Assessment of Nutritional And Free Radical Scavenging Activity of Selected Genotypes of Cordia Myxa L.: A Potential Underutilized Fruit Crop of Indian Arid Zone


 Cordia myxa is one of the important underutilized fruit plants suitable for arid and semi-arid regions of India having potential for commercial exploitation in vegetable and pickle industry. Fruits of four improved genotypes of Cordia myxa were analyzed for nutritional value, total phenol content and free radical scavenging activity. Acetone extracts from dried fruit samples were prepared to determine total phenol content and free radical scavenging activity using DPPH essay. The fruits harvested at 20 or 30 days after fruit set recorded higher crude fibre and crude protein as compared to at full ripening stage. The total phenol content was found highest in fruits harvested earliest irrespective of genotypes. The correlation coefficient between total phenol content and free radical scavenging activity of fruit extract was found positive and significant. The results suggest that the fruits of Cordia myxa should be harvested at 20 to 30 days after fruit set to get maximum benefits of its anti-oxidant and other nutritional properties.


Introduction
Cordia myxa, known as gonda or lasora is an important multipurpose fruit plant belonging to the family Boraginaceae. It grows well in arid to semi-arid regions of north India and other isoclimatic regions the world over. Its distribution extends in tropical and subtropical regions of India especially in dry deciduous forest of Rajasthan and western ghat in Mynmar. The plant is a small to moderate sized deciduous tree with a short trunk and spreading crown. The owers are short stalked, hermaphrodite and white in color which open in morning around 9.0 hours. It takes about three months from owering to full ripening of the fruits; however, they are harvested at mature green stage much before full ripening upon turning the fruit colour from green to yellowish green or yellowish pink. Though, the fruits are very sweet at full ripening, they hardly have commercial value due to high content of mucilage. The fruit pericarp is edible part while endocarp is viscous. The unripe fruits of C. myxa are used as vegetable and pickled with raw mango. The Fruits can also be preserved after dehydration for subsequent uses. The picking of the fruits at right stage of maturity is necessary not only for better nutrient value, but also for getting higher yield and better market price. The fruit pulp was found rich in crude protein, ber, carbohydrates and minerals (Aberoumand 2011). The fruits and other plant parts also have curative properties in skin diseases, dropsy, dysentery, dyspepsia, cholera and headache etc. Trees are mostly planted around farm as wind break and as shelter belt but for past two decades they are also planted as planned orchards as the fruits fetch high pro ts. Looking to its drought resistance, limited water requirement and multiple uses, systematic evaluation and ex-situ conservation were carried out at Central Arid Zone Research Institute, Jodhpur Rajasthan, India for further improvement of Cordia myxa. Collection was made on selective sampling strategy and each collection was allotted individual accession number. Out of various collections, fourteen diverse accessions were selected for characterization study (Meghwal et al. 2014).
After long term evaluation of vegetatively propagated genotypes along with check population, four elite genotypes i.e. CZCM-2011, CZCM-2021, CZCM-2012, CZCM-2025 were selected as improved genotypes with respect to production and phytochemicals. Most of the accessions did not show signi cant variations with respect to vegetative growth parameters such as plant height, canopy and morphological characteristics of leaves and owers but signi cant variations were recorded in fruit yield in long term evaluation (Meghwal 2018). One of the accessions i.e. CZCM-2025 was released as Maru Samridhi, a new high yielding variety recently (Meghwal et al. 2019). The fruits of other three accessions used in this study were also consistently high yielder as compared to other germplasm accessions. In literature, little or no information exists on nutritional properties, total phenol content and antioxidant activity of fruits of C. myxa at different maturity stages. Hence, this study was undertaken to derive the information on these aspects.
Previous studies on nutritional composition of the fruits of Cordia myxa showed that they contained 8.32% crude protein,57.08% carbohydrate 6.7% ash, 25.7% bre and 2.2% fat (Al-Sna 2016). Mineral analysis of the fruits is reported to contain 1.62mg/g sodium, 7.83 mg/g potassium, 0.46mg/g calcium, 0.35mg/g zinc and 0.51mg/g iron (Aberoumand 2011) The free radicals are molecular species having unpaired electron(s) in their atomic structure which can have independent existence (Jesburgar and Richardson 1991). The presence of an unpaired electron makes these species unstable and strongly reactive. The oxygen free radicals are most abundant free radicals in biological system. Singlet oxygen is another radical that owes its origin from oxygen. In biological system, the reactive oxygen species are generated enroute mitochondria and also as an intermediate product in several enzymatic reaction. These free radicals can be toxic to biomolecules such as lipid, protein and nucleic acids. The natural antioxidants present in the living systems try to neutralize them but if they are not effectively neutralized, they might harm to body leading to disease like conditions (Przedborski and Jackson-Lewis 1998). In recent years, antioxidant properties of fruits, vegetables and medicinal plant are studied with renewed interest for nding solution of several diseases in human beings. Phenolic compounds are naturally produced in the plants during secondary metabolism. The fruits and vegetables also exhibit strong antioxidant and antimicrobial properties due to phenolic substances (Alesiani 2010). Several other compounds such as avonoids, tannins and proanthocyanidins might also act as antioxidant for protection against many diseases. The quantitative and qualitative composition of phenolic compounds in the fruits is determined by genotype, agro techniques and environmental conditions under which they are grown (Miletic et al. 2012). The extracts of different plant parts which are good source of phenolic compounds are nding uses in food and pharmaceutical industries because of their nutraceutical values. Some previous studies report the presence of phenolic and antioxidant activities from accessions from naturally occurring trees C. myxa without any background of fruit yield and growing conditions. However, in our study, we have quanti ed the total phenol content and free radical scavenging activity from well tested improved genotypes (developed and maintained at our research farm under similar agrotechniques) at different stages of fruit maturity.

Sample collection and preparation
Fruits of Cordia myxa were obtained from central research farm, ICAR-Central Arid Zone Research Institute, Jodhpur during the month of April, 2019. So far, no information is available on standard maturity criteria for harvesting the fruits of C. myxa and generally, it is believed that less matured fruits have better culinary values. Therefore, simple criteria of harvesting the fruits after different days of fruit set was considered. The fruits of four accessions viz. CZCM-2011, CZCM-2012, CZCM-2021, and CZCM-2025 ( Fig. 1) were harvested at 20, 30, and 40 days after fruit set to examine the presence of total phenolic content and free radical scavenging activities as in uenced by stages of harvesting and genotypes.
These genotypes were promising selections identi ed after long-term evaluation from previous project based upon consistent higher yield. Flowering and fruit setting dates were recorded to harvest the fruits at speci ed stages. After separating the fruits from the stalks, the individual fruits were destoned and stones (seeds with hard coat) were discarded. The pericarp (the edible portion) was retained and dried in an oven at 55 0 C till it become brittle or with 5-7% moisture. The dried samples were kept in a separate screw cap PVC container with its lid tightly closed and stored in deep freeze in dark till analysis.

Extraction procedure
Dried samples were grounded in a mixture to a ne powder. The extraction procedure for phenolic substances was slightly modi ed as per the available resources. 20 g of powdered fruit sample from each treatment was mixed with 100 ml of acetone (99.5% purity) in 200 ml conical ask and stirred thoroughly and kept overnight to allow solvent extraction. In the next morning, the mixture was stirred over magnetic stirrer for 30 minutes. The mixture of each sample was then ltered. The ltrates were kept in known weight of glass beakers under room temperature for 24 hours to allow evaporation of the solvent. The dried acetone extract was weighed to calculate the yield of total phenolic extracts from each sample on the basis of empty beaker weight.

Determination of free radical scavenging activity
The solutions of fruit extracts (100 & 500 ppm) and synthetic antioxidant i.e. BHT (200 ppm) were prepared in pure methanol. The DPPH solution (0.1mM) was prepared in methanol fresh each time. Free radical scavenging activities of the fruit extracts and synthetic antioxidant (BHT) were determined according to Shimada et al. (1992) which is based on the principle of scavenging the DPPH. In this assay, a volume of 2 mL of methanolic solution of fruit extract was mixed with 2 mL DPPH (0.1 mM). An equal amount of methanol and DPPH was taken as control. The samples were then kept in dark at room temperature for 30 minutes and the absorbance measured at 517 nm using a UV-VIS spectrophotometer (Model TS2080PLUS, Analytical Technologies Limited). The free radical scavenging activity was expressed as percent inhibition calculated using the following formula: Where, A 0 is the absorbance of the DPPH solution and A 1 is the absorbance of the sample.

Determination of total phenolics
The total phenol content from the extract was determined using Folin-Ciocalteu Reagent (McDonald et al., 2001) with slight modi cation. Different concentrations of gallic acid i.e. 25, 50, 75, and 100 ppm were prepared in methanol for preparation of standard curve. To each concentration of gallic acid, 5 mL of Folin-Ciocalteu Reagent was mixed and of 2 mL of Na 2 CO 3 (75g/L) was added after 5 minutes. The solution was kept in dark for two hours and the absorbance measured at 760 nm using a UV-VIS spectrophotometer. The standard curve was plotted from the absorbance values of gallic acid. One mL (1000 ppm) of dried fruit extract was mixed with 5 mL of Folin-Ciocalteu Reagent for determination of total phenol. After 5 minutes, 2 mL of sodium carbonate (75g/L) was added and the mixture was kept in dark for two hours at room temperature and the absorbance was measured at 760 nm. The total phenols in the extracts were measured in terms of gallic acid equivalents (GAE) by the following equation, T = CV/M; where, T = total phenolic contents (GAE) in milligram per gram extract; C = the concentration of gallic acid established from the calibration curve mg/mL; V = the volume of extract in milliliter and M = the weight of sample extract (g).

Determination of nutritional properties
The fruits were harvested at different maturity stages, destoned and dried were grounded in mixer for analyzing of nutritional and mineral analysis. The samples were analyzed on dry matter basis (AOAC 2007) for Ca, Fe, Zn, Cu, Mn, Cr, Pb and Mo using Graphite furnace P.G (PG Instruments Ltd.,UK).

Statistical analysis
The experiments were laid out in Completely Randomized Block Design with three replications and the results were presented as mean ± standard error with CD at 5 % signi cance level. The data in all the experiments were analyzed in Microsoft Excel 2016 for statistical signi cance. The correlation coe cients between total phenol content and free radical scavenging activity were determined using Pearson's correlation.

Results And Discussion
Nutritional and mineral assessment of selected genotypes Result of important nutritional quality traits of four selected genotypes at three maturity stages were analysed (Tables 1 and 2). Wide variability among genotypes vis-a-vis within the genotypes at different maturity stages was recorded for all proximate traits. The range of various parameters recorded are ether extractives (2.16-4.03%), crude protein (6.5-12.21%), total ash (8.8-13.10%), silica (0.50 to 1.3%), crude bre (11.

Extract yield from fruits
The total content of phenolics and free radical scavenging activity might differ in fruits of different genotypes as well as according to fruit maturity stages. Therefore, fruits of four genotypes of C. myxa at three maturity stages were tested for phenolic content and free radical scavenging activity. The acetone extract yield of phenolic substances differed signi cantly with highest value in the fruits harvested at 20 days after fruit set irrespective of genotypes and this declined as the harvesting was delayed with lowest yield in fruits harvested at 40 days after fruit set ( Table 3). The difference between extraction yield of phenolic compounds were not signi cant when compared with different genotypes at same stage of harvesting. This shows that extraction yield is a species-speci c character not in uenced signi cantly due to genotypes/varieties. The extraction yield was lowest in fruits harvested after 40 days of fruit set which indicated lower content of these substances with advancement of fruit maturity. Less matured fruits are considered good for making vegetables or pickles because of lower content of mucilaginous substances. The fact established from this study that fruits harvested at 20 days after fruit set recorded highest value of phenolic compounds which might also have higher anti-oxidant activity.   Preparation of standard curve and calculation of total phenolic content Total phenolic content in fruit extracts was determined by Folin-Ciocalteu method using gallic acid as the standard. The absorbance values obtained at different concentrations of gallic acid were used for the preparation of standard curve (Fig. 2). In this method, electron is transferred from phenolic groups to phosphomolybdic phosphotungstic acid complex. This results in appearance of blue colour which is measured colorimetrically at 760 nm. A regression equation (Y = 0.0113x -0.0055; R 2 = 0.9913) was obtained from the standard curve and the total phenolic content was calculated as mg gallic acid equivalents (GAE) per gram of extract. The phenolic compounds present in fruits or any other plant parts might represent antioxidant activity. Therefore, total phenol content in fruit extracts were determined and given in Table 4. The data indicated that the total phenol content did not vary signi cantly between different genotypes but it differed signi cantly when compared between the data of 30 and 40 days after fruit set irrespective of genotypes. Though, the highest content of total phenol were found in the fruits harvested at 20 days after fruit set in all the genotypes but the differences were non-signi cant between phenol content at 20 and 30 days after fruit set. The total phenol content decreased signi cantly in fruits harvested after 40 days of fruit set irrespective of genotypes. From these data it appears that total phenol content is more in less matured fruits, but it was almost same up to 30 days after fruit set when the fruits also attain optimum size. Therefore, it will be more economical if the fruits are harvested after about 30 days of fruit set when they attain maximum total phenol along with higher fruit yield due to increased fruit size. The presence of polyphenols in C. myxa fruits was also reported by Aberoumand (2011) during phytochemical screening which indicated the presence of alkaloids, saponins, polyphenols and steroids. The presence of phenolic compounds such as phenols, tannins and saponins were also reported positive in fruit extract of different accessions of Cordia dichotoma (Nandekar and Mulani 2013) which is synonymous of C. myxa. In general, the total phenol content at different maturity stages followed similar trend as observed in case of extraction yield of phenolic substances. The trends of phenolic content according to fruit maturity stages, however, cannot be generalized as it may depend upon several other factors such as species besides growing environment. In Eugenia jambolana, for example, the total phenol content decreased from mature green stage to half ripened fruits but it increased again in fully ripened fruits (Balamurugan,2014). Free radical scavenging activity Several methods and modi cations have been used to determine anti-oxidant activity/free radical scavenging activity. DPPH assay is the preferred colorimetric method for the determination of free radical scavenging activity of plant extracts because of its simple and rapid reaction 15 . In this method, the DPPH radicals react readily with the antioxidant compounds in the extract resulting in the disappearance of blue colour.
The present results suggest that the extracts of C. myxa fruits are apparently good free radical scavengers both at 100 and 500 ppm concentration as shown in Fig. 3. The free radical scavenging activity was found maximum in fruits harvested at 20 days (mean values of 41.49 and 82.56 % at 100 and 500 ppm respectively) after fruit set irrespective of genotypes and concentration of the extract and it declined with delay of harvesting stage with the lowest value in the fruits harvested after 40 days of fruit set. This indicated that the presence of total phenolics and its free radical scavenging activity depends more on fruit maturity stages rather than genotypes. Phenolic compounds present in plant extracts make stable phenoxy radicals by reacting with free radicals. The fruits harvested at 20 days after fruit set also had higher extract yield and total phenol content. Therefore, the correlation coe cient between total phenol content (1000 ppm) and free radical scavenging activity of fruit extract of C. myxa (100 ppm) was calculated which showed positive and signi cant correlation with R square value of 0.9667. Earlier studies have also reported the association of phenolic compounds with antioxidant activities (Hantano et al. 1989, Liu et al. 2009). This trend was further strengthened by recent ndings by Hadakar et al. (2020) where in positive correlation was recorded in mango fruits between the total phenol and antioxidant activity both in peel and pulp.
Present study established the fact that stage of harvesting of fruit is very important along with genotypes for harnessing highest quantity of phytochemicals and mineral content. It is very much clear from the study that the harvesting of fruits at about 30 days after fruit set is ideal stage for obtaining highest content of nutrients and thus maximum antioxidant activity and free radical scavenging activity of this important species. The presence of the secondary metabolites i.e. polyphenols, antioxidants have contributed to its medicinal value as well. Many of these effects have been linked to their known functions as strong antioxidant, free radical scavenger. Comparing the fruits nutritional, antioxidants and mineral contents with recommended dietary allowances, Cordia myxa fruits in general and selected genotypes in particular may prove as a good supplement for human health of local inhabitants, if harvested at appropriate stage of maturity. Standard curve for gallic acid