus pyogens (S.p), Staphylococcus aureus (S.au) at various concentration 100µg/L, 75µg/L, 50µg/L, 25µg/L. Gentamycin was used as the standard for antibacterial studies. The media for anti bacterial study is Nutrient Agar (NA).

 

Antifungal activities:

Antifungal activities of extract was carried out against the Candida albicans, at various concentrations 100µg/L, 75µg/L, 50µg/L, 25µg/L. Clotrimazole was used as the standard for antifungal studies. The media for antifungal study is potato dextrose agar (PDA).

 

RESULTS AND DISCUSSION:

Literature survey revealed that curry leaf extracts have large varieties of pharmacological importance. This biological importance of curry leaf prompted our study towards the extraction of its constituents by using chloroform and alcohol, the screening of this crude extracts for its antimicrobial activity.

 

Extracts of curry leaf was obtained by using cold extraction process. In this method the powdered plant material was soaked in 150 ml of solvent in a conical flask. This was placed on a shaker for about 5 days. The resulting extract was passed through muslin cloth and filtered through Whatmann filter paper. The resulting dark green coloured extract was evaporated to dryness. A small quantity of the sample was dissolved in both the solvents and thin layer chromatography was performed to determine the number of constituents present in the crude extract by using 10%, 15%, 20%, 25%, 30% ethyl acetate in petroleum ether as moving phase. From the TLC results while comparing the Rf values, it is clear that mainly four different components are present in the chloroform extract and three in the ethanol extract.

 


Identification of Constituents by Phytochemical Test:

Then we moved our study towards the determination of phytochemical constituents present in the crude extract.

 

Here we performed the test for alkaloids, carbohydrates, Phenolic compounds, glycosides, tannins, quinines, carboxylic acid, flavanoids, Terpenes/steroid, saponnins, Anthraquinones.

 

 Compounds

Ethanol extract

Chloroform extract

Alkaloids

Detected

Detected

Carbohydrates

Detected

Detected

Phenolic compounds

Detected

Not detected

Glycosides

Not detected

Not detected

Tannins

Not detected

Detected

Quinines

Not detected

Detected

Carboxylic acid

Not detected

Not detected

Flavanoids

Not detected

Not detected

Terpenes/steroid,

Not detected

Not detected

Saponnins,

Not detected

Not detected

Anthraquinones.

Not detected

Not detected


Pharmacological activities:

Antimicrobial Susceptibility Testing against Bacteria

 

Pathogen

Zone of Inibition (diameter in mm)

 

1.0mg/mL

0.75 mg/mL

0.5 mg/mL

0.25 mg/mL

 

CHCl3

C2H5OH

CHCl3

C2H5OH

CHCl3

C2H5OH

CHCl3

C2H5OH

STD

1

S, pyogenes

21

16

18

15

16

15

12

≥10

12

2

S. aureus

15

16

12

14

10

13

>10

>10

10

3

E. coli

18

17

14

15

11

12

10

10

13

4

P. Sp

16

20

15

18

13

13

>10

>10

11.5

 


From the table while comparing the activity of both the extracts, it is found that the chloroform extract showed highest activity than that of alcohol extract. And also in chloroform extract against Streptococcus pyogenes s the extract inhibit the growth of this bacterium much more than that of the others and also than that of the standard.

 

SUMMARY:

In this study we have extracted the phytochemical constituents Murayya koenigii by using chloroform and alcohol. Determined the number of chief different components present in extracts of curry leaf by thin layer chromatography. From the TLC results while comparing the Rf values, it is clear that mainly four different components were present in the chloroform extract and three in the ethanol extract. Further the crude extract was screened for antimicrobial activity using Well diffusion method against the Staphylococcus aureus (S.a), Escherichia coli (E.c), Pseudomonas Sp (P.Sp), Streptococcus pyogens (S.p), Staphylococcus aureus (S.au), Candida albicans (C.a) at various concentration 100µg/L, 75µg/L, 50µg/L, 25µg/L. The extract was active towards all the bacterial stains and the result was comparable with the standard but against fungal stain it was not active.

 

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Received on 03.04.2020         Modified on 19.04.2020

Accepted on 09.05.2020  ©AandV Publications All right reserved

Res. J. Pharmacognosy and Phytochem. 2020; 12(2): 101-105.

DOI: 10.5958/0975-4385.2020.00017.5