CHARACTERIZATION AND ANTIOXIDANT ACTIVITY OF SEED EXTRACT OF N. NUCIFERAHTML Full Text
CHARACTERIZATION AND ANTIOXIDANT ACTIVITY OF SEED EXTRACT OF N. NUCIFERA
Saya Tyagi, Anuradha Shukla, Pankaj Jain and Rashmi Tripathi *
Department of Bioscience and Biotechnology, Banasthali Vidyapith, Banasthali, Rajasthan, India.
ABSTRACT: N. nucifera, often recognized as lotus, is an aquatic plant. Extracts of various solvents have shown therapeutic potential against many diseases. This research focused on screening preliminary qualitative phytochemicals and investigating the seed's antioxidant activity. Primary and secondary metabolites were discovered in a hydroethanolic fraction of different extracts of lotus seeds. The extractive value, total ash (3.56±0.36) water-soluble (0.86±0.68), and acid insoluble ash value (2.09±0.88) were determined. The solvents benzene, chloroform, ethyl acetate, n-butanol, and aqueous were used as extracts. TPC and TFC content of ethyl acetate extract of seeds were found to be 366.6±10.77 (mg GAE/ g-1 dry weight) and 344.4±3.71 (mg RE/ g-1 dry weight), respectively. The ethyl acetate fraction of the seed extract showed the lowest value determining the highest free radical scavenging activity (105.30 ± 12.06µg/ml) in DPPH and FRAP (142.2± 0.0509 mgAA gm-1). The values were found to be close as compared to the standard (56.40±23.63µg/ml).The results support high antioxidant content in the seed extract of N. nucifera. FTIR and GCMS did the characterization of compounds. The results conclude that seeds of N. nucifera can be included in medicine as it shows pharmaceutical and therapeutic potential.
Keywords: Antioxidant activity, FTIR, GCMS, Hydroethanol, N. nucifera and phytochemical analysis
INTRODUCTION: Plants have been a part of our lives for ages and are an ancient form of medicine known to human. The various products which are obtained from the plant are not only good for health but also play an important role in our existence. Herbal medicine is a type of medicine that uses natural plant compounds to prevent and treat illnesses 1.
Alkaloids, glycosides, terpenes, steroids, flavonoids, tannins and other secondary metabolites are found in herbal plants. Some chemical substances, such as tannins and phenols or their oxygen-substituted derivatives, whereas others may include nitrogen or sulphur and are physiologically active and beneficial in the treatment and prevention of illness in humans and animals 2, 3.
Antioxidant chemicals are found in many medicinal plants and protect cells from the harmful effects of reactive oxygen species (ROS) 4, 5. When the balance between ROS production and antioxidant defence is disrupted, oxidative stress occurs, triggering a cascade of events that disrupt cellular function and result in diseases such as ageing, arthritis, asthma, liver disorders, carcinogenesis, diabetes, rheumatism and a variety of neurodegenerative diseases 6. The plant N. nucifera has been used for its medicinal properties for ages. It is cultivated in India and is a national flower. In India, N. nucifera is commonly known as lotus, Kamala; the different parts being used in herbal medicines for ages. The seeds of the lotus N. nucifera are edible, medicinally significant, and a key component of age-old conventional medical treatments such as indigenous medicine 7, 8. Seeds are sold as kamal gatta and consumed as a vegetable. The seeds contain hepatoprotective and free radical scavenging properties 9, 10. This research aimed to look into the pharmacognostical and antioxidant properties of N. nucifera seed extract. The presence of secondary metabolites in its extracts was discovered using phytochemical screening. The total flavonoid and phenolic content were calculated using the aluminum chloride colorimetric method and Folin–ciocalteu's, respectively, to determine the extract's quantity of flavonoids and phenols. Various functional groups and chemicals were determined using FTIR and GCMS analysis.
MATERIALS AND METHODS:
Accumulation of Plant Sample: The natural healthy plant seeds of Nelumbo nucifera were acquired from the Drona Sagar, Kashipur, Uttarakhand, India in November 2018. Taxonomists from the department recognized and authenticated the plant, which Dr. P. K. Mishra subsequently confirmed, HOD, Dept. of Botany, VBU, Hazaribagh, and a voucher specimen with the accession no. VBU/BOT/00368/2004 has been reserved in the Department of Botany, Vinoba Bhave University, Hazaribagh.
Plant Material: Fruit is a clump of indigestible nutlets. The ripe nutlets are ovoid, roundish, or oblong in shape, measuring up to 1.0 cm in length, and 1.5 cm in width, with a firm, smooth greyish black pericarp and one seed. The lotus seeds were crushed in a mortar pestle, and the outer seed coat was removed. The seeds collected were grounded in a mixer to get fine powder, then kept in an airtight jar for future testing and analysis.
Phytochemical Profiling of N. nucifera Seed Extracts Prepared in Different Solvents: The pharmacognostic characteristics were investigated 11, 3.
Assessment of Total Ash Content: The total ash value was determined in crucibles. The crucibles were washed, dried, and weighed. About 2gms of the plant material powder was placed in each crucible, weighed, and ashed in a muffle furnace at 450°C for six hours. The crucibles were cooled and weighed.
Total ash value (sample) = (Z-X) × 100 / Y
Estimation of Acid Insoluble Ash Content: The ash obtained after the incineration of the plant material was taken with 25ml of dilute hydrochloric acid in a beaker. The content was boiled for 5 min and then passed through an ‘ash less’ filter paper. The remains on the filter paper were taken in the crucible and kept in the muffle furnace at 450°C for 4-5 h. The acid-insoluble fraction of the ash obtained on the filter paper was then weighed accurately after cooling the crucibles.
Acid insoluble ash value (sample) = a × 100 / y
Determination of Water-Soluble Ash Content: This is determined using the same procedure as acid insoluble ash, with the exception that instead of dil HCl, 25ml of water is used.
Determination of Extractive Value: To determine the extractive value, 5gms of powder was taken and mixed in 100ml of solvents namely benzene, chloroform, n-butanol, ethyl acetate and aqueous. All the beakers were kept on a rocker shaker for 6h and left undisturbed for 18 h at room temperature. The weight of empty glass petriplates was taken and noted. The solutions passing through filter paper was poured into glass petri plates and kept in oven for drying. After drying weight of each petriplates were taken, and moisture was determined using the formula:
% yield = weight of residue × 100 / Sample weight
Fluorescence Characteristics Analysis: To estimate the fluorescence characteristic, the seed powder was taken, and foreign particles were removed with the help of a sieve. The powder was taken on the watch glass, and the solvents were added. Then watch glasses containing samples were observed with UV fluorescence analysis cabinet under different visible wavelengths UV short 254nm and UV long 366 nm12.
Plant Extraction: The fine powder of the seed, about 10 gms was extracted with 100ml of hydro-ethanol solvent, with the proportion of ethanol, and water, respectively, in 70:30 ratios. The extract was then kept on a rotor shaker for 24h at normal temperature and filtered using Whatman filter paper, and further extraction was carried. After completing, the final extract was evaporated and dried at room temperature.
Separation of Extracts in Different Solvents: The separation was performed using a separating funnel, 3 gms of the extract was dissolved properly in 60 ml distilled water. This was introduced into a separating funnel, and 60 ml benzene was added and mixed well. The separation resulted in the formation of two layers, then the upper layer was separated, and the same process was repeated thrice. The same technique was used for all solvents, including benzene, chloroform, ethyl acetate, n-butanol, and aqueous.
Phytochemical Screening: Standard techniques were used to test the phytochemical content of an N. nucifera seed extract. To detect significant natural chemical groups, phytochemical screening was carried out in the extracts using various solvents. Tests for tannins, saponins, quinones, flavonoids, terpenoids, phenols, steroids, coumarins, anthocyanins, betacyanin’s, carbohydrates, and protein were performed following standard protocols 13, 14, 15.
Estimation of Total Phenolic Content: The Folin–ciocalteu’s method was followed to estimate the total phenolic content 16. Added to the sample (1mg/ml) was 500 µl of the previously diluted Folin- Ciocalteu reagent (1:1). It was then incubated for 5min. 1ml of 20% sodium carbonate and 12.5 ml DW were put in and mixed into the mixture. It was left for 30min to 2h in the dark, and observation of the sample was taken at 720nm. TPC was estimated as mg gallic acid/100g as a standard compound. The gallic acid dissolved in methanolin the range 10-50 mg/ml to prepare the standard curve. To calculate the TPC Equation 1 was used.
T= CV / M
Where: T = Total phenolic concentration C = Concentration of gallic acid from calibration curve (mg mlG1) V = Volume of extract (ml) M = Weight of plant extract.
Total Flavonoid Content: The concentration of total flavonoids was measured using the aluminum chloride colorimetric technique (AlCl3) with minor modifications using rutin as a reference 17. Each extract 50 ml at a concentration of (1mg/ml) 1 ml sodium nitrite (5%) and 0.15 ml aluminum chloride (10%) were added. 1 ml 1N NaOH was added and vortexed vigorously. The OD was measured at 415 nm. The rutin standard solution in methanol was used to create the standard curve. The extracts' total flavonoid concentration was measured in milligrams of rutin equivalents per gram of dry weight. The total flavonoid content may be determined using the following formula.
T= CV / M
Where: T = Total phenolic concentration C = Concentration of quercetin from calibration curve (mg mlG1) V = Volume of extract (ml) M = Weight of plant extract
DPPH Assay in Antioxidant Activity Measurement: The radical scavenging action of the stable DPPH free radical was used to test the antioxidant activity of the plant extracts and standard 18. The different concentrations of the fractions of the hydroethanolic extract in different solvents (benzene, chloroform, n-butanol, ethyl acetate) were taken (10µl to 300µl).
The volume was made up to 1 ml with methanol. Then 1.5 ml solution of freshly produced DPPH in methanol was added. After 30 min of incubation at room temperature in the dark, the absorbance of each solution was measured with a spectrophotometer at 517nm. The equivalent blank reading and ascorbic acid were taken, which was used as a standard, in various concentrations ranging from 20µl to120µl. The antioxidant activity was calculated using Equation 3.
Inhibition of DPPH activity (%) = A-B ×100 / A
Optical density of the control (A) and Sample (B)
FRAP in Determination of Antioxidant Activity: The activity of antioxidants was measured spectrophotometrically using the FRAP assay 19. The FRAP reagent was made by combining 300mM acetate buffer, 10mM TPTZ in 40mM HCl and 20mM FeCl3. 6H2O in a 10:1:1 ratio at 37°C.The freshly made FRAP reagent was mixed well with plant extracts.
After 30 min of incubation at 37°C, a strong blue colour complex developed and absorbance was measured at 593nm against blank. All the tests were carried out in triplicates. The FRAP values were computed in mg of ascorbic acid equivalent/ gm of plant material.
Compound Characterization by FTIR: The ethyl acetate fraction (mg/ml) was subjected to IR spectroscopy which showed the presence of functional groups. The transverse frequencies of infrared waves in wave number cm were used to identify the functional categories. Each functional group's transverse frequencies differed from one another. A Perkin-Elmer FT-IR spectrophotometer was used to evaluate the samples' FT-IR spectra.
GCMS Analysis: Using a Perkin Elmer GC clarus 500 system with an AOC-20i autosampler and a gas chromatograph interfaced to a mass spectrometer (GC-MS) instrument, the following parameters were used to perform GC-MS analysis: Column TG 5MS, 5 percent phenyl methyl polysiloxane, operating in electron impact mode at 70 eV; carrier gas was helium (99.999 percent) at a constant flow of 1 ml/min, with an injection volume of 1µl (split ratio of 10:1); injector temperature 200°C; ion source temperature 200°C.
The oven temperature was programmed to climb gradually from 110°C to 200°C (isothermal for 2 min), then 5°C/min to 280°C, with a 9-min isothermal at 280°C.
With pieces spanning from 40 to 70 eV, the mass spectrum was obtained at 70 eV with a 0.5 s scan interval. The temperature of the MS transfer line is 280°C.
Ash Content Determination: The total ash content of N. nucifera seeds was found to be higher than the acid-insoluble and water-soluble ash values in this investigation Table 1. The Total ash (3.56±0.36), acid insoluble ash (0.86±0.68) and water-soluble ash (2.09±0.88) values were estimated to be below the standard values.
TABLE 1: PHYSICO-CHEMICAL EVALUATION OF THE SEEDS OF N. NUCIFERA
|Parameters (% w/w)||N. nucifera (value in %)|
|Acid insoluble ash||0.86±0.68|
|Water soluble ash||2.09±0.88|
(Values expressed as Mean ±SD where n=3).
GRAPH 1: VALUE OF ASH CONTENT IN % (W/W) OF N. NUCIFERA SEEDS
Assessment of the Extractive Value: The active components in plant material, when extracted with different solvents, is determined by extractive value. When compared to other extracts, non-polar extracts had a higher extraction yield.
TABLE 2: EXTRACTIVE VALUES OF SEEDS OF N. NUCIFERA IN DIFFERENT SOLVENTS
|Solvent||Extractive value (%)|
(Values expressed as Mean ±SD where n=3)
Fluorescence Characteristics Analysis: Fluorescence is mostly an inherent and natural property of some natural compounds when exposed to different light conditions, generally the visible daylight. Herbal plants often exhibit fluorescence when their cut surface or powdered form is exposed to UV light.
However, certain compounds which do not fluoresce under the normally visible daylight can produce fluorescence under UV light. Moreover, non-fluorescent substances also get converted into their fluorescent derivatives on treatment with different reagents 20.
TABLE 3: DIFFERENT FLUORESCENCE CHARACTERISTICS OF THE SEED POWDER OF N. NUCIFERA
|S. no.||Treatments||UV short (254nm)||UV long (366nm)||Visible|
|1||Powder||Creamish white||Fluorescent||Whitish cream|
|2||Powder + distilled water||Light green||Fluorescent black||Whitish cream|
|3||Powder + glacial acetic acid (GAA)||Light green||Fluorescent cream||Whitish cream|
|4||Powder + 1N HCl||Light green||Black||Creamish yellow|
|5||Powder +1N H2SO4||Dark green||Fluorescent black||Brownish yellow|
|6||Powder + conc. HNO3||Dark green||Black||Dark yellow|
|7||Powder + FeCl3 (5%)||Green||Black||Yellowish orange|
|8||Powder + iodine solution 5%||Brownish green||Fluorescent black||Dark brown|
|9||Powder + ammonia solution||Dark green||Fluorescent black||Dark Creamish,|
|10||Powder + 1N NaOH||Dark green||Black||Creamish peach|
|11||Powder + potassium dichromate||Green||Black||Yellowish orange|
|12||Powder + HNO3 + NH3 solution||Dark green||Black||Brownish yellow|
|13||Powder + ethanol||Fluorescent light green||Fluorescent black||Creamish brown|
|14||Powder + methanol||Fluorescent light green||Fluorescent black||Creamish brown|
|15||Powder + Toluene||Dark green||Black||Creamish white|
Phytochemical Screening: Phytochemical screening is a useful method in the study of bioactive compounds.
It is a rapid, low-cost, and straightforward process that demonstrates the numerous types of phytochemicals found in plants. Phytochemicals indicate that the plant could be a potential source of precursors for synthetic medicine production. Phytochemicals are physiologically active molecules found in trace levels in plants that are not recognized as nutrients but contribute greatly to preventing degenerative diseases.
Phytochemical testing was completed to screen the existence of phytochemicals in different fractions of the current study's hydroethanolic extracts of N. nucifera seeds.
TABLE 4: PHYTOCHEMICAL SCREENING OF DIFFERENT FRACTIONS OF HYDROETHANOLIC EXTRACTS OF THE SEEDS OF N. NUCIFERA
|S. no.||Secondary metabolites||Phytochemical tests||Benzene||Chloroform||Ethyl acetate||N-butanol||Aqueous|
|1.||Tannins||Ferric chloride test||-||-||-||-||-|
|3.||Quinones||Sulphuric acid test||+++||++||++++||+++||+|
|4.||Terpenoids||Sulphuric acid test||-||-||-||-||-|
|5.||Flavonoids||Sodium hydroxide test||+||+||+++||++||-|
|6.||Phenols||Potassium per magnet test||+||+||+||+||+|
|7.||Steroids||Liebermann- burchard test||-||-||-||-||-|
|9.||Anthocyanin||Sodium hydroxide test||-||-||-||-||-|
|10.||Carbohydrates||Fehling’s test Benedict’s test||-
|11.||Proteins||Millon’s test Biuret test||+++
(Where, + = slightly, ++ = moderate, +++ = significant and – = negative).
Total Phenolic Content: The phenolic compounds have oxidation-reduction potential, allowing them to function as antioxidants.
The free radical scavenging activity is due to the hydroxyl group. Whereas the flavonoids are the secondary metabolites present in the plants, the antioxidant activity depends on the presence of free OH groups. The total phenolic content of the different extracts of seeds of N. nucifera ranged from 56.66±2.25mg GAE/ g-1 dry weight of dried sample for the benzene extract to 366.6±10.77mg GAE/ g-1 dry weight of dried sample for the ethyl acetate extract.
total flavonoids content of the different solvent extracts of seeds of N. nucifera ranged from 124.4±3.02mg RE/ g-1 dry weight in the aqueous extract to 344.4±3.71mg RE/ g-1 dry weight in the ethyl acetate extract.
GRAPH 2: STANDARD CURVE FOR THE ASSESSMENT OF TOTAL PHENOLIC CONTENT
TABLE 5: TOTAL PHENOLIC AND FLAVONOID CONTENT IN THE DIFFERENT FRACTIONS OF THE HYDROETHANOLIC EXTRACT OF SEEDS OF N. NUCIFERA
|Extracts of N. nucifera seeds in different solvents||Total phenolic content
(mg GAE/ g-1 dry weight)
|Total flavonoid content
(mg RE/ g-1 dry weight)
(Values expressed as Mean ±SD where n=3)
GRAPH 3: TOTAL PHENOLIC CONTENT OF DIFFERENT FRACTIONS OF THE HYDROETHANOLIC SEED EXTRACTS OF N. NUCIFERA
GRAPH 4: STANDARD CURVE FOR THE ASSESSMENT OF TOTAL FLAVONOID CONTENT
GRAPH 5: TOTAL FLAVONOID CONTENT OF DIFFERENT FRACTIONS OF THE HYDROETHANOLIC EXTRACT OF THE SEEDS OF N. NUCIFERA
Determination of Antioxidant Activity by DPPH Assay: The principle of the DPPH assay depends on the hydrogen-donating ability of the natural antioxidant compounds present in each plant.
The lowest the IC50 value, the higher the inhibition potential of the extract. The lowest IC50value was determined in Ethyl acetate (105.30 ± 12.06µg/ml), and the highest was determined in N-butanol (555.63 ± 7.56µg/ml) in comparison to the standard ascorbic acid 56.40±23.63µg/ml
TABLE 6: % INHIBITION OF DIFFERENT FRACTIONS OF THE HYDROETHANOLIC EXTRACT OF THE SEEDS OF N. NUCIFERA
|Benzene||537.17 ± 9.27|
|Chloroform||245.5 ± 17.51|
|Ethyl acetate||105.30 ± 12.06|
|N-butanol||555.63 ± 7.56|
|Aqueous||237.29 ± 14.54|
(Values expressed as Mean ±SD where n=3).
GRAPH 6: STANDARD CURVE FOR DPPH ASSAY
Determination of Antioxidant Activity by FRAP: The reducing power assay of the isolated fractions of the seed extract determined by the ferricyanide method also confirmed significant antioxidant activity by the extracts.
The highest value was determined in the Ethyl acetate fraction (142.2±0.51 mg AAgm-1) and the lowest value was reported in the chloroform (34.099±0.39mg AAgm-1).
The non-polar fractions showed the lowest value as compared to the polar fractions.
GRAPH 7: STANDARD CURVE OF THE FRAP ASSAY
TABLE 7: FRAP VALUES OF DIFFERENT FRACTIONS OF HYDROETHANOLIC EXTRACT OF THE SEEDS OF N. NUCIFERA
|Different fractions of the hydroethanolic extract of the seeds of N. nucifera||FRAP values (mg AA/ gm)|
(Values expressed as Mean ±SD where n=3).
GRAPH 8: VALUES OF FRAP IN MG AA/G OF SAMPLE IN DIFFERENT FRACTIONS OF THE HYDROETHANOLIC EXTRACT OF THE SEEDS OF N. NUCIFERA
Compound Characterization by FTIR: FTIR (Fourier Transform Infrared Spectroscopy) is a high-resolution analytical method that may be used to identify chemical components and interpret structural compounds. To fingerprint plant extracts or powders, FTIR provides a quick and non-destructive method 21. The frequency of functional groups found in the Ethyl acetate fraction is shown in Table 8.
TABLE 8: FTIR COMPOUND PEAKS OF ETHYL ACETATE FRACTION OF THE SEEDS OF N. NUCIFERA
|S. no.||Wave number cm-1
|Wave number cm-1
|Functional groups||Phytocompound identified|
980 - 960
|Aliphatic primary amine
GRAPH 9: FTIR SPECTRA OF THE ETHYL ACETATE FRACTION OF THE SEEDS OF N. NUCIFERA
GCMS Analysis: The presence of numerous peaks in the GCMS chromatogram analysis Fig. 9 of the ethyl acetate extract of N. nucifera seeds suggests the presence of many phytochemical constituents. The thirty-eight phytocompounds were described and identified by comparing their mass spectra with Wiley 9 and the NIST library, and their retention time (RT) and molecular weight were calculated.
GRAPH 10: GCMS CHROMATOGRAM OF ETHYL ACETATE FRACTION OF N. NUCIFERA SEEDS
TABLE 9: GCMS ANALYSIS OF ETHYL ACETATE FRACTION OF N. NUCIFERA SEEDS
|S. no.||Compounds||RT||Formula||Probability||MW||Biological activity|
|1||Tungsten, di carbonylbis (η-4-2-methylene cycloheptanone)η-4-2-methylenecycloheptanone)||11.05||C18H24O4W||16.12||488||Antibacterial|
|6||Carda-16,20(η-4-2-methylenecycloheptanone)22)-dienolide, 3-[(η-4-2-methylenecycloheptanone)6-deoxy-3,4-O-methylenehexopyranos-2-ulos-1-yl)oxy]-5,11,14-trihydroxy-12oxo-, (η-4-2-methylenecycloheptanone)3β,5α,11α||24.08||C30H38O11||11.49||547||Hepatoprotectant, Antioxidant Cholesterol antagonist|
|Hepatoprotective Antimicrobial Anti-thrombogenic|
|9||2-Ethyl-acridone||25.07||C15H13NO||11.17||223||Anti-tumor Antiviral Antimicrobial|
DISCUSSION: The results clearly show that the seed is the most effective medication activity. The water-soluble ash value is an important factor when evaluating a drug as it indicates the inorganic compounds present. The ash value accepted should be below the adequate limit 14% recommended by the European Pharmacopoeia 22. The less ash value indicates purity and less contamination in the drug as the higher value indicates the presence of organic matter and impurities 23. The phytochemical analysis shows that the extracts include a high concentration of non-polar molecules such as sugars, proteins, glycosides, organic acids, tannins, salts, and mucus can all be extracted from plant material using non-polar solvents. The varying polarity of different chemicals found in the seeds may account for significant variances in extraction yield. The polarity of the solvents utilized also influenced it. The higher the water-soluble extractive value, the more will be the moisture content leading to the deterioration of drug 23.
The phenomenon of fluorescent analysis can be efficiently used to assess the pharmacognostical parameters of any crude drug qualitatively and could also help in its identification. As the compounds present in the drugs and fluorescent behavior are correlated. These distinct and reproducible colour changes can be well attributed to the solvent properties of the specific phytochemicals 14.
These extracts were made from the plant's various solvent fractionates, including benzene, chloroform, ethyl acetate, n-butanol, and the aqueous. Quinones, flavonoids, phenols, and glucose were found in high concentrations in the several extracts of N. nucifera seed power, while tannins, terpenoids, and saponins were absent as shown in Table 4. These findings backed up claims that its seeds contain active ingredients like carbohydrates, flavonoids, and phenolic compounds that have antioxidant, anti-diabetic, anti-obesity, and anti-fungal properties 24, 25, 26, 27, 28, 29. In epidemiological investigations, the phytochemical components have strong antioxidant activities and produce numerous biological effects. They effectively reduce the number of diseases such as cardio-vascular diseases, and hypercholesterolemia, and possess antibiotic properties. The polarity of the solvents employed for extraction affects the concentration of phenols and flavonoids. Plants' antioxidant capabilities are closely connected to their phenolic and flavonoid content. Phenolic compounds can act as reducing agents, hydrogen donors, and scavengers of free radicals. The presence of phenolics in substantial amounts contributes greatly to the antioxidant capabilities. Phenolic substances have a wide range of antioxidant responses depending on their chemical composition 17. N. nucifera has extensively been demonstrated to have a good antioxidant potential largely due to the synergistic effects of various bioactive compounds found in it. The results exhibited by the ethyl acetate extract show the highest activity among all the solvent fractionates used. Its lower IC50 value of 105.30± 12.06µg/ml clearly resembled a good antioxidant activity attributed to its high DPPH radical scavenging compared to values obtained for the ascorbic acid used as standard (56.40±23.63µg/ml).
The DPPH test, among many others, is one of the most practical techniques for evaluating plant antioxidant potential. The methanolic DPPH solution is decreased owing to the production of non-radicals when antioxidant compounds containing hydrogen-donating groups, such as flavonoids and phenols, are present 30.
The antioxidants act as reductants and contribute to the reducing properties of the plant by acting as hydrogen donors, thereby breaking the free radical chain. These also prevent peroxidation by reacting with the precursors of peroxide. In the FRAP assay, the potent antioxidant activity was indicated by the reducing capabilities of the antioxidants (reductants), based on the ability to reduce and transform Fe3+/ferric cyanide complex to ferrous (Fe2+) form. The colour change measures this transformation at 680nm, directly related to the associated reducing power. The results exhibited by the ethyl acetate extract showed the highest activity among all the solvent fractionates used (142.2±0.509 mg /AA gm). The antioxidants are essential molecules that can provide protection from harm caused by oxidative stress produced by free radicals. The polyphenols function as antioxidants and reducing agents in the body. Their hydroxyl groups have a hydrogen-donating property. The FTIR studies were conducted to confirm the presence of phytocompounds in seeds extract. The spectra showed the presence of the functional groups (C=O, NO, CH, C=N, RX, and OH group), which significantly contribute to the pharmaceutical activity of the plant. The C=O stretching is predominantly found in the seeds of the plant. Carboxylic acid, found in medicinal plants, is the primary pharmaceutical product used to treat ulcers, jaundice, and liver discomfort31. The absence of absorbance in the range 2220-2260 cm-1 shows the absence of cyanide group and, therefore, toxic substance. As a result, the functional group is in favor of hepatoprotective action.
Only a few of the compounds have been reported to have biological activity due to the different functional groups. Table 9 lists the numerous phytochemicals that contribute to the therapeutic properties of N. nucifera seed. Among all the compounds Pyrazine, 2, 5-bis(η-4-2-methylenecycloheptanone) p-fluorophenyl)-3,6-diphenyl (Probability 42.69) and Carda-16,20(η-4-2-methylenecycloheptanone)22)-dienolide, 3-[(η-4-2-methylenecycloheptanone) 6-deoxy-3,4-O-methylenehexopyranos – 2 – ulos – 1 - yl) oxy]-5, 11, 14 – trihydroxy – 12 oxo -, (η-4-2-methyl-enecycloheptanone)3β,5α,11α (Probability 11.49) shown in the Table 9 are interesting findings as because of their hepatoprotective role. These phytochemical constituents can be isolated and can be potential novel drugs against various hepatoprotective disorders.
CONCLUSION: The findings of this study backed up the traditional use of N. nucifera seeds, which contain bioactive chemicals that could be employed as active agents in novel medicines to treat a variety of metabolic issues. The nutritional composition data analysis revealed that seed could be used as a significant source of protein and carbohydrates for humans and livestock and contain significant amounts of phytochemicals, suggesting that lotus seeds may have scientific support for their use in traditional medicine for the treatment of metabolic disorders. Because of the low ash content and the presence of medicinal compounds, the seeds have been discovered to be useful against various ailments. Antioxidant properties are enhanced by the presence of phenolic chemicals in significant levels.
When compared to the standard, the antioxidant activity was determined to be high, indicating that it provides protection against free radicals. The presence of functional groups (C=O, NO, CH, C=N, RX, and OH) contribute considerably to the plant's medicinal activity. The presence of Pyrazine, 2,5-bis (-4-2-methylene cycloheptanone) p-fluorophenyl)-3,6-diphenyl (Probability 42.69) in the crude extract was found by GC-MS. These are noteworthy discoveries because of their hepatoprotective effect. These phytochemical ingredients can be extracted and used to develop new medications to treat various hepatoprotective conditions. Finally, the findings show that ethyl acetate in N. nucifera seeds is the most efficient against hepatic ailments and a variety of other illnesses.
ACKNOWLEDGEMENT: Author’s take this opportunity to express their gratitude and thanks to the Former Hon’ble VC Prof. Aditya Shastri, Banasthali Vidyapith, Banasthali, Rajasthan, for providing us the research facilities, encouragement, and support. Author’s also thank Hon’ble Vice-Chancellor, Prof. Ina Shastri, Banasthali Vidyapith, Banasthali, Rajasthan, for being a consistent support source providing the necessary research facilities. The authors also thank Prof. Dipjyoti Chakraborty, Head of the Department of Bioscience and
Biotechnology, Banasthali Vidyapith, Banasthali, for supporting and encouraging the research work. The authors would like to acknowledge DST for providing networking support through the FIST program at the Department of Bioscience and Biotechnology, Banasthali Vidyapith, Banasthali.
CONFLICTS OF INTEREST: The authors have no conflicts of interest regarding this investigation.
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How to cite this article:
Tyagi S, Shukla A, Jain P and Tripathi R: Characterization and antioxidant activity of seed extract of N. nucifera. Int J Pharm Sci & Res 2022; 13(11): 4608-19. doi: 10.13040/IJPSR.0975-8232.13(11).4608-19.
All © 2022 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
Saya Tyagi, Anuradha Shukla, Pankaj Jain and Rashmi Tripathi *
Department of Bioscience and Biotechnology, Banasthali Vidyapith, Banasthali, Rajasthan, India.
27 March 2022
15 May 2022
06 June 2022
01 November 2022