PHYTOCHEMICAL AND ANTIOXIDANT STUDIES ON AN IMPORTANT INDIGENOUS MEDICINAL PLANT-ANDROGRAPHIS PANICULATA (BURM.F) NEES

PHYTOCHEMICAL AND ANTIOXIDANT STUDIES ON AN IMPORTANT INDIGENOUS MEDICINAL PLANT-ANDROGRAPHIS PANICULATA (BURM.F) NEES

U. Umadevi and M. Kamalam

Department of Botany, PSGR Krishnammal College for Women, Coimbatore, Tamil Nadu, India

ABSTRACT: Andrograhis paniculata is a wonder drug using in traditional systems of medicine for multiple clinical applications. This research article deals with the phytochemical and antioxidant properties of A. paniculata leaves.   Qualitative and quantitative phytochemical analyses were carried out by TLC and HPLC method.  Physico-chemical analysis was done to standardize the plant drug material so as to ensure the use of genuine material. The result of phytochemical studies confirmed the presence of various secondary metabolites like alkaloids, glycosides, flavonoids, proteins, tannins, phenols, saponins, terpenoids, quinones and steroids.  TLC studies of chloroform extracts showed 3 spots and methanol extracts showed 5 spots at different Rf values.  HPLC analysis confirmed the presence of the major active constituent andrographolide. Effect of age on quantity of andrographolide content was studied and it was found to be higher in 120 day old plants. Antioxidant property was determined by reducing power method. Maximum antioxidant activity was observed in pure andrographolide followed by methanol and water extracts of leaf powder.

Androgrpahis paniculata, Physico-chemical analysis, pharmacogonstical studies, TLC, HPLC, Andrographolide, Antioxidant property

INTRODUCTION: Andrographis paniculata, commonly known as Siriyanangai in Tamil, belongs to the family Acanthaceae is widely used in the Indian traditional system of medicine. The major component of A. paniculata is andrographolide is a bitter, colorless, and crystalline in appearance, is called diterpene lactone¹.  It is used to treat poisonous bites, diabetes and respiratory tract infection. The plant possesses anti-inflammatory, antipyretic, antiviral, immune stimulatory, anticancer, antihyperglycemic and antioxidant properties².  Many of the plants are rich in secondary metabolites and are potent source of drugs.

They have been widely screened for their antioxidant properties in order to find out an efficient remedy for diseases associated with oxidative stress and infections.  Hence, the work is undertaken with the aim of studying the phytochemical constituents and antioxidant property of A. paniculata.

 MATERIALS AND METHODS:

The leaves of A. paniculata were collected from Kanjampatty village of Coimbatore (Dt), Tamil Nadu. They are shade dried, powdered and extracted using soxhlet apparatus with different solvents like petroleum ether, benzene, chloroform, acetone, methanol and water and stored in refrigerator in air tight containers for further studies.

 Qualitative phytochemical analysis

Qualitative phytochemical analysis of alkaloids, glycosides, flavonoids, terpenoids, tannins, phenols, fixed oils, fats, gums, mucilage and saponins were carried out following the procedure of Harborne³ (1998).

 TLC studies

Chloroform and methanol extracts of leaf powder and standard andrographolide were used for TLC studies⁴.  Silica gel was used as an adsorbent. The mobile phase of Chloroform: methanol: Ethyl acetate, used at the ratio of 80:15:10.  Phytochemical constituents were identified based on their Rf values.

 HPLC

HPLC studies were carried out to confirm the presence of Andrographolide compound using the method of Manoj kumar pandey et al.,⁵. The system used for analysis was Shimadzu SP 20A model with C-18 column and Methanol: water (65:35) used as a mobile phase. The injector volume was 20μl and the data is reported.

 Quantitative estimation of Andrographolide

Estimation of Andrographolide is done following the method prescribed by Azhar Ali farooq and Sree Ramu⁶. The seeds were collected from pasulamali hills, Theni (Dt), Tamil Nadu and raised the seedlings in the plains of Kanjampatty village in Coimbatore (Dt.). The Andrographolide content was estimated in fresh and dried leaves of 30, 60, 90, 120, 150 and 180 day old plants and the values are expressed in percentage.

 Study of antioxidant activity

The antioxidant property of the plant was studied by reducing power assay method^7. Methanol and water extracts of leaf powder and pure Andrographolide (dissolved in methanol and water) were used to determine the reducing power.1 ml of each extract was taken in different concentration (20, 40, 60, 80 and 100µg/ml) mixed with 2.5ml of phosphate buffer (0.2M, pH 6.6) and 2.5 gm of potassium ferricyanide. The mixture was incubated at 50^oC for 20 minutes. 2.0 ml of TCA was added to the mixture and centrifuged at 3000 rpm for 10 minutes.  2.5 ml of upper layer of solution was taken and mixed with 2.5 ml of distilled water and 5ml of ferric chloride solution ie., in the ratio of 1:1:2 and absorbance were measured at 700nm in UV- Visible spectrophotometer. Ascorbic acid used as a standard reference and phosphate buffer used as a blank solution. Increase in the absorbance value signified the increase in reducing power.

 RESULTS AND DISCUSSION:

Qualitative phytochemical analysis

Chloroform, methanol and water extracts of leaf powder of A. paniculata showed positive result for the presence of most of the secondary metabolites (Table 1). Except fixed oil and fats, all other phytochemical constituents like alkaloids, glycosides flavonoids, tannins, phenols, saponins, terpernoids and    steroids.  Earlier reports were also confirmed this result^8.  Generally, plant contains the above said phytochemicals, will have high medicinal value. The flavonoids are reported to possess anti-allergic, anti-inflammatory, anti-microbial and anti-cancer activities⁹.

TABLE 1: QUALITATIVE PHYTOCHEMICAL ANALYSIS OF LEAF POWDER OF A. PANICULATA

--Not performed, + present, - Absent

Alkaloids have been used as ant malarial, pain killer and to manage heart diseases.  Glycosides serve as defense mechanism against predation by many microbes¹⁰.  Steroids are known for their cardiotonic activities, insecticidal and antimicrobial properties¹¹. Phenols and tannins have antioxidant propertiesand Saponins were used in hypercholesterolemia, hyperglycaemia, antioxidant, anticancer, anti-inflammatory and weight loss.  Apart from this A. paniculata is found to contain the major components like andrographolide, neoandrographolide and andrographanin are reported to have medicinal property¹².

TLC Studies

Qualitative phytochemical analysis of chloroform and Methanol extracts revealed the presence of many secondary metabolites in the leaf materials.  Methanol extract of A. paniculata showed a maximum of five different compounds than chloroform extract (Table 2). The andrographolide was spotted out with the Rf value of 0.53 with other derivatives but in earlier report, the andrographolide was detected with the Rf value 0.58¹³.

TABLE 2: TLC STUDIES OF A. PANICULATA

 HPLC studies

In HPLC analysis, the methanol extract of the selected plant material was run along with the standard andrographolide, showed the presence of 14 different compounds (Fig1 & 2). Among that, andrographolide was detected at the retention time of 2.5 minutes. Similar study was conducted by Meenu Sharma et al., ¹⁴ in the same plant and reported the andrographolide content with the retention time of 2.8 minutes.

FIG 1: HPLC CHROMATOGRAM OF STANDARD ANDROGRAPHOLIDE

 Peak Table

Quantitative estimation of Andrographolide

The quantitative estimation of andrographolide content was studied in fresh and dried leaves of 30, 60, 90, 120, 150 and 180 day old plants. The andrographolide content was higher in 120 day old plants (Fig 3). Significant differences were observed in andrographolide content of fresh and dried leaves. The fresh leaves have higher andrographolide content than dried leaf powder. Similar result was reported in the earlier studies¹⁵.

FIG 3: EFFECT OF AGE ON ANDROGRAPHOLIDE CONTENT OF A. PANICULATA

Antioxidant activity

Antioxidant may be defined as radical scavengers which protect the human body against free radical that cause pathological condition¹⁶. When the mechanism of antioxidant protection becomes unbalanced deterioration of physiological functions may occur requiring the system to depend on exogenous antioxidants from natural sources¹⁷.  In this study, antioxidant property of methanol and water extracts of leaf powder and pure Andrographolide compound was studied and the results are recorded in Table 3 and Fig 4.

Among the two extracts, methanol extract exhibited higher antioxidant property than water extract. Comparatively, the antioxidant activity was higher in pureandrographolide component than leaf powder extracts. However, the standard ascorbic acid showed the highest reducing power. As the concentration of the extracts increased the reducing power also increased. The reducing ability of a compound depends on the presence of reductants (antioxidants). The presence of antioxidants in A. paniculata causes the reduction of the Fe³⁺ ferricyanide complex to the ferrous form exhibiting its antioxidant potency¹⁸.

TABLE 3: ANTIOXIDANT ACTIVITY OF LEAF POWDER OF A. PANICULATA

FIG 4: ANTIOXIDANT ACTIVITY OF LEAF POWDER OF A. PANICULATA

CONCLUSIONS: The efficacy of any drug depends on the scientific validation of the material. In the present study, proper time of collection of plant material for drug preparation is identified and reported. The correct age and time for collecting the material to   obtain maximum amount of andrographolide content is 120 day old plants (just before flowering). The antioxidant potency of the plant is also found to be good.  Therefore, it is concluded that this scientific methods can be conveniently adopted for validating the plants for drug preparation.

 ACKNOWLEDGEMENTS: The Authors thank UGC for providing financial support (JRF) for this work.

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    How to cite this article:

    Umadevi U and Kamalam M: Phytochemical and Antioxidant Studies On an Important Indigenous Medicinal Plant-Andrographis Paniculata (Burm.F) Nees.Int J Pharm Sci Res2014; 5(12): 5240-44.doi: 10.13040/IJPSR.0975-8232.5 (12).5240-44.

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