INTRODUCTION:

Free radical may be defined as any molecular species that is capable of independent existence and contains an unpaired electron in an atomic orbital¹. Several important oxygen containing free radicals formed in the body are: Hydroxyl radical (OH^⃰), superoxide anion radical (O₂^⃰), Hydrogen peroxide (H₂O₂), Oxygen singlet (O₂^⃰), Hypochlorite (ClO^⃰), Nitric oxide radical (NO^⃰) and peroxynitrite radical (ONOO⃰). They are responsible for development of various diseases². These are highly reactive species, capable of damaging DNA, proteins, carbohydrates and lipids in the nucleus and cell membranes³. Anti-oxidants are the substances which protect the cell membranes and other body compartments against oxidants by scavenging free radicals produced in it.

They act by reducing reactive oxygen concentration, inhibiting lipid chain oxidation, inhibiting free radical production³. The need for anti-oxidants is more with increased exposure to free radicals, pollution, cigarette smoking, stress and disease⁴. Free radicals are the major contributors of aging, degenerative disease of aging including cancer, cardiovascular disease, immune system dysfunction and brain dysfunction³. Therefore, the attenuation of these free radicals may be a profound approach to diminish the development of diseases.

P. crispum (Parsley) is a bright green biennial herb belonging to family Apiaceae. Parsley fruits or seeds contain volatile oil in larger proportion including apiole and terpenes. It also contains psoralen, xanthotoxin, ficusin, bergapten, majudin, heraclin, furanocoumarins include 8-methoxypsoralen, 5-methoxypsoralen, oxypuecedanin, isopimpinellin⁵. The seeds also contain myristicin, myrcene, fat, beta-phelandrene, cafeiolene. Iron, calcium, phosphorus and anti-oxidants like luteolin, vitamin C, vitamin A and zinc are also present⁶. Parsley has been used as an anti-cancer⁷, anti-diabetic⁸, diabetic complications⁹, anti-inflammatory¹⁰, inflammatory bowel disease¹⁰, anti-bacterial¹¹, Immunomodulatory action¹¹, cardiovascular and anti-hyperlipidemic agent¹². It is one of the most used plants to treat hypertension, diabetes, cardiac and renal diseases¹³.

Considering this view point, present study was designed to estimate the anti-oxidant potential of hydro-alcoholic extract of seeds Petroselinum crispum.

MATERIALS AND METHODS:

Plant material used:

Seeds of plant P. crispumwere collected from local market, Ludhiana, Punjab, India and were authenticated by National Institute of Science Communication and Information Resources (NISCAIR), New Delhi.

Preparation of extract:

The dried seeds were grounded to coarse powder and 80 g of powdered drug was extracted with hydro-alcohol (aqueous ethanol) (40%) using soxhlet apparatus. The extract was then filtered and concentrated using rotary evaporator (40°C) under reduced pressure.This crude extract was used for the preliminary phytochemical analysis and in-vitro anti-oxidant assay.

Phytochemical screening:

The following procedures were used to test the presence of various chemical constituents in the extract:

1. Test for Terpenoids:

a) Salkowski’s Test:

The extractwas mixed with chloroform and filtered. The filtratewas treated with few drops of Conc. Sulphuric acid, shaken and allowed to stand. Golden yellow colour indicates presence of triterpenes.

b) Copper acetate Test:

The extractwas dissolved in water and 3-4 drops of copper acetate solution were added. Emerald green colour indicates the presence of diterpenes.

2. Test for Flavonoids:

a) Alkaline Reagent Test:

To the extract few drops of sodium hydroxide solution were added. Presence of flavonoids is indicated by intense yellow colour, which becomes colourless on addition of dilute acid.

b) Lead acetate Test:

To the extract few drops of lead acetate solution were added. Presence of flavonoids is indicated by yellow colour precipitate.

3. Detection of Phenols:

a) Ferric Chloride Test: To the extract 3-4 drops of ferric chloride solution were added. Bluish black colour indicates the presence of phenols.

4. Detection of Tannins:

a) Gelatin Test:

1% gelatin solution containing sodium chloride was added to the extract. White precipitate indicates the presence of tannins.

5. Detection of Alkaloids:

Extrac twas dissolved in dilute Hydrochloric acid and filtered.

a) Mayer’s Test:

The filtratewas mixed with Mayer’s reagent (Potassium Mercuric Iodide). Yellow coloured precipitate indicates the presence of alkaloids.

b) Dragendroff’s Test:

The filtrate was mixed with Dragendroff’s reagent (solution of Potassium Bismuth Iodide). Red precipitate indicates the presence of alkaloids.

6. Detection of Glycosides:

Extract was hydrolyzed with dil. HCl and then subjected to test for glycosides.

a) Modified Borntrager’s Test:

The extract was mixed with Ferric Chloride solution and heated in boiling water for about 5 minutes. Mixture was cooled and extracted with equal volumes of benzene; benzene layer was separated and treated with ammonia solution. The presence of anthranol glycosides in the ammonical layer is indicated by rose-pink colour.

b) Legal’s Test:

Extract was treated with sodium nitropruside in pyridine and sodium hydroxide. Pink to blood red colour indicates presence of cardiac glycosides.

7. Detection of Saponins

a) Froth Test:

Distilled water was added to the extract and shaken in a graduated cylinder for 15 minutes. Formation of foam indicates presence of saponins.

8. Detection of Proteins and Amino acids

a) Xanthoproteic Test:

Few drops of conc. Nitric acid were added to the extract. The presence of proteins is indicated by the formation of yellow colour.

b) Ninhydrin Test:

0.25% w/v ninhydrin reagent was added to the extract and boiled for few minutes. The presence of amino acids is indicated by the formation of blue colour.

9. Test for Fats and Fixed oils

a) Solubility Test:

Extract was dissolved in ether, benzene, chloroform, 90% ethanol and water. Solubility of extract in ether, benzene and chloroform, insolubility in ethanol and water shows the presence of fats and fixed oils.

b) Filter paper test:

Put a drop of extract on filter paper. Permanent stain on filter shows the presence of fats and fixed oils.

Quantitative estimation of phytoconstituents:

1. Determination of Total Flavonoids:

10gm of powder of seeds was extracted repeatedly with 100ml of 80% aqueous methanol at room temperature. The solution was then filtered through Whatman filter paper (42). The filtrate was later transferred into a crucible and evaporated into dryness and weighed to a constant weight¹⁴.

2. Determination of Total Saponins:

20gm of powdered seeds were taken in conical flask and 100ml of 20% aqueous ethanol was added. The mixture was heated over a hot water bath for 4hr with continuous stirring at about 55°C. The mixture was then filtered and further re-extracted with 200ml of 20% ethanol. The extracts were combined and reduced to 40ml on a water bath. The concentrate was taken into a 250ml separating funnel to it 20ml of diethyl ether was added and then shaken vigorously. The aqueous layer was taken while the ether layer was discarded. The purification process was repeated. Then 60ml n-butanol was added. The n-butanol extracts were combined and washed twice with 10ml of 5% aqueous sodium chloride. The solution was further heated on a water bath. The sample was dried in the oven to a constant weight, and the saponin content was calculated as percentage¹⁵.

3. Determination of Terpenoids:

Powder of seeds of P. crispum was dissolved in methanol and water in ratio 4:1. Solution was filtered and evaporated. Remaining filtrate was acidified with 2M sulphuric acid followed by chloroform extraction. Gently, stirred and allowed to stand, the non-aqueous layer was extracted and then dried¹⁶.

In-vitro anti-oxidant assays:

1. Estimation of Total phenolic content:

1ml of extract solution (1g/ml) was taken in a volumetric flask and diluted to 46ml with methanol. 1ml of Folin–Ciocalteau reagent was added and mixed thoroughly. After 3 minutes 3ml of 2% sodium carbonate was added and the mixture was allowed to stand for 3h with intermittent shaking. The absorbance of the blue color that developed was measured at 760nm. Gallic acid was taken as a standard. Total content of phenolic compounds in plant extract was determined as μg of gallic acid equivalents (GAE). All determinations were performed in triplicate¹⁷.

2. Reducing Power assay:

The reducing power of the extract was determined using Gallic acid as a standard. 1ml of different concentrations of stock i.e. 50 - 2000μg/ml were taken and the volumes were made up to 2ml with distilled water and were mixed into the mixture of 2.5ml of 0.2M phosphate buffer (pH 6.6) and 2.5ml of 1% potassium ferricyanide. The mixture was then incubated at 50⁰C for 20 min. 2.5ml of 10% trichloro acetic acid was added to the mixture, which was then centrifuged at 3000r.p.m for 10 min. The upper layer of the solution (2.5ml) was mixed with distilled water (2.5ml) and FeCl₃ (0.5ml, 0.1%) and the absorbance was measured at 700nm¹⁸.

Absorbance of Control - Absorbance of test Sample

Reducing power (%) =––––––––––––––––––––––––––––––––––x 100

Absorbance of Control

3. Hydrogen peroxide-scavenging activity:

The extract solution (20-320μl/ml) was dissolved in 3.4 mL of 0.1M phosphate buffer (pH 7.4) and mixed with 600 μL of 43 mM solution of hydrogen peroxide. The absorbance value (at 230 nm) of the reaction mixture was recorded at 10 min intervals between 0 and 40 min. The percentage of hydrogen peroxide scavenging effect was calculated using following formula¹⁹.

A₀ – A₁

Percentage H₂O₂ scavenging effect =–––––––––––––––––––––– X 100

A₀

Where, A₀ = absorbance of blank, A₁ = absorbance standard or extract.

4. Determination of DPPH (1-1-diphenyl 2-picryl hydrazyl) radical-scavenging activity:

0.1 mM solution of DPPH in ethanol was prepared and 1ml of this solution was added to 3ml of extract solution (10 - 320 μg/ml). 30 minutes later, the absorbance was measured at 517 nm (Prakash, 2001). Ascorbic acid was used as the reference compound. Radical-scavenging activity was expressed as the inhibition percentage of free radical by the sample and was calculated using the following formula:

(A₀ - A_t)

% inhibition =–––––––––––––––––––––x 100

A₀

Where A₀is absorbance of the control (blank, without extract) and A_tis absorbance in presence of extract. All the tests were performed in triplicates and the graph was plotted with the mean values.

5. Superoxide anion scavenging activity:

The superoxide scavenging activity was determined by the slightly modified PMS-NADH superoxide generating system (13). About 1ml ofnitro blue tetrazolium (NBT) solution (156μMNBT in 100mM phosphate buffer, pH 7.4) 1ml NADH solution (468μM in 100mM phosphate buffer, pH 7.4) and 0.1ml of extract solution (5μg/ml to 80μg/ml) in water were mixed and treated with 100μl of phenazine methosulphate (PMS) solution (60μM PMS in 100mM phosphate buffer, pH 7.4). The reaction mixture was incubated at 25°C for 5 min, and the absorbance at 560nm was measured against blank sample²⁰.

6. Phosphomolybdenum free radical scavenging activity:

0.1ml of sample solution was mixed with reagent containing 0.6M sulphuric acid, 28mM sodium phosphate and 4mM ammonium molybdate. Tube was capped and incubated in boiling water bath at 95⁰ for 90 min. The mixture was cooled at room temperature and absorbance was measured against blank at 695nm in UV spectrophotometer⁴.

7. Nitric oxide scavenging activity:

2ml of 10mM sodium nitroprusside was dissolved in 0.5ml phosphate buffer saline and was mixed with samples at various concentrations. Mixture was incubated at 25⁰C. After 150 minute of incubation, 0.5ml of incubated solution was withdrawn and mixed with 0.5ml of Griess reagent. Mixture was incubated at room temperature for 30 min and the absorbance was measured at 546nm²¹.

RESULTS AND DISCUSSION:

The results of phytochemical screening of hydro-alcoholic extract of seeds of P. crispumare given in the following table:

Table 1: Qualitative analysis of hydro-alcoholic extract of P. crispumseeds

Phytochemical Test

Hydro-alcoholic extract

Alkaloids

· Mayer’s test

· Dragondroff’s test

Flavanoids

· Alkaline reagent test

· Lead acetate

Phenols and Tannins

· Ferric chloride test

· Gelatin test

Terpenoids

· Salkowski test

· Copper acetate

Saponins

· Froth test

Glycosides

· Modified Borntrager’s Test

· Legal’s Test

Amino acids

· Xanthoproteic test

· Ninhydrin test

Fats and Fixed oils

· Solubility Test

· Filter paper test

The phytochemical screening indicated that flavanoids, triterpenoids, phenols, tannins, saponins and amino acids are present in hydro-alcoholic extract of P. crispumseeds.

Quantitative phytochemical analysis:

1. Flavonoids: The amount of flavonoids was found to be 1.97gm.

2. Terpenoids: The amount of terpenoids was found to be 0.47gm.

3. Saponins: The amount of saponins was found to be 0.59gm.

In- vitro anti-oxidant assay:

1. Total phenolic content

The amount of total phenolic content in the hydro-alcoholic extract of P. crispumseeds was found to be 1740 mg/g gallic acid equivalent.

2. Reducing power:

The reducing power increases with increase in the concentration. The EC₅₀ of hydro-alcoholic extract of seeds of P. crispumwas found to be 11.66 μg/ml at 320μg/ml. Ascorbic acid was used as standard and the EC₅₀was found to be 21.42 μg/ml (Figure 1).

3. Hydrogen peroxide scavenging activity:

The % inhibition of free radicals in hydro-alcoholic extract of P. crispumseeds was found to be 75.25% at 320 μg/ml. Ascorbic acid was used as standardand the % inhibition was found to be 89.49% at 320 μg/ml concentration. The IC₅₀value of hydro-alcoholic extract was found to be 71.91 μg/ml (Figure 2).

4. DPPH scavenging activity:

The % inhibition of hydro-alcoholic extract of P. crispumseeds was found to be 72.25% at 10μg/ml while that of ascorbic acid was found to be 84.98% at 10 μg/ml. The IC₅₀ value of hydro-alcoholic extract of P. crispumseeds was found to be 45.77 μg/ml (Figure 3)

5. Superoxide radical scavenging activity:

The % inhibition of free radicals in hydro-alcoholic extract of P. crispumwas found to be 75.89% at 320 μg/ml and IC₅₀value was found to be 63.23μg/ml. Ascorbic acid was used as standard and the % inhibition was found to be 78.94% at 320μg/ml.

6. Phosphomolybdenum activity:

The IC₅₀of hydro-alcoholic extract of P. crispumseeds was found to be 24.4μg/ml and % content was found to be 0.138μM/g ascorbic acid equivalent.

7. Nitric oxide scavenging activity:

The % inhibition of free radicals in hydro-alcoholic extract of P.crispum, using sodium nitroprusside and Griess reagent was found to be 73.83% at 320 μg/ml. Ascorbic acid was used as standard and % inhibition was found to be 79.74% at 320 μg/ml. The IC₅₀value was found to be 116.87μg/ml. (Figure 4)

Figure 1: Reducing power assay of hydro-alcoholic extract of P. crispumseeds.

Figure 2: Hydrogen peroxide radical scavenging activity of hydro-alcoholic extract of P. crispumseeds.

Figure 3: DPPH radical scavenging activity of hydro-alcoholic extract of P. crispumseed

Figure 4: Nitric oxide radical scavenging activity of hydroalcoholic extract of P. crispumseeds

DISCUSSION:

Phytochemical evaluation of hydro-alcoholic extracts of P. crispumseeds revealed the presence of flavonoids, saponins, tannins and glycosides. Flavonoids like luteolin, apiin and apigenin act as anti-oxidants and protect the arteries from oxidative stress and damage (Meyer et al., 2006). Parsley contains chlorophyll which alkalizes the body and purifies the blood vessels. In-vitro anti-oxidant activity was measured in terms of total phenol content, reducing power assay, hydrogen-peroxide scavenging activity, DPPH scavenging assay, superoxide scavenging activity, phosphomolybdenum scavenging activity and all the tests revealed a significant anti-oxidant potential of hydro-alcoholic extract of P. crispumseeds.

CONCLUSION:

From the study it can be concluded that hydro-alcoholic extract of P. crispumseeds possess significant anti-oxidant activity.

ACKNOWLEDGEMENTS:

Authors are thankful to the Shri Baba Mastnath Institute of Pharmaceutical Sciences and Research, Baba Mastnath University, Rohtak, Haryana for providing necessary facilities to carry out this work.

CONFLICT OF INTEREST:

Authors declare no conflict of interest.

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Received on 27.06.2020 Modified on 19.10.2020

Research J. Pharm. and Tech 2021; 14(10):5391-5396.

DOI: 10.52711/0974-360X.2021.00940