Article History

Received 10 September 2013
Received in revised form 13 September 2013
Accepted 15 September 2013
Available online 28 September 2013

Keywords

Antimicrobial activity, Indigofera suffruticosa, சீமைநீலி Medicinal plants, Alkaloid.

IDENTIFICATION OF PHYTOCHEMICAL CONSTITUENTS AND ANTIMICROBIAL ACTIVITY OF INDIGOFERA SUFFRUTICOSA LEAVES

D.Vijisaral Elezabeth and Arumugam Subramanian*

PG & Research Department of Chemistry,

Nehru Memorial College, Puthanampatti - 621 007,

Tiruchirappalli, Tamilnadu, India

*Corresponding author: nmcaaru@gmail.com

Abstract:

Indigofera suffruticosa (சீமைநீலி) is an Indian herb used for various ailments by traditional healers. The present study is carried out to identify the phytochemical constituents of Indigofera suffruticosa plant leaves. Secondary metabolites like total phenolic and flavonoid content were analyzed by UV spectroscopy and antimicrobial activity of ethanol extracts of the leaves were detected. Phytochemical studies revealed the presence of reducing sugar, tannin, flavonoid, phenol, alkaloid, glycoside, and steroid. Susceptibility testing by disc diffusion assay revealed significant antimicrobial activity of ethanol extracts of leaves against bacteria such as Staphylococcus aureus (22mm), Escherichia coli (14mm), Pseudomonas aeruginosa (16mm) and fungi against Klebsiella pneumonia (20mm), Aspergillus niger (22mm) and Candida albicans (38mm).  These findings provide supportive evidence for the use of Indigofera suffruticosa in traditional medicines.

 

Introduction:

India has a rich culture of medicinal herbs and spices, which includes about more than 2000 species and has a vast geographical area with high potential abilities for Ayurvedic, Unani, Siddha traditional medicines but only very few have been studied chemically and pharmacologically for their potential medicinal value. Human beings have used plants for the treatment of diverse ailments for thousands of years (Sofowara, 1993). According to the World Health Organization, most populations still rely on traditional medicines for their psychological and physical health requirements, since they cannot afford the products of Western pharmaceutical industries, together with their side effects and lack of healthcare facilities. Rural areas of many developing countries still rely on traditional medicine for their primary health care needs and have found a place in day-to-day life. These medicines are relatively safer and cheaper than synthetic or modern medicine. People living in rural areas from their personal experience know that these traditional remedies are valuable source of natural products to maintain human health, but they may not understand the science behind these medicines, but knew that some medicinal plants are highly effective only when used at therapeutic doses. Herbal medicines are in great demand in both developed and developing countries as a source of primary health care owing to their attributes having wide biological and medicinal activities, high safety margins and lesser costs. Herbal molecules are safe and would overcome the resistance produced by the pathogens as they exist in a combined form or in a pooled form of more than one molecule in the protoplasm of the plant cell. Traditional use of medicine is recognized as a way to learn about potential future medicines. Researchers have identified number of compounds used in mainstream medicine which were derived from "ethnomedical" plant sources. Plants are used medicinally in different countries and are a source of many potent and powerful drugs. Indigo obtained from species of Indigofera suffruticosa was used in the Old World, the use of wild indigo by pre-Columbian natives of Mexico to dye cloth and paint in various shades of blue was passed down to the Spanish colonists (Haude, 1997). Indigofera suffruticosa species became important commercial crops in various tropical and subtropical areas. The blue dye was produced by fermentation of the leaves, usually with caustic soda or sodium hydrosulfite, and the exudates processed into dry cake. The blue color developed as the cake was exposed to the air (Simon et al., 1984). Indigo was used as a bluing to counter the yellowing in clothes from washing with soap (Velez and van Overbeek, 1950). In the last few decades, natural indigo has been almost wholly replaced by synthetic dyes. Poultices and extracts of wild Indigofera suffruticosa leaves, alone or in combination with other ingredients, are used in herbal medicine to treat fever, headaches, hemorrhages, convulsions, acute cough, skin parasites, and boils (HealthLink, 2001).

  

Materials and Methods:

Collection of Plant Material:

The leaves of Indigofera suffruticosa were collected from Vaithiynathpuram Village in Perambalur District of Tamilnadu, India during January to December, 2012 and authenticated by the Director of the Rapinat Herbarium and Centre for Molecular Systematics, St.Joseph’s college campus, Trichirappalli, Tamilnadu, India. Fresh leaves were cleaned with running tap water and dried under the shade (sunlight). Then the dried plant leaves were ground to fine powder mechanically and preserved in airtight containers for further analysis.

 

Preparation of Plant Extract:

The powdered leaves were extracted in a Soxhlet apparatus with ethanol and water successively. The resultant extracts were evaporated under reduced pressure in a rotary evaporator to obtain the crude extract of Indigofera Suffruticosa.

 

Test for Phytochemical Analysis:

The extracts were analyzed for the presence of alkaloid, terpenoid, reducing sugars, tannin, flavonoid carbohydrate, phenol, anthocyanin, protein and steroid (Sofwora, 1993; Harborne, 1973).

 

Test for Alkaloid:

About 0.2 g of plant extract was weighed in separate test tube and warmed with 2% Sulphuric acid for 2 minutes. And it was filtered in separate test tube and few drops of Dragencloffs reagent were added and observed for the presence of orange red precipitate for the presence of alkaloid.

 

Test for Glycoside:

Legal’s Test:

About 0.5 g of plant extract was weighed in a separate test tube with pyridine and sodium nitroprusside reagent was added and made alkaline with NaOH solution and observed for the presence of pink to red color solution which indicates the presence of glycoside.

 

Test for Terpenoids:

About 0.5 g of plant extract in separate test tube was taken with 2 ml of chloroform; 5 ml of concentrated sulphuric acid was carefully added to form a layer and observed for presence of reddish brown color interface to show positive results for the presence of terpenoid.

 

Test for Reducing Sugars:

Two ml of crude plant extract and add 5ml distilled water were stirred and filtered. The filtrate was boiled with 3-4 drops of Fehlings solution A and B for 2 minutes and observed for orange red precipitate which indicates the presence of reducing sugars.

 

Test for Tannin:

Small quantity of plant extract was mixed with 5ml of distilled water and heated on water bath. The mixture was filtered and ferric chloride was added to the filtrate and observed for dark green solutions that indicate the presence of tannin.

 

Test for Flavonoid

About 0.2 g of plant extract was weighed in separate test tubes and dissolved in diluted sodium hydroxide and diluted Hydrochloride added and observed for yellow solutions that turn colorless. This indicates the presence of flavonoid.

 

Test for Steroid:

Two ml of acetic anhydride was added to 0.5 g extract with 2ml of Sulphuric acid and observed for the color change from violet to blue or green in samples indicating the presence of steroid.

 

Test for Anthocyanin:

Sodium Hydroxide Test:

About 0.2 gm of plant extract was weighed in separate test tube, 1ml of 2N Sodium hydroxide was added, and heated for 5 minutes at 100 2C and observed for the formation of bluish green color which indicates the presence of anthocyanin.

 

Test for Phenol:

Ferric Chloride Test:

About 0.2 g of plant extract was weighed and treated with 5% ferric chloride and observed for the formation of deep blue color which indicates the presence of phenol.

 

Test for Amino acid:

Ninhydrin Test:

About 0.2g of plant extract was weighed and treated with Ninhydrin solution and observed for a characteristic purple color which indicates the presence of amino acid.

 

Test for Protein:

Million’s Test:

Small quantity of plant leaf extract was treated with few drops of Million’s reagent and observed for the formation of white precipitate which indicates presence of protein.

 

Estimation of Secondary Metabolites:

Determination of Total Phenolic Content:

The total phenolic content of Indigofera suffruticosa leaves was determined in two different solvent extracts spectrophotometrically according to the Folin-Ciocalteu method (Singleton et al., 1999) using Gallic acid as a standard (the concentration range; 0.25 to 0.5mg mL-1). The total phenolic content was expressed as milligram per gram dry extract.

 

Determination of total Flavonoid Content:

The total flavonoid content was determined according to the aluminum chloride colorimetric method (Lin and Tang, 2007). Rutin was chosen as a standard (the concentration range; 0.005 to 0.1mg mL-1) and the total flavonoid content was expressed as milligram per gram of dry extracts.

 

Antimicrobial Assay:

The following organisms were employed for this study as test organisms; bacteria such as Staphylococcus aureus, E.coli, Pseudomonas aeruginosa and Klebsiella pneumonia, fungi such as Aspergillus niger and Candida albicans. The test microbial pathogen cultures were obtained from National Chemical Laboratory (NCL) Pune, maintained by periodical sub culturing on Nutrient agar and Sabouraud dextrose agar medium.

 

Antimicrobial and antifungal activity of ethanol extracts were tested using the disc diffusion method described by Indian pharmacopoeia, (1996, Vol II A-105). All the above mentioned bacteria were inoculated into nutrient agar medium and fungi inoculated to Sabouraud dextrose agar medium. The well of 8mm diameter was punctured in the culture medium using sterile cork borer. Ethanol extracts were administered to fullness in each well. Culture plates were incubated at 37 2 for 24 h for bacteria and incubated at 37 2 for 4 days for fungi. Bioactivity was determined by measuring diameter of inhibition zones in mm. Solvents used for extraction served as control.

                                                      

Results:

Phytochemical Analysis:

Ethanolic extracts of the leaves of Indigofera suffruticosa showed the presence of alkaloid, glycoside, reducing sugar, tannin, flavonoid, steroid and phenol. Amino acid, anthocyanin and protein were not found in ethanolic extract of Indigofera Suffruticosa. Similarly, the aqueous extract of the plant revealed the presence of alkaloid, glycoside, reducing sugar, tannin, flavonoid, steroid and phenol and except anthocyanin, amino acid and protein. Table 1 showed the results of phytochemical analysis of the ethanolic and aqueous extracts of Indigofera Suffruticosa.

  

Table 1: Results of Phytochemical Analysis Ethanolic and Aqueous Extract of Indigofera Suffruticosa

Name of the Phytochemical

        Constituents

 

   Ethanol extract

 

Aqueous extract

 

       Alkaloid    

 

       Glycoside

 

       Reducing sugar

 

       Tannin

 

       Flavonoid

 

       Steroid

 

       Anthocyanin

 

       Phenol

 

      Amino acid

 

      Protein                                

 

            +

 

            +

 

            +

 

            +

 

            +

 

            +

 

            -

 

            +

 

            -

 

            -

 

        +

 

        +

 

        + 

 

        +

 

        +

 

        +

 

        -

 

        +

 

        -

 

        -      

 

+: Indicates the presence and -: Indicates the absence of phytoconstituents

 

Estimation of Secondary Metabolites:

Total Phenolic Content:

Total phenolic content of two different solvent extracts of leaves of Indigofera suffruticosa was determined by spectrophotometricaly according to the Folin-Ciocalteu method and the results are given in Table 2. It is clear from the table that among all the extracts, the aqueous extract showed high amount of phenolic content 0.54% compared to ethanol extracts. The ethanolic extract showed the least phenolic content 0.42%.

 

Total flavonoid content:

The total flavonoid content was determined according to the aluminum chloride colorimetric method in two different solvent extracts of leaves of Indigofera suffruticosa and the results are given in the Table 2. The ethanol extract showed high amount of flavonoid content 0.22% when compared to aqueous extract. The aqueous extract showed the least flavonoid content 0.14%.

 

Table 2: Determination of Secondary Metabolites in two Different Solvent Extracts of Indigofera suffruticosa Leaves

 

Secondary metabolites

 

Ethanol extract (%)

 

Aqueous extract (%)

 

Total Phenolic content

 

Total flavonoid content

 

   0.54

 

   0.22

 

     0.42

 

     0.14

 

Antimicrobial assay:

The results of antimicrobial assay of the ethanolic extracts of the leaves of Indigofera suffruticosa against tested strains were shown in Table 3. The antimicrobial analysis reveals maximum activity against the Staphylococcus aureus and minimum activity was noted against the E.coli and Pseudomonas aeruginosa. Fungal pathogens activity was maximum towards Candida Albicans and minimum activity was seen against Klebsiella pneumonia and Aspergillus niger.

 

Table 3: Antimicrobial Efficacy of Ethanol Extract of Indigofera suffruticosa Leaves.

 

S.No

 

Name of the Microorganism

 

 

          Zone of inhibition in mm

 

 

Ethanol extracts of leaves of Indigofera suffruticosa

 

Solvent                   control

 

  Standard

 

  1

 

Staphylococcus aureus

(NCIM 2079)

 

             20

 

      -

 

     35

 

  2

 

E.coli

(NCIM 2065)

 

             14

 

      -

 

    38

 

  3

 

Pseudomonas aeruoginosa (NCIM 2036)

 

             16

 

      -

 

    35

 

  4

 

Klebsiella pneumonia

(NCIM 2098)

 

             20

 

      -

 

    30

 

  5     

 

 

Aspergillus niger

(NCIM 105)

 

             22

 

      -

 

    35

 

  6

 

Candida albicans

(NCIM 3102)

 

             38

 

 

      -

 

 

    25

 

 

Standard - Ciprofloxacin 5g / disc for bacteria; Nystatin 100 g / disc for fungi. Solvent-DMSO

  

Figure: 1 Antimicrobial Activity of Leaves of Indigofera Suffruticosa

 

 

 

Discussion:

The phytochemical analysis estimated the secondary metabolites such as flavonoid and phenol and revealed the antimicrobial activity of Indigofera suffruticosa leaves. The presence of notable phytochemicals; alkaloid, glycoside, terpenoid, steroid, flavonoid, reducing sugar, tannin and phenol in the plant leaf might have been responsible for its uses in traditional health care. The phenolic compounds are one of the largest and most ubiquitous groups of plant metabolites. They possess biological properties such as antiapoptosis, antiaging, anticarcinogen, antiinflammation, antiatherosclerosis, cardiovascular protection and improvement of endothelial function, as well as inhibition of angiogenesis and cell proliferation activities (Han et al., 2007). Non toxic glycoside can also be hydrolyzed to release phenolic compounds that are toxic to microbial pathogens. These bioactive compounds exert antimicrobial activity through different mechanisms. Natural antioxidants mainly come from plants in the form of phenolic compounds such as flavonoid, phenolic acids, tocopherols etc. (Ali et al., 2008). Tannins bind to proline rich protein and interfere with protein synthesis. There is no report on the total phenolic and flavonoid present in Indigofera suffruticosa leaves extract in previous studies. Derived polyphenols from plants are of great importance because of their potential antioxidant and antimicrobial properties. Phenolic compounds exhibit a considerable free radical scavenging (antioxidant) activity (Wojydylo et al., 2007). Thus the phenolic compound present in the plant Indigofera suffruticosa might contribute various therapeutic uses of the extract’s traditional system of medicine. Antimicrobial assay of ethanolic extracts were carried out against bacteria and fungi. Among the extracts, the leaf extract of Indigofera suffruticosa were effective against bacteria and fungi. The plants are vital source of innumerable number of antimicrobial compounds. Several phytochemical constituents like flavonoids (Tsuchiya et al., 1996), phenolics and polyphenols (Mason and Wassermann, 1987), tannins (Ya et al., 1988), etc. are effective antimicrobial substances against a wide range of microorganisms. One of the largest groups of chemical produced by plant is the alkaloids and their amazing effects on humans have led to the development of powerful painkiller medications (Raffauf, 1996). Alkaloids of Heliotropium indicum and Chorizanthe procumbens are used for the treatment healing, antitumor and analgesic, hence different formulations could be prepared for clinical trials (Boomination and Ramamurthy, 2009).

 

Acknowledgement:        

The authors would wish to acknowledge the management and Principal of Nehru Memorial College for providing research facilities and encouragements.

 

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