Biochemical Changes in Relation to Growth and Ripening of Indian Cherry (Cordia dichotoma): An Underutilized Fruit

Abstract

The underutilized fruit of Cordia dichotoma, which is popularly known as Indian cherry, was studied at its sequential stages of growth and ripening. The biochemical analysis of this fruit revealed that it is a climacteric type and is a rich source of sugars, proteins, and phenolic compounds, indicating its potential as a nutritious food. The change in the specific activities of enzymes, such as amylase, invertase, catalase, peroxidase, pectinmethylesterase, cellulase, and polygalacturonase, was correlated with the compositional changes of the fruit during growth and ripening.

Keywords:

• Cordia dichotoma
• development
• Indian cherry
• Lehsora
• ripening
• underutilized

INTRODUCTION

Due to an ever increasing population and escalating urbanization, highly productive agricultural land is being used for urban development, which in turn places pressure on the remaining agricultural land. The difficulties have become more acute due to the over-dependency of humans on fewer plant species. Over 50% of the daily global requirement of proteins and calories is met by just three crops—maize, wheat, and rice (FAO, 1996). Hence, diversifying production and/or consumption of a broader range of plant species, including those currently identified as ‘underutilized’, can therefore contribute significantly to improve human health, nutrition, income generation, and maintain ecological sustainability (ICUC, 2006).

India is endowed with a wide variety of food plants, which contribute to the diet of the people. Diversity of agro-climatic conditions and people's preference for taste offer great chances to identify the most promising fruits for commercial cultivation. Nutritional contributions and nutritive value of the most common foods have been extensively studied and compiled (ICUC, 2006), but the lack of information regarding the nutritional value of some less commonly consumed foods limits the use of these underutilized fruits.

Indian cherry (C. dichotoma) is a small- to moderate-sized deciduous tree, locally known as Lehsora or Gunda. The fruit of lehsora is underutilized. Its immature fruits are pickled and/or used as a vegetable (Duhan et al., 1992). The globose or ovoid and drupaceous fruit when ripe turns yellow or pinkish-yellow (Fig. 1). The seed kernel of Indian cherry can be used as a potential cattle feed (Singh, 1982). Moreover, the fruit extract of lehsora is known to have various medicinal uses (Warrier et al., 1994).

FIGURE 1 C. dichotoma fruit at successive stages of growth and ripening.

However, a review of the literature reveals that although the fruit of C. dichotoma is known to possess high nutritional and medicinal value, so far due consideration has not been given to understand the compositional changes that occur during the sequential stages of growth and ripening of Indian cherry fruit. Hence, the present study.

MATERIALS AND METHODS

The fruits of C. dichotoma were collected at their successive developmental stages from the University Botanical Garden, Sardar Patel University, Gujarat, India. After recording the measurements of fresh weight, length, and diameter (Table 1), they were subjected to biochemical analyses using the methods as described earlier by Patel and Rao (2009). The statistical analysis of the data obtained was analyzed as per the method of Bliss (1967).

TABLE 1 Measurements of Fresh Weight, Diameter, Length, pH, and Total Acidity of C. dichotoma Fruit at Successive Stages of Growth and Ripening

Stage no.	Stages of development	Fresh weight (gm)	Diameter (cm)	Length (cm)	pH	Total acidity (%)
1	Young	1.29 ± 0.07	1.06 ± 0.11	0.90 ± 0.10	6.12 ± 0.02	0.13 ± 0.06
2	Premature	5.56 ± 0.53	2.06 ± 0.06	1.56 ± 0.06	6.15 ± 0.02	0.23 ± 0.06
3	Mature	10.22 ± 1.51	2.53 ± 0.15	2.13 ± 0.11	6.16 ± 0.01	0.20 ± 0.10
4	Pre-ripened	13.53 ± 0.58	2.83 ± 0.06	2.46 ± 0.06	6.15 ± 0.02	0.26 ± 0.06
5	Ripened	15.39 ± 0.48	3.06 ± 0.06	2.80 ± 0.10	6.16 ± 0.04	0.16 ± 0.11
Values ± standard deviation of five replicates.

RESULTS

The pH of the pulp of the Indian cherry at its young stage was found to be 6.1, but it remained more or less unchanged in all the subsequent developmental stages (Table 1). In contrast, the total acidity of the fruit increased by two-fold with 0.13% citric acid equivalent at the young stage to 0.26% citric acid equivalent in the pre-ripened stage. However, with the onset of ripening, sharp reduction in the total acidity (0.16% citric acid equivalent) was observed (Table 1).

As the fruit of C. dichotoma continues its growth and proceeds towards the ripening stage, a visual change occurs in its color from green to pink. A quantitative analysis of pigments revealed that the amount of chlorophyll ‘a’ and chlorophyll ‘b’ increases by three- and two-fold, respectively, from the young stage to the mature stage, but thereafter it decreases. Also, the amount of total chlorophylls at the mature stage was recorded to be as high as 1.41 mg/100 gm, but subsequently it showed a declining trend (Table 2). In contrast, the quantity of total carotenoids in the fruit measured 0.09 mg/100 gm at the young stage, but increased significantly by two-fold measuring 0.21 mg/100 gm at the ripened stage. Similarly, anthocyanins also accumulated to the highest levels of 1.05 mg/100 gm at the ripened stage (Table 2).

TABLE 2 Changes in the Biochemical Composition of C. dichotoma Fruit at Successive Stages of Growth and Ripening

	Stages of growth and ripening
Parameters	Young	Premature	Mature	Preripened	Ripened
Chlorophyll ‘a’ (mg/100 gm)	0.06^a	0.19^c	0.23^d	0.14^b	0.14^b
Chlorophyll ‘b’ (mg/100 gm)	0.57^b	0.73^c	1.17^e	0.89^d	0.44^a
Total chlorophylls (mg/100 gm)	0.64^a	0.93^b	1.41^c	1.04^b	0.58^a
Total carotenoids (mg/100 gm)	0.09^a	0.05^a	0.13^b	0.17^c	0.21^d
Total anthocyanins (mg/100 gm)	0.46^a	0.57^b	0.77^c	0.90d	1.05^d
Reducing sugars (mg/gm)	12.46^a	24.89^b	44.37^c	47.82^d	53.76^e
Non-reducing sugars (mg/gm)	28.20^b	25.84^a	28.54^b	29.88^b	31.18^c
Total sugars (mg/gm)	40.66^a	50.73^b	72.92^c	77.71^d	84.94^e
Starch (mg/gm)	15.92^e	10.26^d	8.03^c	4.46^b	2.83^a
Free amino acids (mg/gm)	2.33^c	1.39^b	0.94^a	2.95^d	1.20^b
Total phenols (mg/gm)	0.97^b	0.75^a	1.68^c	0.77^a	0.80^a
Total proteins (mg/gm)	19.40^d	17.43^b	13.17^a	18.97^c	14.71^a
Values of means followed by different letters are statistically significant according to Duncan's multiple range test (DMRT) at 5% level. (n = 3).

Sugars are said to play an essential role in imparting attractive flavor, appearance, and texture to the fruits. The quantitative analysis of non-reducing sugars in the fruit exhibited an increase in quantity from 28.2 mg/gm at its young stage to 31.2 mg/gm at the ripened stage, which represents a 25% rise. In contrast, the amount of reducing sugars was found to increase by more than four-fold, being at 12.5 mg/gm at the young stage and 53.8 mg/gm at the ripened stage (Table 2). Starch, which is considered to be the major storage polysaccharide found in the fruits, was at 15.9 mg/gm at the young stage, but decreased by more than five-fold to 2.8 mg/gm at the ripened stage (Table 2).

Proteins are known to occur in low concentrations within the fruits and they are also known to be involved in the metabolism during the growth and ripening of the fruit. The amount of protein in the fruit of Indian cherry is higher at its young stage at 19.4 mg/gm than during its subsequent stages of development and ripening. The protein content of the fruit exhibited inconsistency in its amount with 13.2 mg/gm at mature stage, increasing to 19.0 mg/gm at the pre-ripened stage, and eventually declining to 14.7 mg/gm during ripening (Table 2). The quantity of amino acids in the fruit decreased from 2.3 mg/gm at the young stage to 0.94 mg/gm at the mature stage, but a significant increment in their quantity occurs at the pre-ripened stage (2.95 mg/gm). The phenolic compounds, which are said to be important in determining the flavor and color of fruits, measured 0.97 mg/gm in the young fruit. An increase in their quantity to 1.68 mg/gm at maturity was found, but with the onset of ripening then decreased significantly (Table 2).

The present study, further revealed that the levels of ethylene evolution in the fruit of Indian cherry was 0.23 μL/kg/hr during the young stage, but with the onset of ripening, the rate of ethylene evolution increased by more than three-fold reaching 0.78 μL/kg/hr (Fig. 2). Although the rate of respiration remained more or less consistent from the young stage (0.28 μL/kg/hr) to the pre-ripened stage (0.32 μL/kg/hr), towards ripening it increased by two-fold to 0.62 μL/kg/hr (Fig. 3).

FIGURE 2 Changes in the levels of ethylene in C. dichotoma fruit during successive stages of growth and ripening.

FIGURE 3 Changes in the rate of respiration in C. dichotoma fruit during successive stages of growth and ripening.

Various metabolic changes that occur in the fruit during its growth and ripening are partly or wholly due to the activity of the enzyme systems present in it. During the course of the present study, the specific activity of the amylase enzyme increased from the young stage (0.007 mg maltose released/min/mg protein) to the mature stage (0.07 mg maltose released/min/mg protein), but subsequently it declines to 0.003 and 0.002 mg maltose released/min/mg protein during the pre-ripened and ripened stages, respectively (Table 3). In contrast, a consistent and gradual increase in the specific activity of enzyme invertase was found with 0.008 mg glucose released/min/mg protein at the young stage to 0.019 mg glucose released/min/mg protein at the premature stage. It, however, showed inconsistency in its activity with its decrease to 0.002 mg maltose released/min/mg protein in the mature and pre-ripened stages and a minor increment (0.006 mg maltose released/min/mg protein) with the onset of ripening (Table 3).

TABLE 3 Changes in the Specific Activity of Enzymes in C. dichotoma Fruit During Successive Stages of Growth and Ripening

	Stages of growth and ripening
Enzymes	Young	Premature	Mature	Preripened	Ripened
Amylase (mg maltose/min/mg protein)	0.007^b	0.007^b	0.073^c	0.003^a	0.002^a
Invertase (mg glucose/min/mg protein)	0.008^b	0.019^c	0.002^a	0.002^a	0.006^b
Catalase (units/min/mg protein)	0.0008^a	0.0007^a	0.0006^a	0.0008^a	0.0005^a
Peroxidase (units/min/mg protein)	0.001^a	0.0057^c	0.0014^b	0.0009^a	0.0008^a
Pectinmethylesterase (A620/min/mg protein)	0.004^a	0.003^a	0.006^b	0.005^b	0.006^b
Polygalacturonase (mg glucose/min/mg protein)	1.385^a	1.623^b	2.842^c	3.024^d	2.495^c
Cellulase (mg glucose/min/mg protein)	0.048^c	0.072^d	0.038^b	0.089^e	0.027^a
Values of means followed by different letters are statistically significant according to Duncan's multiple range test (DMRT) at 5% level. (n = 3).

One mechanism by which plants defend against the deleterious effects of free radicals is by the help of catalase, peroxidase, and superoxide dismutase, which prevent the formation of potent free radicals. During the course of the present study, no significant changes are noticed in the specific activity of catalase, while the specific activity peroxidase increases from 0.001 units/min/mg protein at the young fruit stage to 0.057 units/min/mg protein at the premature stage, but, thereafter, the specific activity remarkably decreases to the tune of 0.0008 units/min/mg protein (Table 3).

The enzymes that are involved in fruit cell wall metabolism during ripening, include pectinmethylesterase (PME), polygalacturanase (PG), cellulase, and β-galactosidase. Throughout the course of the currrent study, the specific activity of PME was found to be 0.004 mg A₆₂₀/min/mg protein at the young stage, but it increased significantly during ripening to 0.006 mg glucose released/min/mg protein (Table 3). However, the specific activity of cellulase exhibited inconsistent results (0.048 mg A₆₂₀/min/mg protein at the young stage, 0.072 mg glucose released/min/mg protein at premature stage, 0.038 mg glucose released/min/mg protein at mature stage, 0.089 mg glucose released/min/mg protein at pre-ripened stage, and 0.027 mg glucose released/min/mg protein during ripening). In comparison with the specific activity of other enzymes, the specific activity of PG was higher with 1.385 mg glucose released/min/mg protein at the young stage and also it subsequently increased further at the pre-ripened stage to 3.024 mg glucose released/min/mg protein (Table 3).

DISCUSSION

During the successive stages of growth and development of the Indian cherry fruit, the pH of the fruit pulp remained more or less stable. The total acidity, which measured high at the premature stage, decreased with the onset of ripening. This decrease in the acidity of the fruit changes the flavor of the fruit pulp from sour to sweet. Moreover, ripening is accompanied by changes in acidity, flavor, texture, color, and aroma of the fruit (White, 2002).

The decrease in the quantity of chlorophylls and concurrent increase in the carotenoids and anthocyanins in the fruit of C. dichotoma may be due to the unmasking of previously present pigments by degradation of chlorophyll and dismantling of the photosynthetic apparatus and synthesis of different types of anthocyanins and their accumulation in vacuoles, and accumulation of carotenoids, such as β-carotene, xanthophylls, and lycopene (Tucker and Grierson, 1987; Lizada, 1993).

The results of the present study regarding the starch accumulation during the early stages supports the opinion of Mattoo et al. (1975) that starch is the main carbohydrate present in the fruits and that it decreases to lower levels with the advancement of maturity. This accumulated starch is hydrolyzed into sugars, which is a characteristic event during fruit ripening. The results of the present study also support the view of Hulme (1970) who noted that the sugar levels within the fruit tend to increase progressively at all successive stages of growth and ripening. He suggested that the increase in sugars may be mainly due to the hydrolysis of starch, which generally accumulates during the early stage of growth and with the onset of ripening results in increasing sugar content. In addition, activation or de novo synthesis of hydrolytic enzymes like amylase was suggested to have an active role in starch degradation (Young et al., 1975). Moreover, the increase in the reducing sugar in the fruit of C. dichotoma may be due to more rapid and partial breakdown of non-reducing sugars and other polysaccharides and their subsequent inversion to reducing sugars in the course of fruit ripening (Sagar and Khurdiya, 1996).

The protein content was observed to exhibit inconsistency during the growth and ripening of the fruit. The results of the present study are in agreement with the findings of Gomez-Lim (1997) in several fruits. Tressel et al. (1975) also reported an increase in the amounts of some proteins and enzymes, while Mathooko (2000) suggested that the dramatic increase in protein reflects the enzyme required for the process of ripening. The reduction in the quantity of total free amino acids in the fruit of C. dichotomamay be due to their incorporation into proteins required for the synthesis of various ripening enzymes, and, subsequently, their utilization may also decline causing an increase in their quantity at the later stages (Frankel et al., 1968).

The declining trend of phenols from high levels during early growth to lower levels when the fruit attains maturity and thereafter may be due to phenolic compounds being easily oxidized to quinines (Vicente et al., 2007). Furthermore, Diley (1972) is of the opinion that the biosynthetic mechanisms of certain phenolic compounds are suspected of being involved in some types of stress responses that occur mainly during ripening.

Moreover, the Indian cherry fruit also exhibited an increase in autocatalytic endogenous ethylene production and an increase in respiration during the ripened stage. This kind of increase in ethylene and respiration is thought to be necessary to provide ATP and substrates for various anabolic processes associated with fruit ripening (Blanke, 1991).

Fruit ripening is regarded as an oxidative phenomenon (Brenan and Frenkel, 1977). This oxidation causes an imbalance in the production of reactive oxygen species (ROS) that leads to negative cellular alterations. During the onset of ripening, the ROS, such as singlet oxygen, hydrogen peroxide, superoxide, or hydroxyl radical (Asada, 1999), are removed by the activities of oxygen-detoxifying enzymes, such as catalase, peroxidase, and superoxide dismutase (Bowler et al., 1992). Whereas catalase plays an important role in removing the toxic hydrogen peroxide within the cell (DeDuve, 1983), it also has a role in the removal of electrons that lead to the production of O^2- free-radical (Abassi et al., 1998). Moreover, peroxidase enzyme is said to act during ripening or senescence (Grover and Sinha, 1985) as peroxidases have free radical scavenger properties (Burris, 1960). The results of the present study reveal that the enzyme peroxidase has a more major role than that of catalase in detoxifying the cells.

The major textural changes resulting during the softening of fruit are due to the enzyme-mediated alterations in the structure and composition of the cell wall and partial or complete solubilization of cell wall polysaccharides, such as pectins and cellulose (Tucker and Grierson, 1987). In the fruit of Indian cherry, the enzyme PG exhibits high specific activity followed by cellulase and PME. Similar results of increased pectic solubilization, loss of tissue firmness, and a rapid rise in the PG activity is said to accompany normal ripening in many fruits (Brady, 1987; Tucker, 1993; Fisher and Bennett, 1991).

Since pectic polymers begin to acquire solubility only after PG has become active, it is believed that the enzyme PG is involved in the breakdown of the insoluble complex polysaccharides by reducing the length of the chains cross-linked by calcium (Wong, 1995). PG hydrolyzes the α-1, 4-glycosidic bonds between the galacturonic acid residues in galacturonans, while PME catalyzes the hydrolysis of pectin methyl ester groups, resulting in desterification. As PME is specific for galacturonide esters, its action is to remove methoxyl groups from methylated pectin (Prasanna et al., 2008). This results in the formation of an acyl enzyme intermediate with the release of methanol, followed by hydrolysis to generate the enzyme and a carboxylic acid. Thus, the process of de-esterification appears to proceed linearly along the chain resulting in blocks of free carboxyl groups (Rexova-Benkova and Markovic, 1976).

It appears that PME preferentially attacks the methyl ester bonds of a galacturonate unit next to a non-esterified galacturonate unit (Pilnik and Voragen, 1970). Therefore, they deesterify the esterified pectic substances, making them vulnerable for PG action. Thus, the action of PME may be a prerequisite for the action of PG during ripening.

From the foregoing account, it may be concluded that the fruit of the Indian cherry is a rich source of total sugars, starch, proteins, and phenolic compounds, which indicated its potential as a nutritious food. Moreover, the present study provides the baseline information as a meaningful indicator for commercial exploitation of this underutilized fruit. Hence, utilization of these underutilized fruits would help in diversifying production, which can, therefore, contribute significantly to human health, nutrition, income generation, and maintenance of ecological sustainability.