EFFECT OF DIFFERENT ABIOTIC STRESSES ON ACETYLCHOLINESTERASE INHIBITION, ANTIOXIDANT ACTIVITY TOTAL PHENOL & FLAVONOID CONTENT OF SELECTED ALLIUM SPECIES: RESULTS FROM A 6-MONTH FIELD STUDY

EFFECT OF DIFFERENT ABIOTIC STRESSES ON ACETYLCHOLINESTERASE INHIBITION, ANTIOXIDANT ACTIVITY TOTAL PHENOL & FLAVONOID CONTENT OF SELECTED ALLIUM SPECIES: RESULTS FROM A 6-MONTH FIELD STUDY

Hurmat, Richa Shri *and Gulshan Bansal

Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala, Punjab, India.

ABSTRACT: Environmental factors influence plant growth, morphology, as well as nature and quantity of secondary metabolites. Modification of abiotic factors during plant growth can impact the production of bioactive phytoconstituents. A six-month field study was carried out to assess the effect of different abiotic stresses on bioactive compounds (flavonoids, phenols, quercetin), acetylcholine sterase (AChE) inhibition and antioxidant capacity of two selected Allium species i.e. Allium cepa L. (AC) & Allium sativum L. (AS; family Amaryllidaceae). AC and AS plants were grown for six months (from November 2018 to April 2019) on three different soil types (i.e. black, red, clay-loamy) and various stresses (salinity, water deprivation, flooding, fertilizer, metal, shade) were applied on the plants. At the end of the season, plants were collected, dried and plant yield was determined. Hydro alcoholic extracts of all samples were prepared. Extract yield, total flavonoids (TFC), total phenols (TPC), AChE inhibition, and antioxidant activity were determined in all the extracts. The results show the varied response of AC & AS plants to different soil types and stresses. AS plants grown in red soil with salt stress and AC plants on red soil with fertilization give higher biomass yield. AC plants grown in black soil and AS in red soil under metal stress have the highest TFC, TPC, AChE inhibition, and antioxidant activity. Hence these conditions may be recommended for incorporation in cultivation practices of these valuable medicinal plants. This would ensure a commercial supply of plants with higher phenol and flavonoid content and better activity.

Keywords: Abiotic stress, Allium species, Antioxidant activity, Acetylcholine sterase inhibition, Total phenol content, Total flavonoid content

INTRODUCTION: Onions Allium cepa L. (AC) and garlic Allium sativum L. (AS) are highly valued members of the genus Allium (Family Amaryllidaceae) ¹. AC and AS are considered imperative dietary components due to their unique flavors and nutritional value. Both plants have been traditionally used as a functional food to enhance physical and mental health ^{2, 91}. These were used as popular remedies for many diseases like cold, flu, asthma, ophthalmic problems, inflammatory disorders, etc. ^{3, 4, 5, 92}.

Several commercial formulations (i.e., extract, essential oil, macerate powder) have gained popularity due to their remarkable culinary and therapeutic properties ^{6, 7, 93}. Both plants have been extensively investigated phytochemically and pharmacologically. Both have shown diverse and significant biological activities like antioxidant, antibacterial, antiviral, anticancer, immuno-modulatory effects ⁹⁵. These reduce the risk of cardiovascular disorders and are strong neuroprotectants in various neurodegenerative disorders ^{8, 9, 10, 11}. These effects are attributed to their high organosulphur, phenolic, and flavonoid content ^{12, 13, 96}. These are also rich in anthocyanins, tannins, proteins, sterols, glycosides, saponins, and carbohydrates. Apart from these, phytoconstituents AC and AS are also rich sources of vitamins i.e., vitamin B-6, vitamin B-12, vitamin C, Riboflavin, Niacin, Folate, Vitamin A, E, K, etc., minerals i.e., Potassium, Selenium, water, Calcium, Iron, Magnesium, Zinc, Copper, protein, carbohydrates, non-dietary fibers, etc. ^{14, 15, 16, 17}.

Both plants were amongst the earliest to be cultivated 1. The cultivation practices for AC and AS are well documented ^{18, 19, 94} but the emphasis has been on plant growth and yield rather than bioactive constituents and related biological activities. Studies show that abiotic and biotic factors influence not only plant growth but also the nature and amount of phytoconstituents present in plants ²⁰. These factors can be modified in cultivation practices to enhance plant growth and production of valuable active constituents/markers and their activity. Considering the medicinal importance of both plants, i.e., AC & AS, it was thought worthwhile to identify the environmental factors during cultivation that enhance biomarker production and augment the plants' biological activities. The objective of the present study was to understand the effect of abiotic factors (season, soil type, metal, salinity, flooding, drought, fertilizer, shade stress) on the production of a marker compound, total flavonoids content (TFC), total phenols content (TPC), acetylcholinesterase (AChE) inhibition and antioxidant activities of both selected Allium species.

MATERIAL AND METHODS:

Field Trial: The field experiment was conducted at the Medicinal Plant Garden of the Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala, India. Onion seeds and garlic cloves were procured from the National Horticultural Research and Development Foundation (NHRDF), Bathinda, Punjab (151005), India in October, 2017.

The plants were authenticated by the National Horticultural Research and Development Foundation (NHRDF), Bathinda, Punjab (151005), (India) with Specimen number: NHRDF/SC/BTI/RD-2/2017-2018/515. Fresh plantlets of AC and AS were prepared before the start of the season. Based on literature reports, the plantlets were sown in the month of November under different conditions and cultivated for six months ^{21, 22, 23, 24}. Fig. 1 summarizes the plan of work of this investigation.

Experimental Design: Season for field study: November 2018 to April 2019.

Propagation: Seven plots (1 × 1m²) were prepared for each soil type. Farmyard manure (FYM) was added 2.5 kg in each plot ²⁵.

The plantlets (20 plantlets) of length 5 cm of both onion & garlic were directly planted in plots (1 ×1m²) at a distance of 10 cm in a row; each row was 10 cm apart and immediately irrigated.

Application of Abiotic Stresses:

Control Plants (No Stress): Plants were allowed to grow on different soils without any stress being applied. For normal growth of plants, irrigation was done at weekly intervals ²⁶.

Fertilization: Plants of onion and garlic grown in different soils were applied with fertilizers (Urea 3 g/m², phosphorus 6 g/m^2, and potash 3 g/m2) and (Urea 6 g/m², phosphorus 5 g/m^2, and potash 6 g/m²) respectively once in a month. According to ratio 30:60:30 and 60:50:60 for onion & garlic respectively ^{25, 27, 28}.

Shade Stress: Plants were cultivated under full shade conditions to determine the effect of shade on growth and secondary metabolite production ²⁹.

Salt Stress: Salt stress was applied by adding 3mM/L sodium chloride (NaCl) solution to each plot at monthly intervals ³⁰.

Metal Stress: Metal stress was applied by adding 75 µM solution of copper sulphate (CuSO₄) to each selected plot at monthly intervals ³¹.

Water Stress: Two types of water stress were applied:

Drought: Plants were irrigated after 14 days and these plants were grown under water scarcity conditions.

Flooding: Plants were kept submerged underwater ³².

Collection of Plant: According to literature, the bulbs of both plants exposed to different conditions were collected after 6 months, shade dried and weighed ^{21, 22, 33, 34}.

FIG. 1: PLAN OF WORK IN THE PRESENT INVESTIGATION

Preparation of Extracts: Onion and garlic bulbs were harvested at the maturity stage from all stress treated plots separately, dried, and weighed. Outer scales & edible portions were separated from both plants. The edible portion of AC & AS were ground with 80% methanol and filtered. The filtrate was collected, concentrated and dried to prepare extracts, and yield (% w/w, dry weight basis) was recorded for all plants ^{8, 35}. Outer scales of AC & AS were ground with 90% methanol and allowed to stand with solvent for 2-3 days for exhaustive maceration and filtered. The filtrate was collected, concentrated, and dried to prepare extracts, and yield (% w/w, dry weight basis) was recorded for all plants ^{8, 35, 36}.

Phytochemical Screening: Phytochemical evaluation of prepared extracts was carried to determine the presence or absence of flavonoids, saponins, steroids, carbohydrates, triterpenoids, tannins and proteins as per standard tests ^{36, 37, 38, 39, 40}.

Standardization of Extracts of AC and AS Affected by Different Stresses:

Estimation o Total Phenol Content: Total phenol content (TPC) analysis of hydro-methanol extracts was determined by Folin-Ciocalteu procedure using the standard plot of Gallic acid ^{36, 41}.

Results were expressed as percentage w/w and calculated using the following formula:

Total phenolic content (% w/w) = GAE × V × D × 10^–6 × 100/W,

GAE - Gallic acid equivalent (μg/ml), V - Total volume of sample (ml), D - Dilution factor, W - Sample weight (g).

Estimation of Total Flavonoid Content: Total flavonoid content (TFC) of hydro-methanol extracts was determined by using the Aluminium chloride method using the standard plot of quercetin ^{36, 41}.

Results were expressed as percentage w/w and calculated by using the following formula:

Total flavonoid content (% w/w) = QE × V × D × 10^–6 × 100/W,

QE - quercetin equivalent (μg/ml), V - total volume of sample (ml), D - dilution factor, W - sample weight (g).

In-vitro Evaluation of Biological Activities of the Extracts:

In-vitro Antioxidant Activity: The antioxidant activity of various plant extracts was determined by the DPPH (2, 2-diphenyl-1-picrylhydrazyl) assay. The DPPH scavenging activity was evaluated as described by Blois (1958), Kamboj and Rana, (2014), and Singh et al., 2018 with slight modifications ^{42, 43, 44}. The IC₅₀ value of each extract was calculated by using linear regression analysis and expressed in µg/ml. All readings were taken in triplicate.

In-vitro Acetylcholinesterase (Ache) Inhibitory Activity: The AChE inhibitory effect of each test extract was evaluated using the method of Ellman et al., (1961) ⁴⁵. The enzyme acetylcholine stress hydrolyzes the substrate acetylcholine, which results in the production of thiocholine. The latter reacts with 5, 5’-dithiobis (2-nitrobenzoic acid) (DTNB), thereby producing 2-nitrobenzoate-5-mercaptothiocholine and %-thio-2-nitrobenzoate, which can be detected at 412 nm. All the readings were taken in triplicate ^{11, 45, 46}.

RESULTS AND DISCUSSION:

Effect of Abiotic Stresses on Plant Growth: AC and AS are herbaceous, perennial, flowering plants, which are monocots in the order Asparagales. Both plants can be easily propagated by seeds, cloves, direct sowing or bulbs ⁴⁷.

In this study, propagation was done by plantlets which spread fast and evenly. The plants grown under different conditions were collected at the end of the study period, shade dried, and weighed separately. Fig. 2 and 3 summarizes the amount of plant collected per unit area expressed as g/m².

FIG. 2: PLANT YIELD OF ALLIUM CEPA GROWN UNDER DIFFERENT STRESS CONDITIONS WS- water scarcity; MS- metal stress; SS- salt stress.

FIG. 3: PLANT YIELD OF ALLIUM SATIVUM GROWN UNDER DIFFERENT STRESS CONDITIONS WS- water scarcity; MS- metal stress; SS- salt stress

According to literature, for the cultivation of onion and garlic, plantlets or medium-sized bulbs can be used for sowing during two seasons in a year i.e. April to May and October to November ^{25, 48}. These grow well in pH range of 6-7 and in a mild season without extremes of heat and cold. The recommended soils for their cultivation are red soil, black soil, red loam soil, and clay loam soil with good drainage facilities. These plants require sufficient soil moisture during their growth period, but heavy rains during bulb germination and bulb formation affect the crop growth ^{48, 49, 50, 51}. The results found in our study are in consonance with the previous studies as the plantlets grew well in all three soil types with weekly irrigation (except for the water stress groups). Reports on crop yields show that yield of onion bulbs (25-40 ton/hectare) is generally higher than that of garlic (50-70 quintal / hectare) ^{52, 53, 54, 98}. In this investigation, also yield of AC was significantly higher than the yield of AS in all cultivation conditions. Nitrogen, phosphorus, and potassium are the primary nutrients necessary for plant growth. Nitrogen fertilizers improve plant vigor, water utilization, number of seeds, size of leaves and stems, number of roots ⁵⁵. It is well documented that the application of nitrogen fertilizer increases the bulb yield of onion and garlic significantly ^{18, 56, 97, 98}.

In our study yield of AC was maximum with fertilizers irrespective of soil type.

Effect of Abiotic Stresses on AC and AS Extract Yield: By reviewing the literature, it was found that onion and garlic have been used in folk medicines since ancient times in different forms, e.g., Juice, paste, poultice, powder, and in food items. Various methods such as maceration, Soxhlet extraction, Freeze-drying, Pressurized Liquid Extraction, Subcritical water extraction, etc., are reported for the preparation of extracts from AC and AS ^{57, 58, 59, 60, 61, 62, 63}. Alcoholic or aqueous extracts of both plants are generally prepared since these contain higher quantities of flavonoids and phenols ³⁶. Thus based on literature, in our study, hydro-methanol extracts of AC and AS were prepared by maceration. Numerous studies show phytochemical variations in outer scales and edible portions of both plants; therefore, it results in changes in pharmacological activities as well ^{1, 64, 65, 66, 67, 68, 69, 70}. So, the edible portion and outer scales were separated, and their hydro-methanol extracts were prepared.

Table 1 summarizes the yield (% w/w, dry weight basis) of hydro-methanol extract prepared from plants grown under different conditions.

TABLE 1: YIELD OF METHANOL EXTRACTS OF AC & AS PLANTS GROWN UNDER DIFFERENT ABIOTIC STRESSES

N.A - Not applicable

The edible portion of AC plants grown in red soil under fertilization gave the highest yield of methanol extract. It is emphasized here that red soil is porous and rich in iron content with a pH range of 6.6 to 8.0. Red soil is loose, aerated and responds well to fertilizers. It contains salt in a lower quantity. It has been reported that red soil increases the biomass yield of onion ⁷¹.

Phytochemical Screening of Extracts: The results of phytochemical screening revealed the presence of flavonoids, carbohydrates, tannins, steroids, saponins, and triterpenoids in both parts of the plants, while edible portions of the onion and garlic also contain proteins and amino acids. These results are in agreement with earlier reports ^{37, 38, 39, 40, 72}.

Standardization of AC and AS extracts: Phytochemical screening, in accordance with the literature, revealed the presence of phenols and flavonoids in hydro-methanol extracts of onion and garlic. Therefore, all the prepared hydro-methanol extracts of AC and AS were standardized with respect to TPC and TFC.

Estimation of Total Phenol Content: Quantification of total phenol in hydro-methanol extract of all the stress affected plants was done on

the basis of a standard curve of gallic acid. The standard curve of absorbance of gallic acid was prepared Fig. 4. Table 2 presents the TPC of all the prepared extracts.

FIG. 4: STANDARD CURVE OF GALLIC ACID

FIG. 5: STANDARD CURVE OF QUERCETIN

TABLE 2: TOTAL PHENOLIC CONTENT IN EXTRACTS OF AC AND AS

N.A - Not applicable, n=3,* on dry weight basis. a p < 0.05 vs. black soil control of AC edible portion; b p < 0.05 vs. red soil control of AC edible portion; c p < 0.05 vs. clay loamy soil control of AC edible portion. p p < 0.05 vs. red soil control of AC outer scales; q p < 0.05 vs. red soil control of AC outer scales r p < 0.05 vs. clay loamy soil control of AC outer scales. x p < 0.05 vs. black soil control of AS edible portion; y p < 0.05 vs. red soil control of AS edible portion; z p < 0.05 vs. clay loamy soil control of AS edible portion. $ p < 0.05 vs. black soil control of AS outer scales; # p < 0.05 vs. red soil control of AS outer scales; @ p < 0.05 vs. clay loamy soil control of AS outer scales.

The total phenol content of AC and AS plants grown under different abiotic stresses is shown in Table 3. The data is expressed as Mean ± S.D. (n=3) and analyzed by Two way ANOVA followed by Tukey’s post-hoc analysis test.

TABLE 3: TOTAL FLAVONOID CONTENT OF THE EXTRACTS OF AC AND AS

N.A - Not applicable, n=3,* on dry weight basis. a p < 0.05 vs. black soil control of AC edible portion; b p < 0.05 vs. red soil control of AC edible portion; c p < 0.05 vs. clay loamy soil control of AC edible portion. p p < 0.05 vs. red soil control of AC outer scales; q p < 0.05 vs. red soil control of AC outer scales r p < 0.05 vs. clay loamy soil control of AC outer scales. x p < 0.05 vs. black soil control of AS edible portion; y p < 0.05 vs. red soil control of AS edible portion; z p < 0.05 vs. clay loamy soil control of AS edible portion. $ p < 0.05 vs. black soil control of AS outer scales; # p < 0.05 vs. red soil control of AS outer scales; @ p < 0.05 vs. clay loamy soil control of AS outer scales.

Total flavonoid content of AC and AS plants grown under different abiotic stresses is shown in Table 4. The data is expressed as Mean ± S.D. (n=3) and analyzed by Two way ANOVA followed by Tukey’s post-hoc analysis test. According to the literature, there is variation in the phytochemical distribution in bulb and outer scales of onion and garlic. It is well documented that the edible portion of AS contains a higher amount of TPC and TFC as compared to outer scales, and in contrast, outer scales of AC contains a higher amount of TPC and TFC as compare to the edible portion of the plant ^{65, 69, 37, 28, 63, 73}.

From the results shown in Tables 3 & 4, it is evident that the outcomes of this study are consonant with previous findings. Secondary metabolites are biosynthesized to help plants to cope with any change or stress. It is evident from Tables 3 & 4 that in response to different stresses applied in this study, the TPC and TFC increases. The most marked increase in TPC and TFC was observed in plants with mental stress. Literature shows that plants exposed to heavy metal stress show differential responses in synthesis and accumulation of pharmacologically active molecules. Usage of heavy metals in optimum concentration acts as abiotic elicitors that improve the biosynthesis of specific bioactive compounds ^{74, 99, 100}.  It is reported that heavy metal treatment increases the biosynthesis of secondary metabolites in many plant species.

In red cabbage stress of copper metal increased the level of phenolic and flavonoid compounds as well as antioxidant activity ⁷⁵. In case of Cajanus cajan L. it was found that under the Zn and Ni treatment, amount of ascorbic acid has increased owing to which the antioxidant activity enhanced 76. When Matricaria chamomilla plants were exposed to Cd and Cu (60 μM to120 μM) during cultivation, the amount of ferulic acid, cinnamic acid, caffeic acid, p-coumaric increased significantly ⁷⁷.

Similarly, in our study, it is found that metal stress showed the most marked effect on total phenol content and total flavonoid content of onion and garlic plants. Our results showed:

Metal Stress in Black Soil Resulted in Highest Total Phenol Content, Total Flavonoid Content in Onion Extracts:

Metal Stress in Red Soil Resulted in Highest Total Phenol Content, Total Flavonoid Content in Garlic Extracts:

In-vitro Evaluation of Bioactivities:

Antioxidant Activity of Prepared Extracts: The antioxidant activity of extracts of different stress-affected plants of AC and AS (edible portion and outer scales) were evaluated using in-vitro DPPH assay. The IC₅₀ values are reported in Table 4.

• Metal stress-affected AC plants grown in black soil have the highest antioxidant potential.
• Metal stress affected AS plants are grown in red soil have highest antioxidant potential.
• Higher TPC and TFC is strongly correlated with antioxidant activity ^{65, 78, 79, 80, 101, 102, 103}.

In this study also plants extracts with higher TPC and TFC had better antioxidant activity.

Acetylcholinesterase Inhibitory Activity: The IC₅₀ values of the extracts in the Ellman assay are reported in Table 5.

TABLE 4: THE IC₅₀ VALUES OF EXTRACTS OF AC AND AS IN DPPH ASSAY

NA - Not applicable, n=3, * on dry weight basis. a p < 0.05 vs. black soil control of AC edible portion; b p < 0.05 vs. red soil control of AC edible portion; c p < 0.05 vs. clay loamy soil control of AC edible portion. p p < 0.05 vs. red soil control of AC outer scales; q p < 0.05 vs. red soil control of AC outer scales r p < 0.05 vs. clay loamy soil control of AC outer scales. x p < 0.05 vs. black soil control of AS edible portion; y p < 0.05 vs. red soil control of AS edible portion; z p < 0.05 vs. clay loamy soil control of AS edible portion. $ p < 0.05 vs. black soil control of AS outer scales; # p < 0.05 vs. red soil control of AS outer scales; @ p < 0.05 vs. clay loamy soil control of AS outer scales.

Comparison of antioxidant activity i.e., IC₅₀ values of AC and AS of control plants grown in soil A, B, and C with plants grown under different abiotic stresses shown in Table 5.

The data is expressed as Mean ± S.D (n=3) and analyzed by Two way ANOVA followed by Tukey’s posthoc analysis test.

TABLE 5: THE IC₅₀ VALUE OF EXTRACTS OF AC AND AS IN ELLMAN ASSAY

NA - Not applicable, n=3, * on dry weight basis. a p < 0.05 vs. black soil control of AC edible portion; b p < 0.05 vs. red soil control of AC edible portion; c p < 0.05 vs. clay loamy soil control of AC edible portion. p p < 0.05 vs. red soil control of AC outer scales; q p < 0.05 vs. red soil control of AC outer scales r p < 0.05 vs. clay loamy soil control of AC outer scales. x p < 0.05 vs. black soil control of AS edible portion; y p < 0.05 vs. red soil control of AS edible portion; z p < 0.05 vs. clay loamy soil control of AS edible portion. $ p < 0.05 vs. black soil control of AS outer scales; # p < 0.05 vs. red soil control of AS outer scales; @ p < 0.05 vs. clay loamy soil control of AS outer scales.

Comparison of acetylcholinesterase inhibitory activity i.e. IC₅₀ values of AC and AS of control plants grown in soil A, B and C with plants grown under different abiotic stresses shown in Table 6. The data is expressed as Mean ± S.D (n=3) and analyzed by Two way ANOVA followed by Tukey’s post-hoc analysis test.

• The metal stress effected AC plants grown in black soil have the highest acetylcholine stress inhibitory potential among all the stress-affected plants.
• The metal stress effected AS plants has the highest acetylcholine stress inhibitory potential among all the stress-affected plants.

Phenols and flavonoids are well known for their antioxidant and neuroprotective potential ^{79, 81, 82, 83, 84, 85, 104, 105}. Therefore, the presence of the higher amount of phenol and flavonoid compounds in hydro-methanol extracts of metal stress treated plants might be the possible reason for the observed higher antioxidant and acetylcholine stress inhibitory potential ^{86, 87, 88, 89, 90, 106}.

CONCLUSION: Environmental factors have a strong impact on the growth of plants as well as the biosynthesis of plant metabolites. Based on the results of this investigation, it may be recommended that for higher biomass yield, plants may be cultivated in red soil with fertilization for AC and salt treatment for AS. Secondary metabolites are produced by plants to protect them from any change or stress. In this field experiment, too, it is evident that as compared to control-group plants, the plants subjected to different stresses had higher TPC, TFC content. Consequently, the antioxidant activity and AChE inhibition were also more distinct. The impact was most prominent with mental stress as metal act as elicitors and enzyme inducers. Therefore for higher total phenol content, total flavonoid content and better antioxidant and acetylcholinesterase inhibitory activity AC and AS plants should be grown in black and red soil, respectively and with metal stress.

ACKNOWLEDGEMENT: The authors are grateful to the UGC for MANF (F1-17.1/2017-18/MANF-2017-18-PUN-83294 /(SA-III/Website), for support during the course of this study.

CONFLICTS OF INTEREST: There is no conflict of interest to declare.

REFERENCES:

1. Bisen SP and Emerald M: Nutritional and therapeutic potential of garlic and onion (Allium species). Current Nutrition & Food Science 2016; 12(3): 190-99.
2. Bahaeddin Z, Yans A, Khodagholi F and Sahranavard S: Neuroprotection and anxiety like behavior reduction of Allium hirtifolium and Astragalus hamosus in the Aβ - injected rat. Research Journal of Pharmacognosy 2016; 3: 39-49.
3. Sima Nasri MA and Khatami N: Evaluation of analgesic and anti- inflammatory effects of fresh onion juice in experimental animals. African Journal of Pharmacy and Pharmacology 2012; 6(23): 1679-84.
4. Arshad MS, Sohaib M, Nadeem M, Saeed F, Imran A, Javed A, Amjad Z and Batool SM: Status and trends of nutraceuticals from onion and onion by-products: A critical review. Cogent Food & Agriculture 2017; 3(1): 1-14.
5. Teshika JD, Zakariyyah AM, Zaynab T, Zengin G, Rengasamy KR, Pandian SK and Fawzi MM: Traditional and modern uses of onion bulb (Allium cepa L.): a systematic review. Critical Reviews in Food Science and Nutrition 2019; 59(1): 39-70.
6. Ioku K, Aoyama Y, Tokuno A, Terao J, Nakatani N and Takei Y: Various cooking methods and the flavonoid content in onion. Journal of Nutritional Science and Vitaminology 2001; 47(1): 78-83.
7. Chanda S, Kushwaha S and Tiwari RK: Garlic as food, spice and medicine: A perspective. Journal of Pharmacy Research 2011; 4(6): 1857-60.
8. Bhanot A and Shri R: A comparative profile of methanol extracts of Allium cepa and Allium sativum in diabetic neuropathy in mice. Pharmacognosy Research 2010; 2(6): 374-79.
9. Kumar A, Bora KS, Jaggi AS and Shri R: Comparative evaluation of neuroprotective effect of three varieties of Allium cepa in chronic constriction injury induced neuropathic pain. Thai Journal of Pharmaceutical Sciences 2016; 29: 40(1): 9-20.
10. Singh V, Krishan P and Shri R: Antioxidant-mediated neuroprotection by Allium schoenoprasum L. leaf extract against ischemia reperfusion-induced cerebral injury in mice. Journal of Basic and Clinical Physiology and Pharmacology 2018; 29(4): 403-10.
11. Kaur A, Randhawa K, Singh V and Shri R: Bioactivity-Guided isolation of Acetylcholinesterase Inhibitor from Ganoderma mediosinense (Agaricomycetes). International Journal of Medicinal Mushrooms 2019; 21(8): 739-48.
12. Kim JM, Chang HJ, Kim WK, Chang N and Chun HS: Structure activity relationship of neuroprotective and reactive oxygen species scavenging activities for Allium organosulfur compounds. Journal of Agricultural and Food Chemistry 2006; 54(18): 6547-53.
13. Shri R and Bora KS: Neuroprotective effect of methanolic extracts of Allium cepa on ischemia and reperfusion-induced cerebral injury. Fitoterapia 2008; 79(2): 86-96.
14. Fossen T, Slimestad R and Andersen ØM: Anthocyanins with 4′-glucosidation from red onion, Allium cepa. Phytochemistry 2003; 64(8): 1367-74.
15. Franke AA, Custer LJ, Arakaki C and Murphy SP: Vitamin C and flavonoid levels of fruits and vegetables consumed in Hawaii. Journal of Food Composition and Analysis 2004; 17(1): 1-35.
16. Bonaccorsi P, Caristi C, Gargiulli C, Leuzzi U. Flavonol glucoside profile of southern Italian red onion (Allium cepa L.). Journal of Agricultural and Food Chemistry. 2005; 53(7): 2733-2740.
17. Corea G, Fattorusso E, Lanzotti V, Capasso R and Izzo AA: Antispasmodic saponins from bulbs of red onion, Allium cepa L. var. Tropea. Journal of Agricultural and Food Chemistry 2005; 53(4): 935-940.
18. Abdissa Y, Tekalign T and Pant LM: Growth, bulb yield and quality of onion (Allium cepa L.) as influenced by nitrogen and phosphorus fertilization on vertisol I. growth attributes, biomass production and bulb yield. African Journal of Agricultural Research 2011; 6(14): 3252-58.
19. Yayeh SG, Alemayehu M, Haileslassie A and Dessalegn Y: Economic and agronomic optimum rates of NPS fertilizer for irrigated garlic (Allium sativum L) production in the highlands of Ethiopia. Cogent Food & Agriculture 2017; 3(1): 1-10.
20. Hurmat, Shri R and Bansal G: Does abiotic stresses enhance the production of secondary metabolites A review. The Pharma Innovation Journal 2020; 9(1): 412-22.
21. Bindu B and Bindu P: Performance evaluation of onion (Allium Cepa L. Var. Cepa) varieties for their suitability in. Kollam District 2015; 1(1): 18-20.
22. Manna D, Maity TK and Ghosal A: Influence of foliar application of boron and zinc on growth, yield and bulb quality of onion Allium cepa L. Journal of Crop and Weed. 2014; 10(1): 53-55.
23. Kumar A, Kumar S, Kumar A and Singh KV: Response of different organic and inorganic fertilizers on growth and yield of Garlic (Allium Sativum L.). Haryana Journal of Agronomy 2013; (291/2): 77-79.
24. Ibrahim ND and Adesiyun AA: Seasonal abundance of onion thrips, Thrips tabaci Lindeman in Sokoto, Nigeria. Journal of Agricultural Science 2010; 2(1): 107-14.
25. Horticulture crops. Crop production techniques of horticulture crops. [cited 2015 June 10]. 2013.
26. Pelter GQ, Mittelstadt R, Leib BG and Redulla CA: Effects of water stress at specific growth stages on onion bulb yield and quality. Agricultural Water Management 2004; 68(2): 107-15.
27. Islah ME: Response of garlic (Allium Sativum L.) to some sources of organic fertilizers under North Sinai conditions. Research Journal of Agriculture and Biological Sciences 2010; 6(6): 928-36.
28. Mogren LM, Olsson ME and Gertsson UE: Quercetin content in field-cured onions (Allium cepa L.): effects of cultivar, lifting time and nitrogen fertilizer level. Journal of Agricultural and Food Chemistry 2006; 54(17): 6185-91.
29. Semchenko M, Lepik M, Götzenberger L and Zobel K: Positive effect of shade on plant growth: amelioration of stress or active regulation of growth rate. Journal of Ecology 2012; 100(2): 459-66.
30. Hirrel MC and Gerdemann JW: Improved growth of onion and bell pepper in saline soils by two vesicular-arbuscular mycorrhizal fungi 1. Soil Science Society of America Journal 1980; 44(3): 654-55.
31. Meng Q, Zou J, Zou JH, Jiang WS and Liu DH: Effect of Cu2+ concentration on growth, antioxidant enzyme activity and malondialdehyde content in garlic (Allium sativum L. Acta Biologica Cracoviensia Series Botanica 2007: 49(1): 95-101.
32. Jackson MB, Colmer TD. Response and adaptation by plants to flooding stress. Annals of Botany 2005; 96(4): 501-05.
33. Naik VR, Patel PB and Patel BK: Study on effect of different organics on yield and quality of organically grown onion. The Bioscan 2014; 9(4): 1499-03.
34. Rosen CJ and Tongn CB: Yield, dry matter partitioning, and storage quality of hardneck garlic as affected by soil amendments and scape removal. Horticulture Science 2001; 36(7): 1235-39.
35. Bozin B, Mimica-Dukic N, Samojlik I, Goran A and Igic R: Phenolics as antioxidants in garlic (Allium sativum L., Alliaceae). Food Chemistry 2008; 111(4): 925-9.
36. Singh V, Chauhan G, Krishan P and Shri R: Allium schoenoprasum L: a review of phytochemistry, pharmacology and future directions. Natural Product Research 2017; 2202-16.
37. Lanzotti V: The analysis of onion and garlic. Journal of Chromatography A 2006: 21; 1112(1-2): 3-22.
38. Ly TN, Hazama C, Shimoyamada M, Ando H, Kato K and Yamauchi R: Antioxidative compounds from the outer scales of onion. Journal of Agricultural and Food Chemistry 2005; 53(21): 8183-8189.
39. Miean KH and Mohamed S: Flavonoid (myricetin, quercetin, kaempferol, luteolin and apigenin) content of edible tropical plants. Journal of Agricultural and Food Chemistry 2001: 49(6): 3106-12.
40. WHO: Quality Control Methods for Medicinal Plant Material. World Health Organization Geneva 1998; 10-11.
41. Madaan R, Bansal G, Kumar S and Sharma A: Estimation of total phenols and flavonoids in extracts of Actaea spicata roots and antioxidant activity studies. Indian Journal of Pharmaceutical Sciences 2011; 73(6): 666-71.
42. Blois MS: Antioxidant determinations by the use of a stable free radical. Nature 1958; 181(4617): 1199.
43. Kamboj S and Rana V: Physicochemical, rheological and antioxidant potential of corn fiber gum. Food Hydrocolloids 2014; 39: 1-9.
44. Singh V, Krishan P and Shri R: Antioxidant-mediated neuroprotection by Allium schoenoprasum L. leaf extract against ischemia reperfusion-induced cerebral injury in mice. Journal of Basic and Clinical Physiology and Pharmacology 2018; 29(4): 403-10.
45. Ellman GL, Courtney KD, Andres Jr V and Featherstone RM: A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology 1961: 7(2): 88-95.
46. Randhawa K and Shri R:  Comparison of antioxidant and anticholinesterase activities of selected Pleurotus species (Agaricomycetes) from India. International Journal of Medicinal Mushrooms 2018; 20(8): 739-48.
47. Koller M and Vieweger A: Pest and disease management strategies-vegetable crops-weed management in organic onion production: optimizing cultivation technique and mechanical weed control to reduce hand labour. Acta Horticulturae 2012; (933): 391-98.
48. Barakade AJ, Lokhande TN and Todkari GU: Economics of onion cultivation and it's marketing pattern in satara district of Maharashtra. International Journal of Agriculture Sciences 2011: 3(3): 110-17.
49. Block E: Garlic other alliums. Cambridge RSC publishing 2010.
50. Tsiropoulos NG and Miliadis GE: Field persistence studies on pendimethalin residues in onions and soil after herbicide postemergence application in onion cultivation. Journal of Agricultural and Food Chemistry 1998; 46(1): 291-95.
51. Corgan JN and Kedar N: Onion cultivation in subtropical climates. CRC Press Boca Raton 1990; 2: 31-48.
52. Ouzounidou G, Giannakoula A, Asfi M and Ilias I: Differential responses of onion and garlic against plant growth regulators. Pakistan Journal of Botany 2011; 43(4): 2051-57.
53. Ghaffoor A, Jilani MS, Khaliq G and Waseem K: Effect of different NPK levels on the growth and yield of three onion (Allium cepa L.) varieties. Asian Journal of Plant Sciences 2003; 2(3): 342-46.
54. Bhuiya MA, Rahim MA and Chowdhury MN: Effect of planting time, mulch and irrigation on the growth and yield of garlic. Asian Journal of Plant Science 2003; 2(8): 639-43.
55. Mithöfer A, Schulze B and Boland W: Biotic and heavy metal stress response in plants: evidence for common signals. Federation of European Biochemical Societies letters. 2004; 566(1-3): 1-5.
56. Nasreen S, Haque MM, Hossain MA and Farid AT: Nutrient uptake and yield of onion as influenced by nitrogen and sulphur fertilization. Bangladesh Journal of Agricultural Research 2007; 32(3): 413-20.
57. Farías-Campomanes AM, Horita CN, Pollonio MA and Meireles MA: Allicin-rich extract obtained from garlic by pressurized liquid extraction: quantitative determination of allicin in garlic samples. Food and Public Health 2014; 4(6): 272-78.
58. Kumar U, Singh J, Naresh P and Singh R: Management of Stemphylium blight of garlic through chemicals. Annals of Plant Protection Sciences 2011; 19(1): 126-28.
59. Ko MJ, Cheigh CI, Cho SW and Chung MS: Subcritical water extraction of flavonol quercetin from onion skin. Journal of Food Engineering 2011; 102(4): 327-33.
60. Lu X, Wang J, Al-Qadiri HM, Ross CF, Powers JR, Tang J and Rasco BA: Determination of total phenolic content and antioxidant capacity of onion (Allium cepa) and shallot (Allium oschaninii) using infrared spectroscopy. Food Chemistry 2011; 129(2): 637-44.
61. Lee SN, Kim NS and Lee DS: Comparative study of extraction techniques for determination of garlic flavor components by gas chromatography–mass spectrometry. Analytical and Bioanalytical Chemistry 2003; 377(4): 749-6.
62. Banerjee SK and Maulik SK: Effect of garlic on cardiovascular disorders: a review. Nutrition Journal 2002 (1): 1-14.
63. Patil BS, Pike LM and Yoo KS: Variation in the quercetin content in different colored onions (Allium cepa L.). Journal of the American Society for Horticultural Science 1995; 120(6): 909-13.
64. Sun W, Shahrajabian MH and Cheng Q: The insight and survey on medicinal properties and nutritive components of shallot. Journal of Medicinal Plants Research 2019; 13(18): 452-57.
65. Ariyanti NA, Torikai K, Kirana RP, Hirata S and Sulistyaningsih E, Ito SI, Yamauchi N, Kobayashi N and Shigyo M: Comparative study on phytochemical variations in Japanese F1 varieties of bulb onions and South-East Asian shallot landraces. The Horticulture Journal 2018; 87(1): 63-72.
66. Major N, Goreta Ban S, Urlić B, Ban D, Dumičić G and Perković J: Morphological and biochemical diversity of shallot landraces preserved along the Croatian coast. Frontiers in Plant Science 2018; 9: 1-14.
67. Ferioli F and D’Antuono LF: Evaluation of phenolics and cysteine sulfoxides in local onion and shallot germplasm from Italy and Ukraine. Genetic Resources and Crop Evolution 2016; 63(4): 601-14.
68. Chope GA, Cools K and Terry LA: Alliums (Onion, Garlic, Leek and Shallot). Health-Promoting Properties of Fruits and Vegetables 2011; 1: 5-28.
69. Leelarungrayub N, Rattanapanone V, Chanarat N and Gebicki JM: Quantitative evaluation of the antioxidant properties of garlic and shallot preparations. Nutrition 2006; 22(3): 266-74.
70. Fattorusso E, Iorizzi M, Lanzotti V and Taglialatela-Scafati O: Chemical composition of shallot (Allium ascalonicum Hort. Journal of Agricultural and Food Chemistry 2002; 50(20): 5686-90.
71. Patel N and Rajput TB: Dynamics and modeling of soil water under subsurface drip irrigated onion. Agricultural Water Management 2008; 95(12): 1335-49.
72. Shenoy C, Patil MB, Kumar R and Patil S: Preliminary phytochemical investigation and wound healing activity of Allium cepa Linn (Liliaceae). International Journal of Pharmacy and Pharmaceutical Sciences 2009; 2(2): 167-175.
73. Yang J, Meyers KJ, van der Heide J and Liu RH: Varietal differences in phenolic content and antioxidant and anti-proliferative activities of onions. Journal of Agricultural and Food Chemistry 2004; 52(22): 6787-93.
74. Namdeo AG: Plant cell elicitation for production of secondary metabolites: a review. Pharmacognosy Review 2007: 1(1): 69-79.
75. Posmyk MM, Kontek R and Janas KM: Antioxidant enzymes activity and phenolic compounds content in red cabbage seedlings exposed to copper stress. Ecotoxicology and Environmental Safety 2009; 72(2): 596-602.
76. Rao KM and Sresty TV: Antioxidative parameters in the seedlings of pigeonpea (Cajanus cajan (L.) Millspaugh) in response to Zn and Ni stresses. Plant Science 2000; 8: 157(1): 113-28.
77. Kováčik J, Klejdus B, Hedbavny J, Štork F and Bačkor M: Comparison of cadmium and copper effect on phenolic metabolism, mineral nutrients and stress-related parameters in Matricaria chamomilla plants. Plant and Soil 2009; 320(1-2): 231-42.
78. Granato D, Shahidi F, Wrolstad R, Kilmartin P, Melton LD, Hidalgo FJ, Miyashita K, van Camp J, Alasalvar C, Ismail AB and Elmore S: Antioxidant activity, total phenolics and flavonoids contents: Should we ban in-vitro screening methods. Food Chemistry 2018; 264: 471-75.
79. David AV, Arulmoli R and Parasuraman S: Overviews of biological importance of quercetin: a bioactive flavonoid. Pharmacognosy Reviews 2016; 10(20): 84-89.
80. Stanković MS, Petrović M, Godjevac D and Stevanović ZD: Screening inland halophytes from the central Balkan for their antioxidant activity in relation to total phenolic compounds and flavonoids: Are there any prospective medicinal plants. J of Arid Environments 2015; 120: 26-32.
81. Khan H, Ullah H, Aschner M, Cheang WS and Akkol EK: Neuroprotective effects of quercetin in Alzheimer’s disease. Biomolecules 2020; 10(1): 1-20.
82. Gezici S and Sekeroglu N: Neuroprotective potential and phytochemical composition of acorn fruits. Industrial Crops and Products 2019; 128: 13-17.
83. Senol FS, Sekeroglu N, Gezici S, Kilic E and Orhan İE: Neuroprotective potential of the fruit (acorn) from Quercus coccifera L. Turkish Journal of Agriculture and Forestry 2018; 42(2): 82-87.
84. Shahidi F and Ambigaipalan P: Phenolics and polyphenolics in foods, beverages and spices: Antioxidant activity and health effects-A review. Journal of Functional Foods 2015: 18: 820-97.
85. Kumar GP and Khanum F: Neuroprotective potential of phytochemicals. Pharmacognosy Reviews 2012; 6(12): 81-90.
86. Wan C, Yu Y, Zhou S, Liu W, Tian S and Cao S: Antioxidant activity and free radical-scavenging capacity of Gynura divaricata leaf extracts at different temperatures. Pharmacognosy Magazine 2011; 7(25): 40-46.
87. Saeed N, Khan MR and Shabbir M: Antioxidant activity, total phenolic and total flavonoid contents of whole plant extracts Torilis leptophylla L. BMC Complementary and Alternative Medicine 2012; 12(1): 1-12.
88. Russo D, Valentão P, Andrade PB, Fernandez EC and Milella L: Evaluation of antioxidant, antidiabetic and anticholinesterase activities of Smallanthus sonchifolius landraces and correlation with their phytochemical profiles. International journal of molecular sciences. 2015; 16(8): 17696-18.
89. Hwang H, Kim YJ and Shin Y: Influence of ripening stage and cultivar on physicochemical properties, sugar and organic acid profiles, and antioxidant compositions of strawberries. Food Science and Biotechnology 2019; 28(6): 1659-67.
90. Bakoyiannis I, Daskalopoulou A, Pergialiotis V and Perrea D: Phytochemicals and cognitive health: Are flavonoids doing the trick. Biomedicine and Pharmacotherapy 2019; 109: 1488-97.
91. Desai G, Schelske-Santos M, Nazario CM, Rosario-Rosado RV, Mansilla-Rivera I, Ramírez-Marrero F, Nie J, Myneni AA, Zhang ZF, Freudenheim JL and Mu L: Onion and garlic intake and breast cancer, a case-control study in puerto rico. Nutrition and Cancer 2020; 72(5): 791-800.
92. El-Saber Batiha G, Magdy Beshbishy A, G Wasef L, Elewa YH, A Al-Sagan A, El-Hack A, Mohamed E, Taha AE, M Abd-Elhakim Y and Prasad Devkota H: Chemical constituents and pharmacological activities of garlic (allium sativum L.): A review. Nutrients 2020; 12(3): 872-93.
93. Teshika JD, Zakariyyah AM, Zaynab T, Zengin G, Rengasamy KR, Pandian SK, Fawzi MM. Traditional and modern uses of onion bulb (Allium cepa L.): a systematic review. Critical reviews in food science and nutrition. 2019; 59(1):39-70.
94. de Freitas Souza M, Silva TS, dos Santos JB, Carneiro GD, Reginaldo LT, Bandeira JN, dos Santos MS, Pavão QS, de Negreiros MZ and Silva DV: Soil water availability alter the weed community and its interference on onion crops. Scientia Horticulturae 2020; 272: 109573-83.
95. Petropoulos S, Fernandes Â, Barros L, Ciric A, Sokovic M and Ferreira IC: Antimicrobial and antioxidant properties of various Greek garlic genotypes. Food Chemistry 2018; 245: 7-12.
96. Virshette S, Patil M and Shaikh JR: A review on pharmacological properties and phytoconstituents of indigenous carminative agents. Journal of Pharmacognosy and Phytochemistry 2020; 9(3): 142-45.
97. Brewster JL and Rabinowitch HD: Garlic agronomy. In Onions and Allied Crops 2020; 23: 147-57.
98. Taha NM and Helal NA: Effect of nitrogen fertilization and some foliar applications on growth, yield and quality of two garlic (allium sativum l.) Cultivars. Current Science International 2019; 8(1): 212-20.
99. Malik MQ, Mujib A, Gulzar B, Zafar N, Syeed R, Mamgain J and Ejaz B: Enrichment of alliin in different in-vitro grown tissues of Allium sativum through CdCl2 elicitation as revealed by high performance thin layer chromatography (HPTLC). Industrial Crops and Products 2020; 158: 113007(1-11).
100. Madhu PM and Sadagopan RS: Effect of heavy metals on growth and development of cultivated plants with reference to cadmium, chromium and lead–a review. Journal of Stress Physiology & Biochemistry 2020; 16(3): 84-102.
101. Noreen H, Semmar N, Farman M and Mc Cullagh JS: Measurement of total phenolic content and antioxidant activity of aerial parts of medicinal plant Coronopus didymus. Asian Pacific Journal of Tropical Medicine. 2017; 10(8): 792-801.
102. Afshar FH, Delazar A, Nazemiyeh H, Esnaashari S and Moghadam SB: Comparison of the total phenol, flavonoid contents and antioxidant activity of methanolic extracts of Artemisia spicigera and A. splendens growing in Iran. Pharmaceutical Sciences 2019; 18(3): 165-70.
103. Chanioti S and Tzia C: Optimization of ultrasound-assisted extraction of oil from olive pomace using response surface technology: Oil recovery, unsaponifiable matter, total phenol content and antioxidant activity. LWT Food Science and Technology 2017; 79: 178-89.
104. Antognoni F, Potente G, Mandrioli R, Angeloni C, Freschi M, Malaguti M, Hrelia S, Lugli S, Gennari F, Muzzi E and Tartarini S: Fruit quality characterization of new sweet cherry cultivars as a good source of bioactive phenolic compounds with antioxidant and neuroprotective potential. Antioxidants 2020; 9(8): 677.
105. Sánchez-Martínez JD, Bueno M, Alvarez-Rivera G, Tudela J, Ibañez E and Cifuentes A: In-vitro neuroprotective potential of terpenes from industrial orange juice by-products. Food and Function 2021; 12(1): 302-14.
106. Silva J, Alves C, Freitas R, Martins A, Pinteus S, Ribeiro J, Gaspar H, Alfonso A and Pedrosa R: Antioxidant and neuroprotective potential of the brown seaweed Bifurcaria bifurcata in an in-vitro Parkinson’s disease model. Marine Drugs 2019; 17(2): 85:1-16.
     

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     Hurmat RS and Bansal G: Effect of different abiotic stresses on acetylcholine sterase inhibition, antioxidant activity total phenol & flavonoid content of selected allium species: results from a 6-month field study. Int J Pharm Sci & Res 2022; 13(5): 2048-60. doi: 10.13040/IJPSR.0975-8232.13(5). 2048-60

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