Pharmaceutica Analytica Acta

The antioxidants are a variety of Vitamins, minerals and enzymes that help to protect the body from the formation and disposal of free radicals. Some people have the idea that an antioxidant is a specific nutrient, yet it actually refers to any nutritional compound that has these qualities. They are useful in the fight against ageing and degenerative diseases, it must be kept in mind that they have a wide sphere of influence on the body, and they can positively influence your general wellbeing.

The big source of antioxidants is the green belt in the form of plants, which make life possible on this planet. The use of herbal medicine for the treatment of diseases and infections are as old as mankind. The World Health Organization supports the use of traditional medicine, provided they are proven to be efficacious and safe (WHO 1985). In developing countries, a huge number of people lives in extreme poverty and some are suffering and dying for want of safe water and medicine, they have no alternative for primary health care.

Dandelion is considered to be an excellent general tonic and a “natural” diuretic. Dandelion tea has shown to be very helpful as a liver detoxicant. It also improves functions of gallbladder, pancreas, spleen and intestines. Dandelion can reduce inflammations in cases of hepatitis and cirrhosis, help gallstone dissipation and improve kidney functions.

An antitumor action of the aqueous extract of Taraxacum officinale has been reported in the scientific literature [1]. Dandelion’s active ingredients are found in both the roots and leaves. The leaves contain bitter sesquiterpene lactones such as taraxinic acid and triterpenoids such as cycloartenol. The roots contain these compounds as well as phenolic acids and inulin [2,3]. Potassium is present in the leaves at 297 mg per 100 grams of leaves [3]. The leaves also contain substantial amounts of Vitamin A (14,000 units per 100 grams of leaves, compared with 11,000 units per 100 grams of carrots) [3]. The sesquiterpene lactones found in both leaves and roots have demonstrated diuretic effects [4]. They also stimulate bile flow from the liver. A Chinese case series reported that an herbal combination including dandelion was helpful in treating 96 adults with chronic hepatitis B infection [5]. In Chinese, Arabian and Native American traditional medicine it is used to treat a variety of diseases including cancer [6,7].

Evidence suggests dandelion may influence nitric oxide production [8]. Nitric oxide is important for immune regulation and defense; however, this molecule can be inhibited by cadmium. Classically listed as a cholagogue, dandelion root is approved by the German Commission E for the treatment of disturbances in bile flow, stimulation of diuresis, loss of appetite, and dyspepsia [9].

Dandelion root contains an abundance of sesquiterpene lactones, also known as bitter elements principally taraxacin and taraxacerin [10]. Other related compounds include beta-amyrin, taraxasterol, and taraxerol, as well as free sterols (sitosterin, stigmasterin, and phytosterin). Other constituents include polysaccharides (primarily fructosans and inulin), smaller amounts of pectin, resin, and mucilage, and various flavonoids. Three flavonoid glycosides – luteolin 7-glucoside and two luteolin 7-diglucosides – have been isolated from the flowers and leaves. Hydroxycinnamic acids, chicoric acid, monocaffeyltartaric acid, and chlorogenic acid are found throughout the plant, and the coumarins, cichoriin, and aesculin have been identified in the leaf extracts [11]. Dandelion leaves are a rich source of a variety of vitamins and minerals, including beta carotene, nonprovitamin A carotenoids, choline, iron, silicon, magnesium, sodium, potassium, zinc, manganese, copper, and phosphorous.

The present research work was carried out about the antioxidant potential determination of the dandelion. As per the research methodology, the plant is heavily commenced with phytochemical, so could be associated with high potential of antioxidants, therefore the determination of antioxidant potential of the plant became a compulsory issue, so was carried out the concerned research work.

Plant materials

The plant material was collected from Kupwara region of Kashmir and was authenticated from FRI Dehradun. The collection process was preferably done in the dry condition. Plant was weighed before and after the removal of unwanted material kept under shade at room temperature for the removal of extra moisture. The plant samples were air dried and grounded into uniform powder with a grinder. All the plants parts i.e. stem, flowers and roots were collected separately and were subjected to different operations individually.

Extraction

The extraction procedure was carried out with water. The extraction was done by Soxhlet extraction method. A thimble was used in order to get the purest form of extract. 90 g of the root material was used for extraction, 80 g of flower and 105 g of stem plant material was used for extraction purpose. The percentage yields of various extracts are mentioned in table 1.

Table 1: Percentage yield of various plant extracts.

Antioxidant activity determination: The antioxidant property of plant extracts were determined by two given below mentioned methods.

The antioxidant activities as measured by FRAP method (Ferric reducing ability of plasma or plant) according Benzie and Strain, (1996-19990).

DPPH free radical scavenging assay was measured using DPPH free radical test, employing method of Wong et al. (2005).

Frap- working solution: 25 ml acetate buffer, 2.5 ml TPTZ solution (2, 4, 6- Tripyridyl-S- triazine) and 2.5 ml FeCl₃.6H₂ O solution was freshly prepared. Aqueous solution of known FeCl₄.7H₂ O was used for calibration.

DPPH method: DPPH Scavenging activity was measured by the spectrophotometric method. A stock solution of DPPH (1.5 mg/ml in methanol) was prepared such that 75 μl of it in 3 ml of methanol. Decrease in the absorbance in presence of sample extract at different concentration (10-100 μg/ml) was noted after 15 min. IC₅₀ was calculated from % inhibition.

Protocol for DPPH Free radical scavenging activity

Preparation of stock solution of the sample: 10 mg of extract was dissolved in 10 ml of methanol to get 1000 μg/ml solution.

Dilution of test solution: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μg/ ml solution of test were prepared from stock solution.

Preparation of DPPH solution: 15 mg for DPPH was dissolved in 10 ml of methanol. The resulting solution was covered with aluminum foil to protect from light.

Estimation of DPPH scavenging activity: 75 μl of DPPH solution was taken and the final volume was adjusted to 3 ml with methanol, absorbance was taken immediately at 517 nm for control reading. 75 μl of DPPH and 100 μl of the test sample of different concentration were put in a series of test-tubes and final volume was adjusted to 3 ml with methanol. Absorbance at zero time was taken in UV-Visible at 517 nm for each concentration. Final decrease in absorbance of DPPH with sample of different concentration was measured after 15 minute at 517 nm.

Percentage inhibitions of DPPH radical by test compound were determined by the following formula.

% Reduction=Control absorbance – Test absorbance/ Control absorbance X 100.

Calculation of IC₅₀ value using graphical method.

Percentage yield of various plants extracts (Table 1)

Table 1: Percentage yield of various plant extracts.

Preparation of standard solution:- 0.01 ml of FeSO₄, solution was mixed with 1.5 ml of FRAP reagent and volume was made up to 5 ml with distilled water, rest of dilutions were prepared by varying the volume of ferrous sulphate solution with distilled water. Monitor up to 5 mM/cm path length 37°C. Absorbance was recorded (Tables 2-5 and Figures 1-4).

Table 2: Preparation of standard solution.

Table 3: Data sheet for the Antioxidant properties of the flower extract.

Table 4: Data sheet for the Antioxidant properties of the whole plant extract.

Table 5: Data sheet for the Antioxidant properties of the root extract.

Figure 1: Standard curve for FRAP reagent solution.

Figure 2: Line Graph showing the variation of antioxidant potential of the flower extract with concentration.

Figure 3: Line Graph showing the variation of antioxidant potential of the whole plant extract with concentration.

Figure 4: Line Graph showing the variation of antioxidant potential of the root extract with concentration.

Absorbance was recorded at 593 nm/cm and from the standard graph curve the value of (Ɛ) comes out to be

ε = 1.5 x 10^-4 Lmol^-1cm^-1.

Antioxidant activity testing by DPPH method

DPPH Free Radical Scavenging Activity of Ascorbic Acid (Table 6).

Table 6: DPPH Free Radical Scavenging Activity of Ascorbic Acid.

Absorbance of the sample at 517 nm

Absorbance of Control = 0.490

Antioxidant Power of Taraxacum officinale (Stem) (Table 7)

Table 7: Antioxidant Power of Taraxacum officinale (Stem).

Absorbance of the sample at 517 nm

Absorbance of Control = 0.490

Antioxidant Activity of Root Extract of T. officinale (Table 8)

Table 8: Antioxidant Activity of Root Extract of T. officinale Absorbance of the sample at 517nm Absorbance of Control = 0.490.

Absorbance of the sample at 517 nm Absorbance of Control = 0.490

Antioxidant Activity of Flower Extract of T. officinale (Table 9)

Table 9: Antioxidant Activity of Flower Extract of T. officinale Absorbance of the sample at 517 nm Absorbance of Control = 0.490.

Absorbance of the sample at 517 nm Absorbance of Control = 0.490

Reactive oxygen species (ROS) are involved in the pathogenesis of various diseases. Uncontrolled oxidation is caused by free radicals. Natural antioxidants that are present in herbs and spices are responsible for inhibiting or preventing the deleterious consequences of oxidative stress. Spices and herbs contain free radical scavengers like polyphenols, Flavonoid and Phenolic compounds. The effect of antioxidants on DPPH radical scavenging is thought to be due to their hydrogen donating ability. The DPPH assay is technically simple, but some disadvantages limit its applications. Besides the mechanistic difference that normally occurs between antioxidants and peroxyl radicals, DPPH is long-lived nitrogen radical, which bears no similarity to the highly reactive and transient peroxyl radicals involved in lipid peroxidation. Many antioxidants that react quickly with peroxyl radicals may react slowly or may even be inert to DPPH [12].

Phenolic compounds are known as powerful chain breaking antioxidants. Phenols are very important plant constituents because of their scavenging ability due to their hydroxyl groups. The Phenolic compounds may contribute directly to antioxidative action (Table 10, Figure 5).

Table 10: showing % Inhibition of various plant extracts & Ascorbic Acid.

Figure 5: Graph Showing the Comparative Study of Antioxidant Potential of Ascorbic Acid and Various plant extracts.

Ferric reducing antioxidant power (FRAP) measures the ability of antioxidants to reduce ferric 2, 4, 6- Tripyridyl-s-triazine complex to intensively blue colored ferrous complex in acidic medium. Hence any compound which is having redox potential lower than that of redox pair Fe (III)/Fe (II) can theoretically reduce Fe (III) to Fe (II) [12].

Taraxacum officinale showed strong antioxidant activity by inhibiting DPPH, and reducing power activities when compared with standard L-ascorbic acid. In addition, all the extracts of plant was found to contain a noticeable amount of total phenols, which play major role in controlling oxidation. The results of this study show that the extracts of Taraxacum officinale can be used as easily accessible source of natural antioxidant. However, the chemical constituents present in the extract, which are responsible for this activity, like flavonoid, alkaloids, steroids, terpenoids, tannins, reducing sugars and proteins present in the extract may be responsible for such activity. Some of these constituents have already been isolated from this plant. Hence, the observed antioxidant activity may be due to the presence of any of these constituents.