Studies on Pigments from Fruits Of Rivina Humilis L.: Characterisation, Elicitation, Stability and Safety.

This thesis work was conceived to carry out systematic studies towards introduction of
an unexplored fruit into commercial mainstream as an alternative source of betalains. The aim
of this thesis was to characterise pigments, enhance pigment accumulation, assess stability and
safety of juice of ripened berries of Rivina humilis L. (Phytolaccaceae), commonly called as
pigeon berry. Total betalains content was 0.35% fresh weight, and 1.7% dry weight. Notably,
betaxanthins content was more than that of betacyanins. Ten betalains (two betacyanins and
eight betaxanthins) were successfully identified viz. betanin, isobetanin, indicaxanthin,
dopaxanthin, vulgaxanthin I, glutamine-betaxanthin, aspartic acid-betaxanthin, humilixanthin,
tyrosine-betaxanthin and miraxanthin V based on their absorbance and mass spectral
characteristics. Crude pigment extract was partially purified to betacyanins (70% pure, 23.5
mg/100 g fresh weight) and betaxanthins (95% pure, 6.1 mg/100 g fresh weight) fractions. In
vitro antioxidant activity studies revealed EC50 against DPPH· (0.29, and 0.11 g/mL), and
reducing power (2.8, and 1.3 g/mL) of betacyanins and betaxanthins, respectively. The
activities were higher than that of gallic acid and ascorbic acid. Pigment rich crude extract and
betacyanins did not exhibit in vitro cytotoxicity against HepG2 cell line, whereas betaxanthins
exhibited cytotoxicity (IC50 12.0 g/mL) after the cells were exposed for 24 h. After 48 h of
exposure, betacyanins and betaxanthins showed elevated cytotoxicity (IC50 17.5 and 2.0
g/mL, respectively). Nutritionally important metabolites of R. humilis berries from different
developmental stages viz. early stage (green), intermediate (pink) and matured (red) were
determined. Betalains accumulation increased exponentially during berry ontogeny, however,
mRNA levels of a putative R. humilis betanidin-5-O-glucosyltransferase (RhBGT2) remained
unchanged. Antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) relative
expression decreased. The pattern in pigment increase suggested that, probably, betacyanins
and betaxanthins are synthesised from the same pool of betalamic acid. In red berries, total
carbohydrates (6.2 g), proteins (2.6 g), oil (0.7 g), phenols (105.7 mg gallic acid equivalent),
niacin (5.3 mg), tocopherols (0.8 mg), and organic acids (196.7 mg) in 100 g fresh weight
were determined. The extracted oil had fatty acid composition of 43% unsaturated and 18%
polyunsaturated fats. Among the elements quantified, K content was the highest and Cu
content was the lowest. Experiments were conducted to enhance betalains content in Rivina
berries. It was observed that application of salicylic acid (0.1 mM) and chitosan (0.5%, w/v) to
Rivina flowers resulted in significant (P < 0.05) increase (about 1.3-fold) in betalains content
in berries, probably by inducing betalamic acid formation. Putative RhBGT2, SOD and CAT
mRNA levels were not significantly affected by the treatments except that gene expression
pattern of SOD was significantly (P < 0.01) reduced when chitosan level was increased to 1%
ii
(w/v). Ripened Rivina berry juice (RBJ) betalains were comparatively more stable at pH 5,
low temperature (5C), and under nitrogen atmosphere in dark. RBJ betacyanins degraded
upto 95% and 96% on treatment for 36 min at 90C, and storage for 48 days at 25C,
respectively, whereas only 15% pigment was lost on storage at 5C over a period of 90 days.
Presence of ascorbic acid protected RBJ betacyanins from degradation at 25C and 90C,
whereas it did not show significant effect at 5C. During heating at 90C, Se4+ (upto 40
g/mL) had mild bleaching effect on RBJ betacyanins, whereas Zn2+ and Cu2+ (upto 40
g/mL) degraded 33% and 96% of the pigments, respectively, compared to control. Ascorbic
acid protected the pigments from metal–induced bleaching and stabilising effect of ascorbic
acid (0.25%, w/v) was significantly more than 0.5% ascorbic acid (w/v). Interestingly,
presence of ascorbic acid (0.25%, w/v) along with Se4+ (40 g/mL) enhanced the halflife
time of betacyanins at 90C by five–fold. Betaxanthins degraded rapidly in all the samples.
Ascorbic acid (0.25%, w/v) with Se4+ (40 g/mL) regenerated betacyanins efficiently after
thermal destruction, on storage at 5C. Samples containing both ascorbic acid (0.25%, w/v)
and Se4+ (40 g/mL) produced an orange tinge resulting in the lowest values of colour
parameters (Hunter’s Lab) compared to other samples, suggesting a possible complex
formation, which was further supported by bathochromic shift in the max of betanin and
isobetanin as determined by HPLC. To assess safety, RBJ was fed to adult male rats
(singledose/acute: 1, 2 and 5 g RBJ/kg bw) and growing male rats (repeated dosing/subacute:
2.5 and 5 g RBJ/kg bw/d for 35 days; dietary feeding/subchronic: 0.5, 1.0 and 2.0% RBJ in
diet, w/w, for 90 days). In all the three feeding studies, RBJ was well tolerated and also, the
feed intake, body and organ weights of RBJ administered groups were comparable to that of
control rats. Data on hematology, histology of vital organs, biochemical measurements in
serum and liver homogenates of RBJ treated rats were comparable to that of control in
subacute and subchronic dietary studies. Rivina-banana spread, prepared to evaluate the
feasibility of RBJ as colourant, quality parameters were not significantly affected and pigment
retention was 68% after six months of cold storage. Thereafter, sensory analysis and consumer
acceptability tests revealed that the product had good overall quality and was liked by
consumers. From this thesis work, it could be concluded that R. humilis berry juice is likely to
be a safe dietary source of a wide spectrum of betalain pigments, which could be 1) elicited in
planta through salicylic acid and chitosan application, and 2) stabilised in vitro in presence of
ascorbic acid (0.25%, w/v) along with Se4+ (40 g/mL).