INTRODUCTION:

Medicinal plants are useful for the treatment of various human diseases from ancient times. It is a well-known fact that human beings depend on nature either directly or indirectly for food and shelter. Among that, the use of medicinal herbs has a very important role in the cultures of civilizations. Throughout the ages, the utility of traditional plants for the huge proportions of the world populations is always a dominant criterion in the healthcare system of developing countries.

Inspite of various advancements, the contribution of traditional system of medicines is still increasing and provides a tremendous contribution to the pharmaceutical industry. India is the largest producer of medicinal herbs and a rich heritage of traditional medicine constituting with its different components like ayurveda, siddha, unani and homoeopathy¹. All parts of medicinal plants are effective in the treatment of diseases and help to discover new kinds of drugs².

Phytochemicals present in these plants exhibits a strong physiological action on the human body. The most important property of these bioactive constituents of plants is that they are more effective with little or no side effects when compared to the commonly used synthetic agents^3,4. Medicinal Plants acts as an important source of antioxidants, which act as a major defence against radical mediated toxicity by protecting the damages caused by free radicals⁵. Currently, 25% of herbal drugs in modern pharmacopoeia are plant based and several synthetic drugs are manufactured by using chemical substances isolated from plants⁶. The scientific search for effective, nontoxic compounds with antioxidant activity has been increasing in recent years. Various reports have been evaluated for the antioxidant activities of various herbal and synthetic antioxidant substances⁷. The secondary metabolites from the plants exerts beneficial health effects to humans, animals, prevents the food deterioration⁸.

Picrosides is the active ingredient of the plant Picrorhiza kurroa. It is a well-known traditional medicine belongs to the genus Picrorhiza, a member of the foxglove family, Scrophulariaceae. It is a hairy herb with bitter roots, well known for its hepatoprotective property. Also proved for its various other pharmacological properties such as antioxidant, anti-inflammatory, cardiotonic, brain tonic. It is also used in dyspepsia, fever, purgative, antiperiodic, cholagogue, cathartic, carminative, anthelminthic, asthma, flatulence, cardiac complaints, and chronic hepatitis^9,10,11.

MATERIALS AND METHOD:

Plant Material Collection:

Picrorhiza kurroa root material was purchased from the local market of T.V.K Nagar, Chennai. It was identified and authenticated by the Plant taxonomist Prof.Dr.P.Jayaraman, Plant Anatomy Research Centre (PARC), Chennai, Tamilnadu (Reg No:PARC/2021/4554). A Voucher specimen has been deposited in the Central Research Lab, Meenakshi Ammal Dental College and Hospital, Chennai for future reference.

Preparation of methanolic fraction of Picrorhiza kurroa:

200gms of the root powder dissolved in 600ml of the methanol (1:3 w/v). Mixture was shaken in orbital shaker at 60 rpm for two successive days. The supernatant was filtered through Whatman filter paper (No 2) on a Buchner funnel, while the residues were used for a second (400ml) and third (200lml) extraction. Each day the dissolved parts were filtered and stored in a preweighed flask before drying. After the third extraction, the filtrates were decanted and combined. The crude extract was collected in a petridish, and used for the phytochemical studies. Total yield of the extract was obtained as 42.179gms (w/v). Refrigerated the Dried extract at 4ºC for further future studies.

Fig 1: Various stages of Picrorhiza kurroa root

Methods of Qualitative Phytochemical analysis:

Qualitative analysis of methanolic root extract was carried out to determine the presence of various bioactive compounds using the standard qualitative procedures.^12,13,14

Test for Alkaloids:

About 2ml of the filtrate taken in three different test tubes and few drops of Dil.HCl were added in each of the tube followed by Mayer’s, Hager’s and Wagner’s reagent. Presence of alkaloids indicated by the appearance of cream precipitation, yellow precipitation, reddish brown colored precipitation after adding Mayer’s, Hager’s and Wagner’s reagent respectively.

Test for steroids: Salkowsky Test:

About 0.5gm of the filtrate dissolved in 2ml of chloroform. 2ml of conc. Sulphuric acid added to form a lower layer (chloroform layer). Appearance of reddish-brown colour at the interface indicates the presence of steroids.

Test for triterpenoids:

(Lieberman Burchardt test): Chloroform solution of the extract added with few drops of acetic acid and one ml concentrated sulphuric acid along the sides of the test tube. Appearance of blue green color indicates the presence of triterpinoids.

Test for phenols:

Filtrate treated with few drops of 15% acetic acid and a few drops of sodium nitrate solution. Muddy or Niger brown color indicates the presence of phenols.

Test for Flavanoids:

About 1 ml of the filtrate treated with few drops of 10% lead acetate. Appearance of yellow orange colour indicates the presence of flavonoids.

Test for Tannins:

About 1 ml of the filtrate treated with 0.5ml of 10% lead acetate. White precipitate appearance indicates the presence of tannins.

Test for saponins:

About 0.5gm of filtrate shaken with water in a test tube. Presence of saponins preliminarily evidenced by Frothing, which persist on warming.

Test for carbohydrates:

About 2ml of filtrate added with 2 drops of molish reagent and few drops of conc sulphuric acid. Appearance of violet or reddish color indicates the presence of carbohydrates.

Test for glycosides:

Dissolve a small amount of filtrate in 1ml water and add sodium hydroxide solution. The presence of glycosides was indicated by Appearance of yellow color

Test for Cardiacglycosides:

About 0.5gms filtrate added with 2ml of glacial acetic acid, ferric chloride and conc sulphuric acid. Appearance of reddish brown colour ring at the junction indicates the presence of cardiacglycosides.

Test for Coumarins:

About 1ml of extract, add 1ml of 10% sodium hydroxide. Appearance of yellow color indicates the presence of coumarins.

Test for Proteins: Millon’ s test:

2ml of plant extract, was added with 2ml of Millon s reagent and observed for two minutes for the formation of white precipitate. On gentle heating, turned to red indicates the presence of proteins in it.

Test for Amino Acids:

Ninhydrin test: 2ml of plant extract was added with 2ml of Ninhydrin reagent. Violet color indicates the presence of amino acid.

Fig 2: Preliminary phytochemical tests of the Methanolic root extract of Picrorhiza kurroa

Table 1: Preliminary Phytochemical Screening of Roots of Picrorhiza kurroa

S. No	Test		Inference	Observation
1	Alkaloids	Mayer’s test	Appearance of cream precipitate	-
		Hager’s test	Appearance of yellow precipitate	-
		Wagners test	Appearance of reddish brown colored precipitation	-
2	Saponins	Foam test	Foam produced persists for 10 min.	-
3	Glycosides	Sodium hydroxide test	Appearance of yellow colour	+
4	Cardiac Glycosides	Keller Killani’s test	Appearance of reddish brown colour ring at the junction	+
5	Phenols	Ellagic test	Muddy or niger brown colour	-
6	Flavonoids	Ferric chloride test	Appearance of green colour	+
6	Flavonoids	Sodium hydroxide test	Appearance of orange colour	+
7	Steroids	Salkowski’s test	On standing yields the appearance of red color	+.
8	Tannins	Lead acetate test	Appearance of white precipitate	+
9	Carbohydrates	Molisch’s test	Appearance of violet/purple colour	-
		Benedict’s test	Formation of reddish brown precipitate	-
		Fehling’s test	Formation of reddish brown precipitate	-
10	Proteins And Aminoaicd	Ninhydrin test	Appearance of blue colour	-
10	Proteins And Aminoaicd	Millon’s test	On heating shows the appearance of red colour	-
11	Terpenoids	Lieberman Burchardt test	Appearance of blue green color	+
12	Coumarins	Sodium hydroxide test	Appearance of yellow colour	+

+ Detected - Not Detected

Determination of In Vitro Antioxidant Activity by DPPH assay:

Several assays were used to evaluate the antioxidant effect of the natural antioxidants such as DPPH assay, ABTS radical scavenging assay, nitric oxide radical scavenging activity, etc., DPPH assay, is the widely used method to evaluate antioxidant activities in a relatively short time compared with other methods. Antioxidants react with DPPH which is a stable free radical and convert it to DPPH hydrazine. The degree of decolorization indicates the scavenging potentials of the antioxidant compounds¹⁵.

The antioxidant ability of the Picrorhiza kurroa root extract against 1, 1- diphenyl-2-picryl hydrazyl (DPPH) (Sigma-Aldrich) was determined by using the method of Harborne. 0.1mM solution of DPPH was prepared in ethanol as stock solution. This solution is added to 3mL of each extract at various concentrations (50, 100, 150, 200, 250μg/mL). 200μl of DPHH solution in 2.7ml of ethanol served as control. 100μl of DPHH solution in 2.7ml of methanol mixed with 100μl of Ascorbic acid which was taken as standard. Standards and plant extracts thus prepared were incubated at 37^oC for 30 minutes. Then, absorbance was measured at 515 nm by using spectrophotometer (Nabi UV/Vis Nano Spectrophotometer)¹⁶. The degree of decolorization of DPPH from purple to yellow indicated the scavenging efficiency of the extract. A lower absorbance value shows the higher radical scavenging activity. The percent DPPH scavenging effect was calculated by using following equation¹⁷:

DPPH scavenging effect (%) or Percent inhibition = A0 - A ₁/ A0 × 100.

Where A0 was the Absorbance of control reaction and A₁ was the Absorbance in presence of test or standard sample

IC₅₀ was calculated by plotting the graph, taking % inhibition on y, axis and concentration on x, axis¹⁸.

Data Analysis:

Phytochemical compound was analyzed by a descriptive method and antioxidant activity assay were analyzed using regression linear equation using MS-Excel and presented in Fig: 4

RESULTS AND DISCUSSION:

Phytochemical Analysis:

In recent days, phytochemicals attracts the attention towards the new findings regarding their biological activities. These active compounds play some metabolic role and control development in a living system¹⁹. So, used in various preparations of recent medications²⁰. Phytochemical data indicates the distinct patterns of chemical compositions in constituents of the Picrorhiza kurroa root extract. Results of phytochemical evaluation are shown in Table 1. The patterns of composition differed considerably in their quantitative values. Presences of tannins, flavanoids, glycosides, terpenoids, coumarins, steroids are the important bioactive agents which might be involved in antioxidant activity against various oxidative systems in vivo and also add up to the other therapeutic potential of this root.

Fig 3: DPPH Radical scavenging activity

Antioxidant activity:

Free radicals induced cell damage appears to be the fundamental cause of various human diseases such as cancer, atherosclerosis, diabetes, neurodegenerative disorders and aging^21,22. It is mainly caused due to imbalance between pro-oxidant antioxidant homeostasis. Naturally occurred antioxidants, from many plants, foods and beverages can treat various diseases by preventing cellular damage caused by free radicals in the body²³. Antioxidant supplementation constitutes important defense against various oxidative stress related disease²⁴. Synthetic antioxidants such as Propyl Galllate (PG), Tertiary Butylated Hydroxy Quinone (TBHQ), Butylated Hydroxy Toluene (BHT) and Butylated Hydroxy Anisole (BHA) are available but exhibits adverse effects. Thus, the researchers changing the path towards the natural antioxidants which is rich in plants²⁵.

Picrorhiza kurroa root extract shown a good antioxidant activity. The decrease in absorbance with increase in concentration of the extract manifested the rapid discolouration of the purple DPPH, indicates that the methanolic root extract has antioxidant activity. This is due to its proton donating ability was measured on the basis of the scavenging activity of the stable DPPH free radical, with concentration ranging from 50-250 µg/ml as shown in Figure. The data pertaining to the activity clearly revealed the concentration providing 50% inhibition (IC₅₀) of 162.73 mg/mL.

Fig 4: DPPH Radical scavenging activities of the methanolic root extract of Picrorhiza kurroa

CONCLUSION:

Plants are the rich source of drug molecules with health protective potential and natural antioxidants of significant impact on the status of human health and disease prevention. In summary, the present work was the investigation of rhizomes of Picrorhiza kurroa which is available in Common herbal drug stores in Chennai. In order to check naturalness of the root, preliminary pytochemical screening and antioxidant activity was done. Methanolic root extract of Picrorhiza kurroa rich in phytochemicals like cardiacglycosides, Tannins, terpenoids, steroids and coumarins, and also possessed strong antioxidant potential which evidences the capable of inhibiting, quenching free radicals to terminate the radical chain reaction. Further biological studies such as quantification investigation, animal and molecular determination may subsequently lead to the drug discovery and various pharmacological formulations.

CONFLICT OF INTEREST:

Authors have no conflict of interest.

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Received on 29.06.2022 Modified on 07.11.2022

Research J. Pharm. and Tech 2023; 16(9):4266-4270.

DOI: 10.52711/0974-360X.2023.00698

Preliminary Phytochemical screening and Antioxidant properties of Methanolic root extract of Picrorhiza kurroa