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Full Research Article

Harvest Timing as a Determinant of Physico-chemical Quality Trajectories in Apple (Malus × domestica Borkh.) Cultivars

A
Anil Kumar Saxena1
0009-0004-1942-4548
S
Suneeta Singh2,*
0000-0003-1385-6505
1Department of Soil Science, School of Agricultural Sciences, Shri Guru Ram Rai University, Dehradun-248 001, Uttarakhand, India.
2Department of Horticulture, School of Agricultural Sciences, Shri Guru Ram Rai University, Dehradun-248 001, Uttarakhand, India.

ABSTRACT

Background: A study was undertaken to evaluate the influence of different days to picking on the physical and biochemical quality attributes of three major apple cultivars grown under mid-hill regions of Uttarakhand.

Methods: An experiment was conducted during 2023-25 in Department of Horticulture, School of Agricultural Sciences, Shri Guru Ram Rai University, Dehradun, Uttarakhand (India). The treatment includes as Factor 1: three dates of picking viz. 120 days after fruit set (D1), 140 days after fruit set (D2) and 160 days after fruit set (D3). Factor 2 represents three cultivars viz. red delicious (C1), golden delicious (C2) and gale gala (C3). The experiment was laid out in a 3 × 3 factorial randomized block design with three replications. Observations were recorded on major physico-chemical parameters.

Result: Fruit size parameters increased consistently from early (D1) to late (D3) pickings with red delicious exhibiting the largest fruit dimensions and highest yield potential across stages. Fruit firmness remained maximum in red delicious, whereas specific gravity and flesh: seed ratio improved markedly at later picking stages. Among all combinations, C3D3 recorded the highest moisture content (89.41%), TSS (14.86°Brix), reducing sugar (10.10%), non-reducing sugar (2.63%), total sugar (12.38%) and ascorbic acid (9.89 mg/100 g) along with the lowest titratable acidity (3.64%). Maximum pectin content was found in C3D2.

INTRODUCTION

Apple (Malus domestica Borkh.), a major temperate fruit crop of the family rosaceae, originated in Central Asia and extensively cultivated in Jammu and Kashmir, Himachal Pradesh, Uttarakhand, Arunachal Pradesh and other North-Eastern states. The fruit is rich in carbohydrates, sugars, organic acids, vitamin C, pectin, phenolics and minerals, making it an important functional food (Mehta et al., 2014). Fruit quality in apple is strongly influenced by cultivar and harvesting stage. Early harvesting often results in poor colour and low sugar accumulation, whereas delayed harvesting may cause over-ripening, reduced shelf life and higher physiological disorders (Kumar and Parmar, 2026; Devi et al., 2018). Variations in physico-chemical traits among cultivars are closely associated with maturity stage (Shilpa et al., 2019) and significant differences in firmness, acidity, TSS and sugar content have been reported under different harvesting periods (Kumar et al., 2017; Zakharov et al., 2026; Sharma, 2017; Kumar, 2020). Popular cultivars such as red delicious, golden delicious and gale gala differ considerably in ripening behaviour and quality attributes, highlighting the need for cultivar-specific harvesting schedules. Therefore, the present study was undertaken to determine the optimum picking stage for improved fruit quality under Himalayan conditions. It was hypothesized that delaying harvest from 120 to 160 days after fruit set would enhance fruit size, sugar accumulation, pigment development and overall fruit quality through continued carbohydrate translocation, cell expansion and maturation-related biochemical changes, although excessive delay may adversely affect firmness and storability. The study therefore aimed to identify the optimum harvest stage for three commercially important apple cultivars under mid-hill conditions of Uttarakhand.

MATERIALS AND METHODS

Present experiment was conducted during 2023-2025 in a commercial apple orchard at Village Dharali near Harshil Valley, district Uttarkashi, Uttarakhand, India (31.0409°N, 78.7815°E; altitude 2,485 m above mean sea level), characterized by a cool temperate climate with an average annual temperature of 8-15°C and annual rainfall of approximately 1,100-1,300 mm. Healthy seven year old trees planted at 6×6 m spacing and maintained under a modified central leader training and pruning system were used, with recommended irrigation and fertilizer practices followed uniformly. Fruits were harvested at 120, 140 and 160 days after fruit set based on commercial maturity (fruit colour and days after fruit set) and ten uniform fruits per replication were randomly collected from all canopy directions and brought to Horticulture Laboratory, School of Agricultural Sciences, Shri Guru Ram Rai University, Dehradun for physico-chemical analysis. The research comprising two experimental factors, namely days of picking and cultivar. The details of the factors and their corresponding levels are presented below:
 
Factor 1: Days of picking (D)
 
D1: 120 days after fruit set.
D2: 140 days after fruit set.
D3: 160 days after fruit set.
 
Factor 2: Cultivar (C)
 
C1: Red delicious.
C2: Golden delicious.
C3: Gale gala.
       
All the nine treatment combinations were laid out in a 3 × 3 factorial randomized block design with 3 replications. From all the three cultivars, uniform size fruits were collected at 120, 140 and 160 DAFS for their physico-chemical analysis. Physical characteristics of fruits were evaluated using standard procedures. Fruit length and diameter were measured with a digital vernier calliper, volume by water displacement method, firmness using a digital penetrometer, shape index through length:diameter ratio and specific gravity by weight:volume ratio, while fruit and seed colour were assessed using the Munsell colour chart. Chemical parameters including moisture, TSS, sugars, titratable acidity, ascorbic acid, anthocyanin, carotenoids, pectin and mineral contents were estimated following standard analytical procedures described by Ranganna (2009); Lane and Eynon (1992) and Piper (1966). The experimental data were statistically analyzed using OPSTAT under Factorial RBD and treatment means were compared at 5% level of significance through analysis of variance.

RESULTS AND DISCUSSION

Fruit characteristics
 
Fruit physical attributes were significantly affected by cultivars, days to picking and their interactions (Table 1). Among cultivars, red delicious (C1) exhibited superior fruit characteristics with the highest fruit length (6.78 cm), diameter (7.00 cm), weight (192.27 g), volume (190.94 ml), firmness (7.89 kg/cm²), shape index (1.08) and specific gravity (0.94), whereas gala gala (C3) generally recorded the lowest values. Delayed harvesting significantly enhanced fruit size, weight and volume, with maximum values observed at 160 days after fruit set (D3). The interaction C1D3 produced the highest fruit length (7.20 cm), diameter (8.88 cm), weight (242.62 g), volume (233.78 ml) and specific gravity (0.97), while minimum values were recorded at earlier harvesting stages. The improvement in fruit dimensions and volume with delayed picking may be associated with continuous cell enlargement, vacuolar expansion and accumulation of carbohydrates, water and assimilates during fruit maturation (Hanrahan et al., 2018 and Li et al., 2021). Progressive reduction in fruit firmness with delayed harvesting may be attributed to natural ripening process involving enzymatic degradation of cell-wall polysaccharides, solubilization of pectic substances and weakening of the middle lamella, leading to softening of tissues. Differences among cultivars are associated with genetic variation in cell wall composition, tissue organization and texture characteristics. Similar reductions in apple fruit firmness with advancing maturity have been reported by Fathizadeh et al., (2021) and Costa et al., (2020). The progressive rise in specific gravity at advanced maturity stages may be attributed to higher dry matter accumulation and increased soluble solids concentration in fruit tissues. Similar observation was also reported by Long et al., (2022).

Table 1: Effect of cultivars and picking dates on fruit physical attributes.


 
Seed and fruit quality traits
 
Seed and fruit quality traits were significantly influenced by cultivars, picking dates and their interactions, except seed number which remained non-significant among cultivars (Table 1). Red delicious recorded the highest seed number (9.54), flesh weight (180.32 g), flesh:seed ratio (1:64.83), core diameter (1.94 cm) and fruit yield (113.25 kg/tree), whereas gale gala exhibited the highest seed weight (2.76 g) and golden delicious showed maximum pedicle length (2.12 cm). Delayed harvesting significantly improved flesh weight, flesh:seed ratio, core diameter, pedicle length and fruit yield, with maximum values generally observed at 160 DAFS, including flesh weight (230.44 g) and core diameter (2.09 cm) under C1D3, while pedicle length reached 2.86 cm at D3. These increases may be attributed to continued cell expansion, vascular development, carbohydrate accumulation, biomass deposition and prolonged fruit filling during maturation (Campos et al., 2023; Ilyas et al., 2022; Kumar et al., 2022 and Bhat et al., 2023). Seed weight increased slightly due to reserve accumulation, whereas seed number remained relatively stable because of genetic regulation (Campos et al., 2023). Fruit colour also improved progressively with delayed harvesting, changing from green to yellow, red or orange-red shades depending on cultivar. Red Delicious developed intense red pigmentation at advanced maturity, while gale gala exhibited golden-yellow colouration, likely due to increased anthocyanin and carotenoid synthesis during ripening (Dwivedi et al., 2022 and Zhang et al., 2022). Similar findings were reported by Li et al., (2021); Sharma and Chauhan (2021); Korban et al., (2023); Mohammed et al., (2023) and Ghosh et al., (2024).
 
Biochemical and nutritional attributes
 
Moisture, TSS, sugars, titratable acidity, ascorbic acid, pectin, pigments and mineral composition were significantly influenced by cultivars, picking dates and their interactions (Table 2). Moisture content increased from 85.27% at D1 to 88.42% at D3, with Gale Gala recording the highest mean moisture (87.57%), likely due to enhanced cell expansion, osmotic adjustment and water accumulation in fruit tissues (Kumar et al., 2023 and Cheng et al., 2022). TSS also increased progressively from 10.19°Brix to 13.92°Brix with delayed harvesting, while Golden Delicious recorded the highest mean TSS (12.70°Brix), reducing sugars (8.94%) and total sugars (10.91%). Gale gala exhibited maximum non-reducing sugars (3.08%). Increased sugar accumulation at advanced maturity stages may result from starch hydrolysis and enhanced assimilate translocation, as also reported by Sharma et al., (2017) and Chalise and Rana (2023). In contrast, titratable acidity declined significantly from 0.68% at D1 to 0.39% at D3, with Gale Gala showing the highest acidity (0.63%), possibly due to utilization of organic acids during respiration and ripening (Toivonen and Brummel, 2008). Ascorbic acid content also decreased from 8.51 to 6.82 mg/100 g with delayed harvesting, although Red Delicious maintained the highest mean value (8.71 mg/100 g), which may be linked to oxidation and enhanced metabolic activity during ripening (Szeto, 2023). Pectin content increased up to D2 and declined slightly at D3, with Gale Gala recording the highest mean value (0.549%), likely because of pectin solubilization and cell wall degradation at full maturity (Mosa et al., 2017 and Devi et al., 2018). Anthocyanin, carotenoid and mineral contents increased consistently with delayed harvesting, indicating improved ripening and nutrient accumulation. Red delicious recorded the highest anthocyanin (0.051 mg/100 g) and carotenoid content (0.495 mg/100 g), whereas gala gala showed maximum potassium (5.65 mg/100 g), calcium (2.57 mg/100 g) and iron (5.92 mg/100 g). Enhanced pigment and mineral accumulation at advanced maturity stages may be associated with increased flavonoid biosynthesis, chloroplast to chromoplast transformation and improved nutrient translocation in mature fruits. Similar findings were reported by Kotiyal et al., (2017); Khan (2022); Lin-Wang (2022); Yildiz (2022); Karlidag (2022) and Pandey (2023).

Table 2: Effect of cultivars and picking dates on chemical attributes.

CONCLUSION

The study demonstrated that physico-chemical characteristics were significantly influenced by cultivar and picking stage. Delayed picking up to 160 days after fruit set improved fruit size, weight, volume, flesh content, colour, TSS, sugars, pigments and mineral composition, while acidity and ascorbic acid decreased with maturity. Among the cultivars, Red Delicious performed best for most physical and colour attributes, whereas Golden Delicious recorded higher TSS and sugars. Overall, harvesting at 160 DAFS significantly improved fruit size, sugars, pigments and mineral composition, whereas earlier harvesting maintained comparatively higher firmness and ascorbic acid. Thus, optimum harvest timing depends on the intended market destination, with later harvest favouring immediate fresh consumption and earlier harvest potentially benefiting longer storage.

ACKNOWLEDGEMENT

The authors are thankful to the School of Agricultural Sciences, Shri Guru Ram Rai University, Dehradun for providing necessary facilities and support for the research work. The authors also express sincere gratitude to Prof. (Dr.) Suneeta Singh and Prof. (Dr.) A. K. Saxena for their valuable guidance and cooperation during the study.

CONFLICT OF INTEREST

The authors affirm that there are no conflicts of interest associated with this study.

REFERENCES


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    Full Research Article

    Harvest Timing as a Determinant of Physico-chemical Quality Trajectories in Apple (Malus × domestica Borkh.) Cultivars

    A
    Anil Kumar Saxena1
    0009-0004-1942-4548
    S
    Suneeta Singh2,*
    0000-0003-1385-6505
    1Department of Soil Science, School of Agricultural Sciences, Shri Guru Ram Rai University, Dehradun-248 001, Uttarakhand, India.
    2Department of Horticulture, School of Agricultural Sciences, Shri Guru Ram Rai University, Dehradun-248 001, Uttarakhand, India.

    ABSTRACT

    Background: A study was undertaken to evaluate the influence of different days to picking on the physical and biochemical quality attributes of three major apple cultivars grown under mid-hill regions of Uttarakhand.

    Methods: An experiment was conducted during 2023-25 in Department of Horticulture, School of Agricultural Sciences, Shri Guru Ram Rai University, Dehradun, Uttarakhand (India). The treatment includes as Factor 1: three dates of picking viz. 120 days after fruit set (D1), 140 days after fruit set (D2) and 160 days after fruit set (D3). Factor 2 represents three cultivars viz. red delicious (C1), golden delicious (C2) and gale gala (C3). The experiment was laid out in a 3 × 3 factorial randomized block design with three replications. Observations were recorded on major physico-chemical parameters.

    Result: Fruit size parameters increased consistently from early (D1) to late (D3) pickings with red delicious exhibiting the largest fruit dimensions and highest yield potential across stages. Fruit firmness remained maximum in red delicious, whereas specific gravity and flesh: seed ratio improved markedly at later picking stages. Among all combinations, C3D3 recorded the highest moisture content (89.41%), TSS (14.86°Brix), reducing sugar (10.10%), non-reducing sugar (2.63%), total sugar (12.38%) and ascorbic acid (9.89 mg/100 g) along with the lowest titratable acidity (3.64%). Maximum pectin content was found in C3D2.

    INTRODUCTION

    Apple (Malus domestica Borkh.), a major temperate fruit crop of the family rosaceae, originated in Central Asia and extensively cultivated in Jammu and Kashmir, Himachal Pradesh, Uttarakhand, Arunachal Pradesh and other North-Eastern states. The fruit is rich in carbohydrates, sugars, organic acids, vitamin C, pectin, phenolics and minerals, making it an important functional food (Mehta et al., 2014). Fruit quality in apple is strongly influenced by cultivar and harvesting stage. Early harvesting often results in poor colour and low sugar accumulation, whereas delayed harvesting may cause over-ripening, reduced shelf life and higher physiological disorders (Kumar and Parmar, 2026; Devi et al., 2018). Variations in physico-chemical traits among cultivars are closely associated with maturity stage (Shilpa et al., 2019) and significant differences in firmness, acidity, TSS and sugar content have been reported under different harvesting periods (Kumar et al., 2017; Zakharov et al., 2026; Sharma, 2017; Kumar, 2020). Popular cultivars such as red delicious, golden delicious and gale gala differ considerably in ripening behaviour and quality attributes, highlighting the need for cultivar-specific harvesting schedules. Therefore, the present study was undertaken to determine the optimum picking stage for improved fruit quality under Himalayan conditions. It was hypothesized that delaying harvest from 120 to 160 days after fruit set would enhance fruit size, sugar accumulation, pigment development and overall fruit quality through continued carbohydrate translocation, cell expansion and maturation-related biochemical changes, although excessive delay may adversely affect firmness and storability. The study therefore aimed to identify the optimum harvest stage for three commercially important apple cultivars under mid-hill conditions of Uttarakhand.

    MATERIALS AND METHODS

    Present experiment was conducted during 2023-2025 in a commercial apple orchard at Village Dharali near Harshil Valley, district Uttarkashi, Uttarakhand, India (31.0409°N, 78.7815°E; altitude 2,485 m above mean sea level), characterized by a cool temperate climate with an average annual temperature of 8-15°C and annual rainfall of approximately 1,100-1,300 mm. Healthy seven year old trees planted at 6×6 m spacing and maintained under a modified central leader training and pruning system were used, with recommended irrigation and fertilizer practices followed uniformly. Fruits were harvested at 120, 140 and 160 days after fruit set based on commercial maturity (fruit colour and days after fruit set) and ten uniform fruits per replication were randomly collected from all canopy directions and brought to Horticulture Laboratory, School of Agricultural Sciences, Shri Guru Ram Rai University, Dehradun for physico-chemical analysis. The research comprising two experimental factors, namely days of picking and cultivar. The details of the factors and their corresponding levels are presented below:
     
    Factor 1: Days of picking (D)
     
    D1: 120 days after fruit set.
    D2: 140 days after fruit set.
    D3: 160 days after fruit set.
     
    Factor 2: Cultivar (C)
     
    C1: Red delicious.
    C2: Golden delicious.
    C3: Gale gala.
           
    All the nine treatment combinations were laid out in a 3 × 3 factorial randomized block design with 3 replications. From all the three cultivars, uniform size fruits were collected at 120, 140 and 160 DAFS for their physico-chemical analysis. Physical characteristics of fruits were evaluated using standard procedures. Fruit length and diameter were measured with a digital vernier calliper, volume by water displacement method, firmness using a digital penetrometer, shape index through length:diameter ratio and specific gravity by weight:volume ratio, while fruit and seed colour were assessed using the Munsell colour chart. Chemical parameters including moisture, TSS, sugars, titratable acidity, ascorbic acid, anthocyanin, carotenoids, pectin and mineral contents were estimated following standard analytical procedures described by Ranganna (2009); Lane and Eynon (1992) and Piper (1966). The experimental data were statistically analyzed using OPSTAT under Factorial RBD and treatment means were compared at 5% level of significance through analysis of variance.

    RESULTS AND DISCUSSION

    Fruit characteristics
     
    Fruit physical attributes were significantly affected by cultivars, days to picking and their interactions (Table 1). Among cultivars, red delicious (C1) exhibited superior fruit characteristics with the highest fruit length (6.78 cm), diameter (7.00 cm), weight (192.27 g), volume (190.94 ml), firmness (7.89 kg/cm²), shape index (1.08) and specific gravity (0.94), whereas gala gala (C3) generally recorded the lowest values. Delayed harvesting significantly enhanced fruit size, weight and volume, with maximum values observed at 160 days after fruit set (D3). The interaction C1D3 produced the highest fruit length (7.20 cm), diameter (8.88 cm), weight (242.62 g), volume (233.78 ml) and specific gravity (0.97), while minimum values were recorded at earlier harvesting stages. The improvement in fruit dimensions and volume with delayed picking may be associated with continuous cell enlargement, vacuolar expansion and accumulation of carbohydrates, water and assimilates during fruit maturation (Hanrahan et al., 2018 and Li et al., 2021). Progressive reduction in fruit firmness with delayed harvesting may be attributed to natural ripening process involving enzymatic degradation of cell-wall polysaccharides, solubilization of pectic substances and weakening of the middle lamella, leading to softening of tissues. Differences among cultivars are associated with genetic variation in cell wall composition, tissue organization and texture characteristics. Similar reductions in apple fruit firmness with advancing maturity have been reported by Fathizadeh et al., (2021) and Costa et al., (2020). The progressive rise in specific gravity at advanced maturity stages may be attributed to higher dry matter accumulation and increased soluble solids concentration in fruit tissues. Similar observation was also reported by Long et al., (2022).

    Table 1: Effect of cultivars and picking dates on fruit physical attributes.


     
    Seed and fruit quality traits
     
    Seed and fruit quality traits were significantly influenced by cultivars, picking dates and their interactions, except seed number which remained non-significant among cultivars (Table 1). Red delicious recorded the highest seed number (9.54), flesh weight (180.32 g), flesh:seed ratio (1:64.83), core diameter (1.94 cm) and fruit yield (113.25 kg/tree), whereas gale gala exhibited the highest seed weight (2.76 g) and golden delicious showed maximum pedicle length (2.12 cm). Delayed harvesting significantly improved flesh weight, flesh:seed ratio, core diameter, pedicle length and fruit yield, with maximum values generally observed at 160 DAFS, including flesh weight (230.44 g) and core diameter (2.09 cm) under C1D3, while pedicle length reached 2.86 cm at D3. These increases may be attributed to continued cell expansion, vascular development, carbohydrate accumulation, biomass deposition and prolonged fruit filling during maturation (Campos et al., 2023; Ilyas et al., 2022; Kumar et al., 2022 and Bhat et al., 2023). Seed weight increased slightly due to reserve accumulation, whereas seed number remained relatively stable because of genetic regulation (Campos et al., 2023). Fruit colour also improved progressively with delayed harvesting, changing from green to yellow, red or orange-red shades depending on cultivar. Red Delicious developed intense red pigmentation at advanced maturity, while gale gala exhibited golden-yellow colouration, likely due to increased anthocyanin and carotenoid synthesis during ripening (Dwivedi et al., 2022 and Zhang et al., 2022). Similar findings were reported by Li et al., (2021); Sharma and Chauhan (2021); Korban et al., (2023); Mohammed et al., (2023) and Ghosh et al., (2024).
     
    Biochemical and nutritional attributes
     
    Moisture, TSS, sugars, titratable acidity, ascorbic acid, pectin, pigments and mineral composition were significantly influenced by cultivars, picking dates and their interactions (Table 2). Moisture content increased from 85.27% at D1 to 88.42% at D3, with Gale Gala recording the highest mean moisture (87.57%), likely due to enhanced cell expansion, osmotic adjustment and water accumulation in fruit tissues (Kumar et al., 2023 and Cheng et al., 2022). TSS also increased progressively from 10.19°Brix to 13.92°Brix with delayed harvesting, while Golden Delicious recorded the highest mean TSS (12.70°Brix), reducing sugars (8.94%) and total sugars (10.91%). Gale gala exhibited maximum non-reducing sugars (3.08%). Increased sugar accumulation at advanced maturity stages may result from starch hydrolysis and enhanced assimilate translocation, as also reported by Sharma et al., (2017) and Chalise and Rana (2023). In contrast, titratable acidity declined significantly from 0.68% at D1 to 0.39% at D3, with Gale Gala showing the highest acidity (0.63%), possibly due to utilization of organic acids during respiration and ripening (Toivonen and Brummel, 2008). Ascorbic acid content also decreased from 8.51 to 6.82 mg/100 g with delayed harvesting, although Red Delicious maintained the highest mean value (8.71 mg/100 g), which may be linked to oxidation and enhanced metabolic activity during ripening (Szeto, 2023). Pectin content increased up to D2 and declined slightly at D3, with Gale Gala recording the highest mean value (0.549%), likely because of pectin solubilization and cell wall degradation at full maturity (Mosa et al., 2017 and Devi et al., 2018). Anthocyanin, carotenoid and mineral contents increased consistently with delayed harvesting, indicating improved ripening and nutrient accumulation. Red delicious recorded the highest anthocyanin (0.051 mg/100 g) and carotenoid content (0.495 mg/100 g), whereas gala gala showed maximum potassium (5.65 mg/100 g), calcium (2.57 mg/100 g) and iron (5.92 mg/100 g). Enhanced pigment and mineral accumulation at advanced maturity stages may be associated with increased flavonoid biosynthesis, chloroplast to chromoplast transformation and improved nutrient translocation in mature fruits. Similar findings were reported by Kotiyal et al., (2017); Khan (2022); Lin-Wang (2022); Yildiz (2022); Karlidag (2022) and Pandey (2023).

    Table 2: Effect of cultivars and picking dates on chemical attributes.

    CONCLUSION

    The study demonstrated that physico-chemical characteristics were significantly influenced by cultivar and picking stage. Delayed picking up to 160 days after fruit set improved fruit size, weight, volume, flesh content, colour, TSS, sugars, pigments and mineral composition, while acidity and ascorbic acid decreased with maturity. Among the cultivars, Red Delicious performed best for most physical and colour attributes, whereas Golden Delicious recorded higher TSS and sugars. Overall, harvesting at 160 DAFS significantly improved fruit size, sugars, pigments and mineral composition, whereas earlier harvesting maintained comparatively higher firmness and ascorbic acid. Thus, optimum harvest timing depends on the intended market destination, with later harvest favouring immediate fresh consumption and earlier harvest potentially benefiting longer storage.

    ACKNOWLEDGEMENT

    The authors are thankful to the School of Agricultural Sciences, Shri Guru Ram Rai University, Dehradun for providing necessary facilities and support for the research work. The authors also express sincere gratitude to Prof. (Dr.) Suneeta Singh and Prof. (Dr.) A. K. Saxena for their valuable guidance and cooperation during the study.

    CONFLICT OF INTEREST

    The authors affirm that there are no conflicts of interest associated with this study.

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