Phytochemical Investigation and Antibacterial Activity of Hemigraphis colorata Leaf Extract

Natural products, especially those from plant sources, including spices, have been investigated for their characteristics and health effects. Plants have formed the basis of sophisticated traditional medicine practices that have been used for thousands of years by people in China, India and many other countries. Nowadays, herbal products have potential for the treatment of many diseases. Cancer is a worldwide disease that caused approximately 22% of deaths up until 1990. In 2000, there were 10 million new cases and over 6 million deaths worldwide. Most plants possess many biological activities, such as antioxidant, anticancer, antimicrobial, antidiabetic, anti-inflammatory, anthelmintic, antinociceptive, antidiarrhoeal, anti-elasticity and antidiuretic activities.

Plant chemicals are regarded as secondary metabolites because the plants that manufacture them may have little need for them. They are synthesized in all parts of the plant body (bark, leaves, stem, root, flowers, fruits, seeds, etc.); that is, any part of the plant body may contain active components. These chemical substances are called secondary metabolites. The most important of these bioactive groups of plant constituents are alkaloids, terpenoids, tannins, saponins and phenolic compounds. Knowledge of the correlation between the phytoconstituents and the bioactivity of a plant is desirable for the synthesis of compounds with specific activities to treat various health ailments and chronic diseases. Generally, the presence of different phytochemicals in crude plant extracts has been linked to the detrimental effects of leachates, root exudates or decomposing residues of such plants on other vegetation or succeeding crops. Owing to this significance, preliminary phytochemical screening of plants is the need of the hour in order to discover and develop novel therapeutic agents with improved efficacy. Phytochemical analyses of several species of medicinal plants and of the allelopathic activities of their crude chemical compounds on crops and plants have yielded positive results. The present study reports the qualitative phytochemistry of Hemigraphis colorata, a medicinal plant used by the people of Kanyakumari District, Tamil Nadu, India.

Mature leaves of Hemigraphis colorata were collected from Kuzhithurai, Kanyakumari District. The freshly collected H. colorata leaves were washed in running tap water for 3 minutes. The plant parts were then rinsed thoroughly with sterile distilled water to remove residues. Excess moisture was removed from the sterilized leaves, and they were then subjected to solvent and crude extraction.

Preliminary qualitative phytochemical screening was carried out using the following methods.

Saponins (froth test): About 2 g of the powdered sample is boiled with 20 mL of distilled water in a water bath and filtered. Then, 10 mL of the filtrate is mixed with 5 mL of distilled water and shaken vigorously to obtain a stable, persistent froth. The froth is mixed with 3 drops of olive oil and shaken vigorously. The formation of an emulsion indicates a positive result.

Steroids (Liebermann–Burchard test): To 0.5 mL of the extract, add 2 mL of acetic anhydride and 2 mL of concentrated H₂SO₄ along the sides of the tube. The formation of a green colour indicates the presence of steroids.

Glycosides (Keller–Kiliani test): The extract (5 mL) is treated with 2 mL of glacial acetic acid containing one drop of ferric chloride solution and 1 mL of concentrated sulphuric acid. A brown ring at the interface indicates the presence of cardiac glycosides.

Terpenoids (Salkowski test): To 5 mL of the extract, add 2 mL of chloroform and 3 mL of concentrated H₂SO₄. The formation of a yellow ring at the interface of the two liquids that turns reddish brown after two minutes indicates the presence of terpenoids.

Phenols (Liebermann’s test): To 1 mL of the extract, add 1 mL of sodium nitrite, a few drops of dilute sulphuric acid and 2 mL of dilute NaOH. The appearance of a deep red, green or blue colour indicates the presence of phenols.

Tannins (modified Prussian blue test): To 1 mL of the extract, add 1 mL of 0.008 M potassium ferricyanide and 1 mL of 0.02 M FeCl₃ in 0.1 M HCl. The appearance of a blue colour indicates the presence of tannins.

Coumarins: To 1 mL of the plant extract, 1 mL of 10% NaOH was added. The formation of a yellow colour indicates the presence of coumarins.

Proteins (xanthoprotein test): The extracts were treated with a few drops of concentrated nitric acid. The formation of a yellow colour indicates the presence of proteins.

Carbohydrates (Molisch’s test): To 2–3 mL of the aqueous extract, two drops of alcoholic α-naphthol solution are added and the mixture is shaken well. Concentrated sulphuric acid is then added along the sides of the test tube. The formation of a violet ring indicates the presence of carbohydrates.¹

The antibacterial activity of the samples was determined by the agar well diffusion method according to the National Committee for Clinical Laboratory Standards (NCCLS). An inoculum containing 10⁶ CFU/mL of each bacterial culture to be tested was spread on nutrient agar plates with a sterile swab moistened with the bacterial suspension. Subsequently, wells of 8 mm diameter were punched into the agar medium and filled with 100 µL (25 mg/mL) of the sample, which was allowed to diffuse at room temperature for 2 h. The plates were then incubated in the upright position at 37 °C for 24 h, with standard antibiotic discs of imipenem (10 µg). After incubation, the diameters of the growth inhibition zones were measured in mm.

The preliminary qualitative phytochemical screening of H. colorata leaves was carried out to assess the presence of bioactive components. The hot water extract of H. colorata leaves showed positive results for carbohydrates and tannins, and negative results for alkaloids, terpenoids, saponins, flavonoids, cardiac glycosides, steroids, phenols, proteins and amino acids (Table 1).

A, aqueous extract; C, chloroform extract; E, ethanol extract; +, present; −, absent.

In the present study, the leaf extract of H. colorata showed antibacterial activity against the tested pathogens Escherichia coli and Bacillus subtilis. The maximum zone of inhibition for E. coli (13 mm) was observed at a concentration of 80 mg/mL, while for B. subtilis the maximum zone was 12 mm, also at a concentration of 80 mg/mL. This shows that the plant has significant antimicrobial activity.

Many plants are known to have beneficial therapeutic effects, as noted in the traditional Indian system of medicine, Ayurveda. The effects of plant extracts on bacteria have been studied by a very large number of researchers in different parts of the world. Hence, the last decade has witnessed an increase in the investigation of plants as a resource for human disease management. Based on the above, H. colorata possesses medicinal properties and can therefore be used to discover bioactive natural products that may serve as leads for the development of new pharmaceuticals that address unmet therapeutic needs. Such screening of various natural organic compounds and identification of active agents is the need of the hour, because successful prediction of lead molecules and drug-like properties at the onset of drug discovery will pay off later in drug development. From the results of this study, it is concluded that H. colorata possesses a considerable level of bioactive compounds and, therefore, this species can be used as a potential source of drugs.