Estimation of Synergistic Antioxidant Effect of Methanolic Extracts of Some Medicinal Herbs


Gurleen Kaur1, Srishti Gautam1, Poonam Arora1*, Neelima Dhingra2

1Chitkara College of Pharmacy, Chitkara University, Rajpura, Patiala -140401, Punjab, India.

2University Institute of Pharmaceutical Sciences, Panjab University -160014, Chandigarh, India.

*Corresponding Author E-mail:



Antioxidant activity encompasses protection against cellular wear and tear by interfering in the formation of free radicals. The majority of the diseases are linked to oxidative stress generated due to these free radicals. It makes the basis for plants containing secondary metabolites showing antioxidant activity as the potential source for development of newer formulations showing action against diseases like infections, cancers and other degenerative diseases. The present study involves the study of methanolic extracts of three medicinal herbs cannabis (Cannabis sativa.), wheatgrass (Triticum aestivum) and carrot (Dascus carota L.) commonly available in Indian cultivation. The antioxidant potential of the combination of these extracts was also studied. The combinations showed a synergistic antioxidant activity suggesting that potential of natural plant extracts against any disease can be increased when used in combinations. 


KEYWORDS: Antioxidant, Synergistic, Plant extract, Wheat grass, Cannabis, Carrot, Phytochemical.




An antioxidant is a molecule proficient of retarding or limiting the oxidation of other molecules1. They tend to shield cells from deterioration led by unstable molecules known as free radicals2. The chain reactions caused by free radicals can be eliminated by antioxidants by scavenging free radical intermediates, and more oxidation reactions are further obstructed due to their self- oxidization3,4. Free radicals are basics to any biochemical procedure and are fundamental piece of oxygen consuming life. Greater parts of the diseases are for the most part are connected to oxidative worry because of free radicals5. It has been implicated as a possible contributor to the pathogenesis of numerous diseases, such as various infections, diabetes, cancers, heart diseases and Alzheimer’s disease6. All biological organisms (animals and plants) have antioxidant mechanisms that defend against oxidative injuries and maintain enzymes to remove impaired molecules.


However, the naturally occurring antioxidant mechanisms can be inefficient to protect the body from severe diseases; hence intake of antioxidant compounds as therapy is important7-9.


Plants are the most abundant source of natural antioxidant compounds which have the capability to act as potential antioxidant agents and this effect is credited to the antioxidant properties of the phytoconstituents (secondary metabolites), such as phenols and flavonoids10-14. These secondary metabolities are found in all the parts of plants like roots, stem, bark, fruits and flowers15,16. The required constituent is extracted using a suitable solvent and evaluated for its activity17-20. Also the combination of different extracts can show synergistic effects which can lead to lowering of dose and less side effects related to the plant extract21.


Thus the objective of this report is to determine the antioxidant properties, and screen the phytochemical constituents of methanolic extracts of cannabis (Cannabis sativa.), wheatgrass (Triticum aestivum) and carrot (Dascus carota L.) extract. All three are rich sources of natural antioxidants. Carrot contains antioxidants like beta carotene, ascorbic acid, tocopherol which act against cancer cells and enzymes that promote the conversion of precarcinogens to carcinogens22. The antioxidants present in cannabis have a dramatic influence over the body against malignancy and other diseases23. Flavanoids present in wheatgrass help body get rid of free radicals which are responsible for cellular DNA damage and subsequently prevent cancer and aging24. All these three plant derived extracts show potential to stave off cancer and other degenerative disorders25. Also the present paper provides, the study of synergistic antioxidant effects of combinations of methanolic extracts of cannabis, wheatgrass and carrot have been evaluated.



i. Plant material:

The root of carrot (Daucus carota L.) were collected from local market, leaves of cannabis (Cannabis sativa L.) and shoot of wheat grass (Triticum aestivum Linn.) were collected from medicinal garden of Chitkara college of Pharmacy. These were air dried in shade for 1 month.


ii. Preliminary phytochemical screening:

Preliminary phytochemical analysis was performed through standard official procedures (Indian Pharmacopeia, IP 2010). The phytochemical screening for alkaloids, steroids, carbohydrates, tannins, flavonoid glycosides were carried out to know the presence of different phytoconstituents in the formulations26.


iii. Solvents and Chemicals:

Methanol, hydrogen peroxide, sodium hydroxide and potassium dihydrogen phosphate were used in the study. All the enlisted chemicals and solvents used for extraction, biological evaluation and analytical procedures were of Analytical reagent (AR) grade. Methanol was used for extraction of coarsely powdered dried carrot, wheat grass and cannabis. 2, 2-diphenyl-1-picrylhydrazyl (DPPH) was procured from Sigma-Aldrich27. Distilled water was used throughout the investigation and reference standard for anti-oxidant activity in present study was ascorbic acid.


iv. Instruments:

All the glass apparatus used in the research study was of borosil grade. Soxhlet apparatus was used for extraction procedure and extracts were dried using rotary evaporator (Eyela, OSB-2100) and lyophilyzer (Virtis, Freezemobile 25SL). Electronic weighing machine (Sartorius, BSA2245 -CW) was used for the weighing of different samples. Whereas lambda max was observed using UV Spectrophotometer (Perkin Elmer, Lambda 25).


v. Preparation and drying of Extract:

The extracts of carrot (Daucus carota) (A), cannabis (Cannabis sativa) (B) and wheat grass (Triticum aestivum) (C), were prepared in methanol. 20 g of the plant material was extracted in a soxhlet apparatus using 200 ml of methanol and combinations of carrot and cannabis (A+B), cannabis and wheat grass (B+C), carrot and wheat grass (A+C) were prepared in equal ratio for further studies.


Methanolic extracts of (A, B and C) were concentrated using rotary evaporator under vacuum. Concentrated extracts were subjected to freeze drying by placing them in freeze dryer at very low temperature for 4 days to gently freeze the product, followed by removal of water using a high pressure vacuum to obtain solid mass.


vi. Biological evaluation (Anti-oxidant study):

Hydrogen peroxide (H2O2) Scavenging Activity: The hydrogen peroxide (H2O2) scavenging activity of methanolic extracts (A, B and C) and their combinations (A+B), (B+C) and (A+C) were determined according to the method of Ruch et al. (1989) 28. Sample solutions (0.5 ml) of different concentrations were reacted with 4mM H2O2 solution which was prepared in phosphate buffer. Absorbance of H2O2 at 230nm was determined 10 minutes later against a phosphate buffer solution without H2O2 which acts as a blank. All the tests were performed in triplicate and the graph was plotted with the mean values. Percentage anti-oxidant activity, calculated by the following equation:


Scavenging (%) = [1-(A/B)] X 100


A = absorbance of extract

B = absorbance of control (ascorbic acid)



Collection of raw material and extraction:

Root of Carrot (Daucus carota L.), Leaves of hemp plant (Cannabis sativa L.) and shoot of wheat grass (Triticum aestivum Linn.) were collected from a positively authentic sources in the month of October, 2019 and extracted with methanol using soxhlet extraction method for further phytochemical and anti-oxidant activity. 20g of the plant material was extracted in soxhlet apparatus using 200 ml of methanol.


Phtochemical Evaluation:

The preliminary phytochemical analysis of all the plant extracts showed the presence of alkaloids, glycosides, tannins, carbohydrates, proteins (Table 1, 2 and 3).


Table 1: Phytochemical screening of carrot extract

S. No.


Carrot Extract



Highly Present


Reducing Sugars

Moderately Present



Highly Present



Highly Present



Highly Present


Table 2: Preliminary phytochemical screening of cannabis extract

S. No.


Cannabis extract



Highly Present






Moderately Present





Cardiac Glycosides

Highly Present








Terpenes & Steroids

Highly Present



Highly Present


Volatile Oils



Table 3: Preliminary phytochemical screening of wheatgrass extract

S. No.


Wheatgrass Juice juice








Amino acids









Phenolics & tannins



Fixed oils and fats












Biological Evaluation:

Anti-oxidant Study

Hydrogen peroxide (H2O2) Scavenging Activity:

H2O2, a strong oxidizing agent, is a stable free radical and causes deleterious effects on living tissues29. H2O2  can be formed in vivo by several oxidizing enzymes like superoxide dismutase imparting strong oxidizing properties30. It slowly oxidizes a number of molecules by producing hydroxyl radicals and causes toxicity31. H2O2 can also lead to hydroxyl radical dependent oxidative DNA damage32. As shown in table 4, all the extracts scavenged hydrogen peroxide which is comparable to that of ascorbic acid and the activity of the extracts was found to increase with an increase in the concentration of all the extracts. These extracts may thus be accelerating the conversion of hydrogen peroxide to (H2O2) to water (H2O). The maximum scavenging activity was found at concentration of 10µg/ml with all the extracts. Maximum anti- oxidant activity was observed in case of carrot (A) and combination of carrot + wheat grass (A+C) with 41.60 and 53.60% inhibition respectively at concentration 10µg/ml.


The activity of these plant extracts depends on the secondary metabolites which exist as complexes in the medicinal plant extracts. These metabolites offer a wide range of functional groups which bind to potential sites in the microbes33,34. Such interactions determine the antioxidant activity of secondary metabolites and it is prejudged that the efficacy is based on the collective action of the mixture of constituents35. Thus a potentiation in the activity of combinations of plant extracts has been observed.


Table 4: Percentage inhibition at various concentrations of carrot, cannabis, wheat grass and their possible combinations in H2O2 scavenging activity

Conc. (µg/ml)

Percent (%) growth inhibition (mean±SEM)



Wheat grass

Carrot + wheat grass

Cannabis +wheat grass

Cannabis + carrot

Ascorbic acid











































Figure 1: Percentage inhibition at various concentrations of carrot, cannabis, wheat grass and their possible combinations in H2O2 scavenging activity


Figure 2: Percentage inhibition at various concentrations of carrot, cannabis, wheat grass and their possible combinations in H2O2 scavenging activity



From the above study, it can be concluded that significant antioxidant activity was observed in the extracts of carrot (Daucus carota L.), wheat grass (Triticum aestivum Linn.) and their combination (A+C) in equal ratios. The findings of this report support the fact that the medicinal plants used in the present study are favourable sources for potential antioxidant agents and can form basis for the novel pharmaceutical products to be used for the treatment of various ailments.



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Received on 01.10.2020            Modified on 23.05.2021

Accepted on 06.08.2021           © RJPT All right reserved

Research J. Pharm.and Tech 2022; 15(3):1042-1046.

DOI: 10.52711/0974-360X.2022.00174