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In vitro Antioxidant and Phytochemical Analysis of Aqueous and Kamadhenu Ark Extracts of Nutricereals Millets

J. Venkata Lakshmi1, C. Appa Rao2, Ch.M. Kumari Chitturi3,*
1Department of Chemistry, Government College for Men (Autonomous), Kadapa-516 004, Andhra Pradesh, India.
2Department of Bio Chemistry, Sri Venkateswara University, Tirupati-517 502, Andhra Pradesh, India.
3Department of Applied Microbiology and Bio Chemistry, Sri Padmavati Mahila Visvavidyalayam, Tirupati-517 502, Andhra Pradesh, India.

ABSTRACT

Background: Globally the usage of Ethnomedicinal herbs to treat various diseases and metabolic disorders has increased over recent years. Intensive efforts are carried out by researchers in the last five decades to identify the phytochemicals responsible for Antibacterial, Antiviral, Antifungal, Antimicrobial, Antibiotic, Anticancer, Antidiabetic, Antihypertensive and Antioxidant Properties. Millets are a group of small-seeded grasses. Although millets are frequently referred to as coarse cereals, they are now known as nutricereals because of their greater levels of nutrients. The present study aims at the phytochemical analysis and evaluation of antioxidant potential of aqueous and kamadhenu ark extracts of few minor millets namely proso, little and kodo millets.

Methods: Proso, little and kodo millets are washed, shade dried and homogenized. The Aqueous and kamadhenu ark extracts were prepared by maceration method. Antioxidant activity was determined using DPPH Assay. Phytochemical Analysis were investigated as per standard procedures.

Result: Kamadhenu ark extract of proso millet has the highest antioxidant activity with IC50 value of 0.0212±0.04 mg/ml and the least activity of 3.1952±0.19 mg/ml is shown by sprouted kodo millet in combination with kamadhenu ark. The Phytochemical analysis manifested the inherence of carbohydrates, diterpenes, glycosides, phenols, phytosterols, saponins, steroids, tannins and triterpenoids.

INTRODUCTION

Medicinal plants are nature’s blessings to Mankind to heal numerous diseases. Globally the usage of Ethnomedicinal herbs to treat various diseases and metabolic disorders has increased over recent years (Archana et al., 2012). A wide number of Indian medicinal plants are used Consistently as antibiotics by clinicians of the Ayurveda and Unani systems of medicine and intensive efforts are carried out by researchers in the last five decades to identify the phytochemicals (Mayba et al., 2019) responsible for antibacterial, antiviral, antifungal, anticancer, antidiabetic, antihypertensive and antioxidant properties Mohan et al., (2021).
 
The chemical process which involves transfer of electrons is oxidation. Free radicals are produced during oxidation reactions (Marak et al., 2019). Free radicals trigger cascade of reactions which are harmful. Antioxidants eliminates the intermediary radicals produced during oxidation processes and stops the cascade of reactions. Antioxidants, which are generally reducing substances like thiols, ascorbic acid, or polyphenols, do this by getting oxidised themselves. All the living organisms possess sophisticated process containing various antioxidants, like vitamin C and E, glutathione, enzymes and various peroxidases. Inadequate antioxidants or suppression of antioxidant enzymes may attribute to oxidative stress which may lead to cell death. The pathophysiology of a various human ailments, including Parkinson’s, Alzheimer’s disease, neurodegenerative illnesses, diabetes, cancer and rheumatoid arthritis are interwined with oxidative stress (Pawle and Singh, 2014). It also plays a role in a variety of renal disorders, including acute renal failure, obstructive nephropathy, hyperlipidaemia and glomerular injury. In acute renal failure patients receiving haemodialysis and urolithiasis, studies demonstrate that with higher lipid peroxidation and decline in antioxidative protection oxidative stress is elevated (Perumal Swamy et al., 2008).  
 
Plant bioactive elements called phytochemicals enhance health and are primarily generated by plants to preserve them. To genetically create designer foodstuffs, herbal preparations and beverages, these phytochemicals are isolated as nutrients, dietary supplements and specialized diets. Some of the phytochemicals usually we analyse are Flavonoids, phytoestrogens, polyphenols, anthocyanidins, fibre, terpenoids, carotenoid, phytosterols and glycosides (Murugan et al., 2022; Moatasem et al., 2023).
 
Millets are a group of small-seeded grasses, commonly farmed as cereals for fodder and human nourishment all over the world (Handique et al., 2023). Millets are major crops in Asia and Africa’s semiarid tropics particularly in India and Nigeria (Chauhan et al., 2018). Millets may have been utilized by humans since seven thousand years. Millets played a “critical role in the establishment of multi-crop agriculture and permanent farming civilizations,” according to the researchers (Vanathi et al., 2023). Sorghum (Great millet), Bajra (Pearl millet), Ragi (Finger millet) and small millets such as Korra (Foxtail millet), Little millet, Kodo millet, Proso millet and Barnyard millet are all important millet crops cultivated in India (Malathi et al., 2016). Although millets are frequently referred to as coarse cereals, they are now known as nutricereals because of their greater levels of nutrients. Himanshu et al. (2018).
 
Although Millets are referred as Nutricereals,(Desai and Dutta, 2023) the phytochemicals and Antioxidant Properties are unexplored. The Present Study aims at the phytochemical analysis and evaluation of Antioxidant activity of Aqueous and Kamadhenu Ark extracts (Ipsita Mohanty et al., 2014) in combination with few millets namely proso, little and kodo millet.

MATERIALS AND METHODS

Preparation of extracts
 
Proso Millet (Panicum miliaceum), Kodo Millet (Paspalum scrobiculatum) and Little Millet (Panicum sumatrense) were washed, shade dried for one day and homogenized for the preparation of raw millet extracts. The Sprouted Millets extracts were prepared by soaking the sample in distilled water for 12 hours, later it was tied in muslin cloth and allowed it to germinate for 12 hours, then the sample was dried under room temperature for one day and homogenized to obtain Aqueous Proso Millet (APM), Aqueous Sprouted Proso Millet (ASPM), Aqueous Kodo Millet (AKM), Aqueous Sprouted Kodo Millet (ASKM), Aqueous Little Millet (ALM) and Aqueous Sprouted Little Millet (ASLM).
 
The early morning void urine of Kapila Cow (certified as healthy by veterinarian) was collected from the goshala for preparation of Kamadhenu Ark Extracts namely Kamadhenu Ark Proso Millet (KPM), Kamadhenu Ark Sprouted Proso Millet (KSPM), Kamadhenu Ark Kodo Millet (KKM), Kamadhenu Ark Sprouted Kodo Millet (KSKM), Kamadhenu Ark Little Millet (KLM) and Kamadhenu Ark Sprouted Little Millet (KSLM), The Aqueous and Kamadhenu Ark Extracts were prepared by Maceration method (Karagoz et al., 2015).
 
Preparation of aqueous and kamadhenu ark extracts are carried out in DST-FIST, Department of Applied Microbiology and Biochemistry, Sri Padmavati Mahila Visvavidyalayam, Tirupati andhra Pradesh, from March 2019 to February 2020.
 
Phytochemical analysis
 
Aqueous extracts and kamadhenu ark extract of raw and sprouted proso, kodo and little millets were subjected to phytochemical analysis. Phytochemical screening for carbohydrates is done by using molisch test, for glycosides using legal test, for amino acids using ninhydrin test, for proteins using biuret test, for fixed oils and fats using spot test, for flavonoid’s using test with sodium hydroxide, for phenols and tannins using neutral ferric chloride test, for alkaloids wagner’s test, for saponins foam test, for steroids, phytosterols and triterpenoids using salkowski test, for coumarins test with alcoholic sodium hydroxide and for quinones test with aqueous sodium hydroxide (Murugan et al., 2022).
 
Determination of total phenolic content
 
Using a 24-well microplate method, total phenolic content (TPC) was determined as per Ainsworth and Gillespie’s protocols with a few small tweaks. Precisely, 100 micro litre sample extracts, standard (gallic acid) and blank (95% (v/v) methanol) dissolved in 400 µl double distilled water, mixed thoroughly with a quick spin.150 µl of Folin-Ciocalteu reagent was added and vortexed. After five minutes, 500 µl of 20% Na2CO3 solution was added, vortexed and incubated for sixty minutes in dark. The absorbance was measured at 650 nm with imark Microplate Reader. The gallic acid standard curve was adopted to calculate the total phenolic content, which was then represented as mg of gallic acid equivalents (GAE) per g of sample (Ofoso et al., 2020).
 
Evaluation of total terpenoids
 
Evaluation of total terpenoids was done using the protocol stated by (Indumathi et al., 2014). 100 mg (W1) of sample is soaked in 9 ml of ethanol for 24 hours. The contents were filtered and extracted and the weight W2 noted. Total terpenoids contents percentage of yield was measured by the formula:
 
                                                                   
                                                                  (Malik et al., 2017).
Evaluation of total saponins
 
Total Saponins was measured adopting modified Vanillin-sulphuric acid method by (Anh et al., 2018). A standard curve was constructed using aescin, a natural triterpenoid saponin. Aescin, 150 mg was vortexed in 10 ml methanol to obtain an aescin stock solution. Methanol was utilised as the solvent blank and for preparation of the serial dilution of the standards in triplicate. 2 ml sample extracts, standard or methanol blank were taken and are placed in a water bath at 65°C for approximately 5 minutes to evaporate the methanol. 0.5 ml of vanillin, 2.5 ml of 72% v/v H2SO4 is added, mixed thoroughly and kept in a water bath at 60°C for 15 minutes for incubation. The absorbance was measured at 560 nm after cooling to room temperature using imark Microplate Reader. The total saponins content was determined utilising Aescin standard curve and represented as mg of Aescin equivalents (AE) per gram.
 
Determination of tannins content
 
The Tannin content was measured utilising the protocol described by Makkar and Goodchild (1995). 200 mg of test sample is soaked in a conical flask containing 10 ml of aqueous acetone solution for fifteen hours. The tannin was collected as a supernatant in a flask using a Whatman filter paper No. 1. Fifty microlitre of supernatant (sample), standard was taken and made up to one ml using milliQ water and added 0.5ml of Folin Ciocalteu reagent, 2.5 ml of twenty percent Na2CO3, spin and incubated for 40 minutes at room temperature. Absorbance was measured at 725 nm using imark Microplate Reader. The Tannin content was determined utilising the Tannic acid standard curve and represented as milligrams of tannic acid equivalents per gram of sample (mg TAE/g) (Nassarawa et al., 2019).
 
Evaluation of antioxidant activity
 
“1, 1-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging Assay” was performed using Dr Prieto’s DPPH Microplate Protocol with minor modifications. 7.88 mg of DPPH powder is dissolved in 100 ml of methanol to obtain 0.2 mM DPPH Solution. Standard, test and control solutions are prepared. Ascorbic acid dissolved in methanol to give concentrations ranging from 0.2 mg/ml to 1.6 mg/ml are standard solutions (Xiang et al., 2019). The extracts were dissolved in double distilled water to give concentrations of 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4 and 1.6 mg/ml. Methanol will act as control. 100 μl of standard/test/ control solutions are taken in a 96 well microplate and added 100 μl of 0.2 mM DPPH. The plate is capped to reduce the evaporation and then covered in foil, kept at room temperature to shield the DPPH radical from deterioration by light, for thirty minutes. The absorbance (515 nm) is then read in imark Microplate reader (Rebai et al., 2023). For each Extract Assay was run in Triplicate (Nguyen et al., 2020). The Antioxidant Activity is determined utilizing the equation
 

Analytical statistics
 
Each experiment was performed in triplets. The data were analysed statistically using the statistical software (SPSS 12.0) and were presented as a mean ±standard deviation (SD). For comparison of concentration dependency, regression analysis was employed and one-way analysis of variance (ANOVA) was utilised for comparison of more than two means (Bhattacherjee et al., 2023). When p value is less or equal to 0.05, a difference was judged statistically significant (Abdulhafiz et al., 2022).
 
Phytochemical screening and analysis, determination of antioxidant activity were performed at DST-CURIE, Sri Padmavati Mahila Visvavidyalayam, Tirupati andhra Pradesh from January 2021 to March 2022.

RESULTS AND DISCUSSION

Phytochemical screening
 
The results of phytochemical screening reveal the inherence of saponins, carbohydrates, phenols, glycosides, tannins, steroids, phytosterols, triterpenoids and diterpenes in all the aqueous and kamadhenu ark extracts of raw and sprouted little, kodo and proso millets. (Table 1 and 2). Alkaloids are present in KPM, KSKM, KLM, ALM and ASLM. Oils and fats are present in KPM, KSPM, KKM, KSKM, KLM and ASPM. The presence of phenols in aqueous and kamadhenu ark extracts of millets is in accordance to Liang et al., (2019).

Table 1: Phytochemical screening of kamadhenu ark extracts of millets.



Table 2: Phytochemical screening of aqueous extracts of millets.


 
Determination of total phenolic content
 
Phenols are potent antioxidants and also possess glucose lowering effect (antidiabetic). Total phenolic content TPC is shown in Table 3. KPM and KSPM has highest TPC of 48.34±1.5 and 32.68±2.2 mg GAE/g respectively. ALM and ASLM has the lowest TPC of 4.62±1.4 and 4.39±2.1 mg GAE/g respectively. Aqueous and kamadhenu ark extracts of proso and kodo millets possess high TPC in comparison to finger millet (2.61±0.02), pearl millet (4.79±0.01) and fonio millet (1.96±0.01) (Nassarawa et al., 2019). Proso millets have more TPC than barnyard millet (16.07) (Ofosu et al., 2020 and Kom et al., 2020). Phenols are not present in foxtail millet (Sangma et al., 2019). TPC of proso millet is in accordance to (Kom et al., 2020). Polyphenols have been shown to reduce hyperglycaemia and enhance acute insulin production and insulin sensitivity in a number of different animal models and a few numbers of human trials. These characteristics signify the antidiabetic nature of millets. Rice samples had lower bound phenolic concentration than millet samples, indicating that millets are a best alternative of nutraceuticals. Antioxidant, anti-diabetic, microbicidal, bactericidal and antitumor properties are reported in bound phenolic compounds.

​

Table 3: Total phenolic content (TPC), total terpenoid content (TTC), total saponin content (TSC) and tannins content (TC) in kamadhenu ark and aqueous extracts of millets.


 
Determination of total terpenoid content
 
Terpenoids are plants synthesized small molecular products and are widespread group of natural products (Murugan et al., 2022). Total terpenoid content TTC is tabulated in Table 3. KPM and KSPM exhibited highest percent of TTC 38.94±3.1 and 36.66±2.9 respectively. ALM and ASLM exhibited least per cent of TTC 3.94±2.2 and 4.68±2.4 respectively. Terpenoids are present in Foxtail millet (Sangma et al., 2019). TTC of Finger millet, Pearl millet and Fonio millet are 11.92±0.01, 8.49±0.06 and 5.71±0.10 mg/g (Nassarawa et al., 2019). Provides evidence for the presence of terpenoids in millets.
 
Determination of total saponin content
 
Saponins lowers the blood glucose levels by activation of glycogen synthesis and modulation of Insulin signalling. Hence millets are consumed by people with diabetes and cardiovascular disorders. Total Saponin Content TSC is shown in Table 3. KPM and KSPM has highest TSC of 9.87±2.2 and 9.78±3.2 mg AE/g respectively. ALM and AKM has the lowest TSC of 2.98±1.2 and 3.41±1.5 mg AE/g respectively. Saponins are not present in foxtail millet Sangma et al., 2019. TSC of finger, pearl and fonio millets are 28.00±0.04, 38.64±0.28 and 23.82±0.18 mg/g (Nassarawa et al., 2019), thus possess rich TSC in comparison to proso, kodo and little millet.
 
Determination of tannins content
 
Medicinal Plants having Tannins Are Astringent, hence used for the treatment of Intestinal Disorders. Tannins Content TC is tabulated in Table 3. KPM and KSPM exhibited highest TC of 8.92±1.6 and 8.66±1.8 mg TAE/g respectively. ALM and ASLM exhibited least TC of 1.98±0.5 and 2.12±1.1 mg TAE/g respectively. Proso millets are rich in tannins in comparative to finger millet, pearl millet and fonio millet with 7.70±0.02, 6.56±0.02 and 5.92±0.01 mg TAE/g (Nassarawa et al., 2019) barnyard millet with 1.05 mg TAE/g (Kom et al., 2020).  
 
Phytosterol esters significantly help to decrease the blood serum LDL cholesterol range by fourteen percent, however they have no impact on the blood serum HDL cholesterol range. The presence of Carbohydrates and Glycosides attribute for the delay in gastric emptying by Millets. Alkaloids have great potential to antimicrobial, anti-malarial and anti-inflammatory activities (Marella et al., 2013). The steroids compounds are used to stress relive, decrease cholesterol level, induce immune system and increase memory power (Sharma et al., 2011).
 
Determination of antioxidant potential
 
Present Study on determination of antioxidant potential adopting DPPH free radical scavenging activity reveals that KPM has the highest Antioxidant Activity of 69.03±0.12% and the least activity is shown by KSKM of 23.59±0.29% at 0.2 mg/ml. The IC50 Values reveals that KPM has good antioxidant potential at very low concentrations of 0.0212 mg/ml and KSKM has least antioxidant activity with IC50 Value of 3.1952 mg/ml (Fig 3). At lower concentrations the sprouted aqueous extracts have good antioxidant potential than raw aqueous extracts, the raw kamadhenu ark extracts have high Antioxidant potential comparative to sprouted kamadhenu ark extracts (Fig 1 and 2). Proso millet exhibits high antioxidant potential in comparative to finger millet, pearl millet and fonio millet with 67.06±0.04, 61.80±0.10 and 60.04±0.07 percent (Nassarawa et al., 2019). Proso millet exhibits excellent antioxidant potential with IC50 value of 0.0212 mg/ml in comparison to Barnyard millet, Italian millet and millet possessing IC50 values of 0.3596, 0.4362 and 0.5543 mg/ml (Ofosu et al., 2020). 

Fig 1: Comparative study of antioxidant potential of various kamadhenu ark extracts of millets with standard ascorbic acid.



Fig 2: Comparative study of antioxidant potential of various aqueous extracts of millets with standard ascorbic acid.



Fig 3: Comparative study of IC50 values of various kamadhenu ark and aqueous extracts of millets with standard ascorbic acid.

CONCLUSION

In the present work antioxidant potential of aqueous and kamadhenu ark extracts of kodo millet, little millet and proso millet, are determined adopting DPPH free radical scavenging assay. At lower concentrations the antioxidant potential is low and activity enhanced with increase in concentration. Kamadhenu ark extract of proso millet exhibited excellent antioxidant potential than kodo, little, barnyard, foxtail, finger, pearl and fonio millets. The Phytochemical analysis manifested the inherence of carbohydrates, diterpenes, glycosides, phenols, phytosterols, saponins, steroids, tannins and triterpenoids in all the aqueous and kamadhenu ark extracts of raw and sprouted kodo millet, little millet and proso millet. Kamadhenu ark extract of proso millet is rich in total phenol content, total terpenoid content and tannins content than kodo, little, barnyard, foxtail, finger, pearl and fonio millets. Present in vitro studies should be confirmed in vivo and the phytochemicals should be isolated and used for further study.

ACKNOWLEDGEMENT

The authors thank DST-FIST, Department of Applied Microbiology and Biochemistry, Sri Padmavati Mahila Visvavidyalayam, Tirupati andhra Pradesh for help in preparation of Extracts and DST-CURIE, Sri Padmavati Mahila Visvavidyalayam, Tirupati andhra Pradesh for help in Phytochemical analysis and DPPH radical scavenging Assay. The authors extend their immense gratitude to Rishivatika Ashramam Trust, Alankhanpalli, Kadapa, YSR Kadapa District for providing Kamdhenu Ark.

CONFLICT OF INTEREST

The authors have no conflicts of interest regarding this investigation.

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