KOBA
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P-ISSN: 2349–8528
E-ISSN: 2321–4902
IJCS 2019; 7(3): 403-409 Determination of functional constituents
© 2019 IJCS
Received: 06-03-2019 (Phytochemicals, micronutrients), anti-nutrients
Accepted: 10-04-2019
and antioxidant activity in commonly grown
Shrawan Singh
Division of Vegetable Science,
genotypes of Basella L.
ICAR-Indian Agricultural
Research Institute, New Delhi,
India Shrawan Singh, DR Singh, V Shajeeda Banu and LB Singh
DR Singh
Abstract
ICAR- National Research Centre
Basella L. is a very important and commonly used leafy vegetable in tropical and sub-tropical region. It
for Orchids, Pakyong, Sikkim,
India is a poor man’s spinach for traditional cuisine and micronutrient source. Naturally, it has three common
types i.e. green, red and intermediate in colours, of them green followed by red are most preferred among
V Shajeeda Banu the consumers. The present study observed significant (p<0.05) variation between three genotypes of
Division of Horticulture & Basella L.. The leaf of red type ‘CIARI Shan’ had highest ascorbic acid (138.0±1.7 mg/100g), and
Forestry, ICAR-Central Island anthocyanin (280.0±2.0 mg/100g), green type ‘CARI Poi Selection’ was rich in chlorophyll (135.0±7.6
Agricultural Research Institute, mg/100g) while flavonoids was highest in leaf of their intermediate type (459.0±3.2 mg/100g). The RP-
Port Blair, Andaman and HPLC analysis of leaf samples identified noticeable difference in three types for major carotenoids
Nicobar Islands, India (Lutein, zeaxanthin, β-cryptoxanthin, α-carotene and β-carotene), phenolics (Caffeic acid, chlorogenic
acid, ellagic acid, sinapic acid and naringin) and anthocyanin (Petunidin, cyandin, petunidin,
LB Singh pelargonidin, peonidin and malvidin). Strong positive correlation was observed between antioxidant
Division of Horticulture & activity and carotenoids (r=0.70, p<0.05 for DPPH; r=0.64, p<0.05 for ABTS), chlorophyll (r=0.78,
Forestry, ICAR-Central Island
p<0.05 for DPPH; r=0.84, p<0.05 for ABTS) and ascorbic acid (r=0.31, p<0.05 for DPPH; r=0.36,
Agricultural Research Institute,
Port Blair, Andaman and
p<0.05 for ABTS). The information highlights nutritive potential of green and red types of Basella L. or
Nicobar Islands, India its further promotion in nutritionally challenged communities in tropical and sub-tropical regions.
Introduction
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are major sources of
oxidative stress in cells which cause damage to proteins, lipids, and DNA. They are causative
factors for cardiovascular diseases, atherosclerosis, cancer, diabetes mellitus, neuro-
degeneration and ageing (Azizova, 2002) [3]. The exogenous antioxidants are crucial for
maintaining in built redox homeostasis of body (Bouayed and Bohn, 2010) [7]. The
phytochemical like anthocyanin, carotenoids, phenolics, flavonoids and ascorbic acid
(Rahman, 2007) [19] are the strong agents for the antioxidant hypothesis which state that
antioxidants can prevent oxidative damages and increased intakes from the diet will also
reduce the risks of chronic diseases (Stanner et al., 2004) [29]. Green leafy vegetables are rich
in antioxidants and dietary minerals (Singh et al., 2011) [26] and relatively inexpensive and
widely consumed foods with ethno-medicinal perceptions (Vishwakarma and Dubey, 2011;
Thongam et al. 2016; Singh et al. 2018) [33, 31, 24].
Indian spinach (Basella spp.; Basellaceae) is one of the leafy vegetables which are very
popular and commonly consumed particularly in tropical regions. It is highly productive leafy
vegetables and known as Poi, Ceylon spinach, Malabar spinach, Indian spinach and poor
man’s spinach. Indian spinach or Basella has two commonly grown species namely Basella
alba L (green leaf and stem, white flowers) and B. rubra L. (green purplish leaves, red vein
and stem, purplish white flowers). There is also an intermediate type which is seen at farmers’
field and vegetable market. Basella L. Leaves are most commonly consumed as boiled, fried
Correspondence or mix vegetable items however, its tender stem part is also being consumed (Singh et al.
Shrawan Singh 2018) [24]. Basella leaves (100g dry) contain 9.01g crude fibre, 7.8g protein, 3.7g ash, 79 mg
Division of Vegetable Science,
ICAR-Indian Agricultural
potassium, 24 mg phosphorus and provide 270.6 calories (Vishwakarma and Dubey, 2011) [33].
Research Institute, New Delhi, The leaves and stem are mucilaginous and consumed as vegetable, soup, roasted, fried or even
India in some traditional health practices (Adhikari et al., 2012). Haskel et al. (2004) [2, 13]
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International Journal of Chemical Studies
mixture was cooled and centrifuged at 5000 rpm for 15 min carotenoids confirmed by retention time of their respective
and supernatant was taken into test tube. 0.2 ml extract was standards.
added with 0.8 ml of 5% (w/v) salicylic acid-sulphuric acid
reagent and mixed thoroughly. After 20 min of incubation, the Phenolics
pH was raised to above 12 using 2 M NaOH solution. The The phenolic content in leaf samples was analysed by
mixture was cooled to room temperature and absorbance was injecting 20 µl sample in RP-HPLC. The flow rate was
taken at 410 nm using spectrophotometer. Concentration of 0.8ml/min and mobile phase was a binary solvent system
nitrate in the samples was extrapolated using calibration curve consisting of solvent A (dilute acetic acid; 0.9%; pH-2.7) and
(made by use of KNO3) and nitrate content in the sample was solvent B (100% acetonitrile) and gradient was 9% (0- 5 min),
calculated by using the formula: nitrate (mg/100 g) = [(titre 11% (5-15 min), 18% (15-22 min), 23% (22-38 min), 90%
valueppm - solution volumeml)/(aliquot volumeml × sample (38-43 min), 80% (43-44 min), 80% (44-45 min), 5% (45-60
weightg) × 100]. Saponin content was determined as per min) at 38°C and 280 nm. Column temperature was 38°C and
AOAC method (1995) [1]. Oxalate content in Basella samples absorbance read at 280 nm and compounds identified using
was determined by titration method described by Day and retention time of the standards.
Underwood (1986). The content was titrated against standard
0.05 M KMnO4 aqueous and oxalic acid was calculated from Anthocyanin
1 ml 0.05 M KMnO4 = 0.00225 g anhydrous oxalic acid Anthocyanin compounds in leaf part were quantified by
equation. The titration method as described by Day, and injecting 20 µl sample in RP-HPLC and flow rate kept at
Underwood, 1986 was followed. For this, 1g of sample in 0.8ml/min. Mobile phase was a binary solvent system
100ml conical flask was added with 75ml 3M H2SO4 and consisting of solvent A (100% acetonitrile) and solvent B (5%
stirred for 1hr with a magnetic stirrer. This was filtered using acetic acid and 10 % acetonitrile) and gradient was 100% (0-
a Whatman No. 1 filter paper. 25ml filtrate was taken and 5 min), 80% (5-20 min), 60% (20-25 min), 100% (25-30 min)
titrated while hot against 0.05M KMnO4 solution until a faint and column temperature was 25°C and absorbance read at 530
pink colour persisted for at least 30 sec. The oxalate content nm. HPLC grade standards were used for identification of
was then calculated by taking 1ml of 0.05m KMnO4 as individual compound. Previously reported retention time of
equivalent to 2.2mg oxalate (Chinma and Igyor, 2007) [8]. anthocyanins was also used as reference.
Helliwell, 2001) [9], Fe for catalase enzymes haemoglobin and types. Anthocyanin had CARI Poi Selection >‘Intermediate
myoglobin, Mn for Mn-SOD enzyme and Zn for over 30 type’ >CIARI Shan order while reverse order was observed
mammalian proteins (Rahman, 2007) [19]. Thus, intake of 125 for chlorophyll, carotenoids, phenolics and tannins.
g of Indian spinach can contribute around 25-30 % of Chlorophyll acts as antioxidant (Ferruzzi et al., 2006) [10] and
recommended dietary allowance (RDA) of Ca and Fe also contributes in synthesis of carotenoids, phenolics and
suggesting Indian spinach as potential source in reducing tannins and these phytochemicals are antioxidants therefore,
micronutrient deficiency in human body. health benefits of Indian spinach could be attributed to high
The results for phytochemical contents in three genotypes of content such compounds. The variation among three different
Basella revealed significant (p<0.05) differences among the coloured genotypes could be due to their difference in
genotypes and also between the leaf and stem parts (Table 1). genotypic levels as they represent different botanical species
Tannin was most important constituent followed by (Besseau et al., 2007; Raju et al., 2007) [4, 20].
carotenoids, flavonoids and anthocyanin in Basella. The leaf Phytate, nitrate and oxalate contents were significantly
part was significantly rich in ascorbic acid content than stem (p<0.05) high in leaf part than stem part of all three genotypes
part and it was highest in leaves of red type ‘CIARI Shan’ (Table 1). ‘CARI Poi Selection’ leaves had higher oxalate
(138.0±1.7 mg/100g) while lowest in red type (9.8±0.3 (40.0±2.2 mg/100g) and phytate (293.8±2.4 mg/100g) while
mg/100g). The leaves of CARI Poi Selection was rich in nitrate was highest in ‘Intermediate type’ leaves (75.0±2.6
chlorophyll (41.6±0.9 mg/100g) followed by ‘Intermediate mg/100g). Nitrate content in Basella genotypes are in
type’ (21.6±1.8 mg/100g) while it was lowest in ‘Intermediate agreement with reports of Sanchez-Echaniz et al. (2001) [23].
type’ (51.0±3.6 mg/100g). Chlorophyll a had higher High content of nitrate and phytate in leaves might be due to
proportion in total chlorophyll in all three types of Indian their role in photosynthetic tissues and activities. Oxalate
spinach. Anthocyanin was highest in leaves of red type storage in leaf portion was in conformity with reports of
(280.0±2.0 mg/100g) and lowest in CARI Poi Selection Noonan and Savage (Noonan and Savage, 1999) [17]. Phytate
(160.0±3.0 mg/100g) which reflected by colour pattern of and oxalate interfere with bioavailability of micronutrients
three types. Stem of red (410.0±3.0 mg/100g) and and enzymes (Evans and Helliwell, 2001) [9] and excess
‘Intermediate type’ (320.0±2.65 mg/100g) had significantly nitrate causes methemoglobinemia (Sanchez-Echaniz et al.,
(p<0.05) higher anthocyanin than their leaves. Flavonoid 2001) [23] but their values were low in Indian spinach than
content was high in leaf (459.0±3.2 mg/100g) than stem their risk levels. It is consumed after cooking which destroy
portion (307.0±2.0 mg/100g) in ‘Intermediate type’ which cell matrix and leach out anti-nutritional compounds (Singh et
was also higher than parts of CARI Poi Selection and red al., 2013; 2015) [27, 28].
Table 1: Phytochemicals (mg/100g FW) and micronutrient (mg/100g DW) contents in leaf and stem of Indian spinach types
Leaves Stem C.D.
Phytochemicals
CARI Poi Selection Intermediate type CIARI Shan CARI Poi Selection Intermediate type CIARI Shan (at 5%)
Anthocyanin (mg/100 g) 160.0±3.0 186.7±2.3 280.0±2.0 120.0±5.0 320.0±2.6 410.0±1.0 5.8
phenols (mg/100 g) 175.3±2.1 150.7±3.6 142.0±1.7 61.0±2.6 67.0±1.7 70.0±1.7 3.7
Carotenoids (mg/100 g) 437.3±5.1 208.7±4.9 179.2±10.3 261.3±22.7 205.6±11.8 155.8±5.6 22.5
Tannins (mg/100 g) 790.0±6.9 782.0±1.7 685.0±5.6 320.0±1.7 385.0±2.6 412.0±2.6 6.0
Ascorbic acid (mg/100 g) 113.6±0.4 100.0±1.5 138.4±1.7 63.2±1.2 60.8±1.1 66.4±0.1 0.4
Flavonoid (mg/100 g) 407.6±4.5 459.0±3.2 453.6±4.0 275.0±2.6 307.0±2.0 268.0±1.0 2.7
Chlorophyll (mg/100g) 41.6±0.9 21.6±1.8 9.8±0.3 12.9±2.6 11.567±0.9 7.067±0.7 2.8
Phytate (mg/100 g) 293.8±2.0 274.0±3.1 287.0±2.6 97.0±2.0 85.3±1.9 72.7±1.5 0.1
Nitrate (mg/100 g) 69.0±3.6 75.0±2.6 45.0±1.7 41.0±1.0 43.1±0.9 36.0±1.0 2.6
Oxalate (mg/100 g) 40.0 ± 2.2 37.6±0.2 29.3±0.1 36.4±1.4 29.7±1.2 23.6±1.1 0.3
Micronutrients (mg/100g DW)
Co 13.8±2.1 13.9±2.5 8.05±2.5 4.61±1.2 5.13±1.2 6.47±1.9 1.8
Mn 7.9±1.5 7.9±1.7 9.0±1.6 8.25±1.2 7.07±1.5 5.32±2.1 0.6
Ca 152.5±2.5 205.5±2.5 202.8±2.4 191.16±2.8 215.96±7.5 118.81±1.7 7.6
Mg 71.8±1.7 61.7±1.3 69.9±4.5 90.55±0.5 63.69±1.7 94.97±4.5 4.4
Fe 5.77±0.4 4.6±0.1 8.4±0.8 12.59±0.4 9.16±0.3 21.59±1.4 1.4
Zn 5.74±0.2 5.9±0.7 4.6±0.1 4.97±0.5 3.71±0.1 3.82±0.3 0.5
Cu 0.43±0.1 0.57±0.02 0.30±0.05 0.48±0.03 0.47±0.04 0.41±0.01 0.1
The results of in vitro study for antioxidant activity of Indian The RP-HPLC chromatogram for carotenoid analysis of leaf
spinach types are presented in Figure 2. Significant (p<0.05) extract of CARI Poi Selection generated more peaks (16) than
difference observed between DPPH and ABTS activities. Leaf ‘CIARI Shan’ (10) and ‘Intermediate type’ (7) (Figure 3a).
fraction had significantly (p<0.05) higher DPPH and ABTS The identified carotenoids were lutein, zeaxanthin, β-
activities than stem extract. Both methods showed higher cryptoxanthin, α-carotene, β-carotene and remaining
activities for ascorbic acid (88.1%; 78.4%) and rutin (78.2%; unidentified peaks. The results support high content of total
70.5%). Among the extracts, it was highest for leaf extract of carotenoids in CARI Poi Selection besides confirming earlier
CARI Poi Selection (DPPH, 83.5%; ABTS, 75.0%) while red reports of Raju et al. (2007) [20] on B. alba and
type had lowest activities (60.5% and 58.9%, respectively). It Lakshminarayana et al. (2005) [15] in B. rubra along with new
might be due to high content of carotenoids, tannins and information on ‘Intermediate type’. In phenolics analysis, the
phenolics in CARI Poi Selection. Ascorbic acid and study observed 10 peaks for red types, seven for ‘Intermediate
chlorophyll also support quenching of free radicals. Low type’ and five for CARI Poi Selection (Figure 3b). The
antioxidant activity of stem fraction of three types could be identified compounds were caffeic acid, chlorogenic acid,
attributed to poor phytochemical profile. ellagic acid and naringin in CARI Poi Selection and
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International Journal of Chemical Studies
‘Intermediate type’ while red type had sinapic acid, rutin, and Poi Selection while 10 peaks in each of ‘Intermediate type’
four unidentified peaks. The RP-HPLC analysis of leaf part and ‘CIARI Shan’ representing cyanidin, petunidin,
for anthocyanin (Figure 3c) identified only petunidin in CARI pelargonidin, peonidin, malvidin and two unidentified peaks.
Fig 2: Antioxidant activity of different parts of Indian spinach genotypes. (GL-CARI Poi Selection leaf, GS- CIARI Poi Selection stem, IML-
Intermediate type leaves, IMS-Intermediate type stem, RL- CIARI Shan leaves, RS- CIARI Shan stem), (Reference Rutein and AA- Ascorbic
acid)
Standard CARI Poi Selection leaf Intermediate type Leaf CIARI Shan Leaf
Fig. 3a. Carotenoid
Fig. 3b. Phenolic acids
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International Journal of Chemical Studies
Fig. 3c. Anthocyanin
Fig 3 (a-c): HPLC chromatogram of leaf samples of three types of Indian spinach types
The correlation between phytochemicals and antioxidant p>0.05) showed negative correlation with DPPH activity.
activity remain good but it depends on nature of sample and Similar observations were made for ABTS activity. Strong
on compounds matrix of extract Moure et al. (2001) [16]. In correlation between ABTS and DPPH methods (r = 0.99,
present study, significantly positive correlation was observed p<0.05) was in the line of reports by Bhoyar et al. (2011) in
between DPPH antioxidant activity and phenolics (r = 0.945, Capparis spinosa. Correlation between antioxidant activities
p<0.01), flavonoids (r = 0.832, p<0.05) and chlorophyll (r = and micronutrients in Indian spinach was in conformity with
0.839, p<0.05) (Table 2). The carotenoids (r = 0.718, p<0.05), reports of Tarwadi and Agte (2007) [30] on fruits of Indian
tannin (r = 635, p>0.05) and ascorbic acid (r = 0.717, p>0.05) sub-continent.
also had positive correlation while anthocyanin (r = -0.630,
The leaves and stem parts of Indian spinach had sufficient Journal of Pharmaceutical Science and Drug Research.
dietary microelements like Mg, Ca, Fe, Zn and natural 2012; 4(2):110-114.
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Acknowledgement among natural populations of Caper (Capparis spinosa)
The authors convey sincere thanks to the director, ICAR- leaves collected from cold arid desert of trans-Himalayas.
Central Island Agricultural Research Institute, Port Blair for Australian Journal of Crop Science. 2011; 5(7): 912-919.
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