INTRODUCTION:

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- oxidization^3,4. 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 important^7-9.

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 flavonoids^10-14. These secondary metabolities are found in all the parts of plants like roots, stem, bark, fruits and flowers^15,16. The required constituent is extracted using a suitable solvent and evaluated for its activity^17-20. 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.

MATERIALS AND METHODS:

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 H₂O₂solution which was prepared in phosphate buffer. Absorbance of H₂O₂at 230nm was determined 10 minutes later against a phosphate buffer solution without H₂O₂which 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

where

A = absorbance of extract

B = absorbance of control (ascorbic acid)

RESULTS AND DISCUSSION:

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.	Phytoconstituents	Carrot Extract
1.	Phylobatannins	Highly Present
2.	Reducing Sugars	Moderately Present
3.	Flavonoids	Highly Present
4.	Alkaloids	Highly Present
5.	Terpenoids	Highly Present

Table 2: Preliminary phytochemical screening of cannabis extract

S. No.	Phytoconstituents	Cannabis extract
1.	Alkaloid	Highly Present
2.	Saponin	Absent
3.	Flavonoids	Moderately Present
4.	Tannins	Absent
5.	Cardiac Glycosides	Highly Present
6.	Balsam	Absent
7.	Phenols	Absent
8.	Terpenes & Steroids	Highly Present
9.	Resins	Highly Present
10.	Volatile Oils	Absent

Table 3: Preliminary phytochemical screening of wheatgrass extract

S. No.	Phytoconstituents	Wheatgrass Juice juice
1.	Carbohydrates	Present
2.	Proteins	Present
3.	Amino acids	Present
4.	Alkaloids	Present
5.	Steroids	Absent
6.	Phenolics & tannins	Absent
7.	Fixed oils and fats	Absent
8.	Triterpenoids	Absent
9.	Glycosides	Absent
10.	Saponins	Present

Biological Evaluation:

Anti-oxidant Study

Hydrogen peroxide (H₂O₂) Scavenging Activity:

H₂O_2,a strong oxidizing agent, is a stable free radical and causes deleterious effects on living tissues²⁹. H₂O₂can 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. H₂O₂can 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 microbes^33,34. 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)
Conc. (µg/ml)	Carrot	Cannabis	Wheat grass	Carrot + wheat grass	Cannabis +wheat grass	Cannabis + carrot	Ascorbic acid
2	11.30±1.75	08.60±1.93	08.30±1.69	18.30±1.66	10.30±1.72	10.60±1.55	24.79±1.34
4	17.60±1.41	14.00±1.28	13.30±1.48	26.60±1.41	13.30±1.96	12.30±1.64	27.81±1.84
6	24.00±1.86	21.60±1.93	19.60±1.34	43.00±1.51	22.00±1.23	20.30±2.59	33.40±2.72
8	35.00±1.68	29.00±1.69	27.00±1.72	40.00±1.68	29.30±1.28	26.00±1.82	36.30±1.26
10	41.00±1.74	36.00±1.23	32.00±1.83	53.60±1.09	38.00±1.87	35.30±2.28	40.10±1.91

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

CONCLUSION:

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

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

DOI: 10.52711/0974-360X.2022.00174