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DETERMINATION OF TOTAL PHENOLIC CONTENT AND TOTAL PROTEIN IN GUAVA (Psidium guajava) AND LEMON (Citrus limon) Sharma Asha, Sindhu Pawan, Devi Annu Department of Botany, M.D.University Rohtak-124001. ABSTRACT Phenolic compounds are a large group of secondary plant metabolites. Fruits are rich with antioxidants that help in lowering incidence of degenerative diseases such as cancer, arthritis, arteriosclerosis, heart disease, inflammation, brain dysfunction and acceleration of the ageing process. Wide adaptation of guava to diverse environmental conditions, along with its unique characteristic of possessing a storage protein. Domestic lemon is generally propagated by grafting, although some are grow readily from seed. Many different phenolic compounds have been identified in guava. The two main subtypes of polyphenols are flavonoids and phenolic acids. The present study reveals the total phenolic content and total protein in guava (Psidium guajava) and lemon (Citrus limon). Key Words: Phenolics, Metabolites, Flavonoid, Total proteins. INTRODUCTION Fruits are major food products and considered as key ingredients in many processed food. Eating fruits and vegetables has long been associated with health benefits. Recent studies indicate that the frequent consumption of fruits is associated with lower risk of stroke and cancer (Beecher, 1999). It is strongly established that fruits are rich source of the antioxidant vitamins β-carotene (provitamin A), α-tocopherol (vitamin E), and ascorbic acid (vitamin C). The high intake of those vitamins is frequently associated with reduced disease risk. For this reason, fruit consumption is considered recently one of the main factors that obviously affect healthy life style beside exercise and genetic impact. Citrus fruits are one of the world’s most important fruit crops, and are known for their nutritive values and special aroma. Citrus is mainly consumed as fresh fruit or juice. Many in-vivo and in-vitro studies have reported Corresponding Author Sharma Asha Email: [email protected]

citrus fruit to be effective against many chronic diseases, such as cancers and vascular diseases (So et al., 1996; Miyagi et al., 2000; Poulose et al., 2006; Vanamala et al., 2006).Citrus is the most produced fruit in the world with over the 116 million tons of production (FAO, 2009). Besides, citrus is an extremely important crop on a worldwide basis, and is grown wherever the climate is suitable. It is widely grown in most areas with suitable climates tropical, subtropical, and borderline subtropical temperate (Kahn et al., 2001). Citrus limon Lemon is cultivated in many countries all over the world. Lemon grow in regions with temperate summers and mild winters, particularly in Mediterranean countries, southern California, and Argentina, whereas limes grow in hot subtropical or tropical regions such as southern Florida, India, Mexico, Egypt, and the West Indies. Lemon (Citrus limon) is the third most important species of citrus after orange and mandarin, with a production total more than 4.4 million tonnes during the 2001/2002 season. Argentina with 1.2 million tonnes is currently the world’s largest producer of lemons (FAO. 2003). In the United States, citrus juice is the most popular type of juice, with annual

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consumption of about 33.6 kg/person/year, or 2.25 times of that of the distant second apple juice. Fresh citrus fruit consumption is about 5.6 kg/person/year (Pollack et al., 2003).The production of citrus juice generates a large quantity of waste including fruit peel ,seeds, high contents of flavonoids, carotenoids, polyphenols and limonoids are left in the fruit peel and pulp (Vikram et al., 2007; Wang et al., 2007). These contents could potentially be developed into value-added products such as food additives and health enhancing drugs. The true lemon tree reaches 10 to 20 ft (3-6 m) in height and usually has sharp thorns on the twigs. The alternate leaves, reddish when young, become dark-green above, light-green below; are oblong, elliptic or longovate, long, finely toothed, with slender wings on the petioles.. The fruit is oval with a nipple-like protuberance at the apex; the peel is usually light-yellow though some lemons are variegated with longitudinal stripes of green and yellow or white; it is aromatic, dotted with oil glands; pulp is pale-yellow, in 8 to 10 segments, juicy, acid. Some fruits are seedless, most have a few seeds, elliptic or ovate, pointed, smooth, white inside. (Morton et al., 1987). According to the classification by Swingle (Swingle WT, 1967) lemons belong to species: Citrus limon (L.) family Rutaceae. Conversely, in the Tanaka system (Tanaka, T. Citologia ,1961) they are divided into several species characterized by botanical variability. The composition of lemon peel oil has been the subject of numerous studies reviewed by Lawrence (Lawrence, 1996). Although most of these studies concerned commercial oils for which neither the species nor the varieties were specified. Lemon is an important medicinal plant of the family Rutaceae. It is cultivated mainly for its alkaloids, which are having anticancer activities and the antibacterial potential in crude extracts of different parts (viz., leaves ,stem, root and flower) of Lemon against clinically significant bacterial strains has been reported (Kawaii et al., 2000).The peel of Citrus fruits is a rich source of flavonoid glycosides, coumarins and volatile oils(Shahnah et al., 2007). Psidium guajava Psidium guajava is a small medicinal tree that is native to South America. Guava is successfully grown in Pakistan, India, Island and Brazil. It is greatly relished for its flavour, delicious taste and high nutrient value. (Gratten and Grieve, 1999), (Niazi et al., 2000) and (Zaka et al., 2008). The Punjab province shares 49.7 thousand hectare in area with a total production of 422.3 thousand tons and 8497 kg yield per hectare. Guava can be grown on a wide variety of soils from heavy clay to light sandy and with a range of pH 4.5 (acidic) to 8.5 (alkaline). Guava is a very productive and highly profitable fruit crop. It is liked by fruit growers due to its wide adaptability and higher returns per unit area.Guava (Psidium guajava) is an important tropical fruit, mostly consumed fresh. The fruit is a berry,

which consists of a fleshy pericarp and seed cavity with fleshy pulp and numerous small seeds. World production of guava was estimated at about 500,000 metric tons. Of the South American countries, Brazil, Colombia, Mexico,and Venezuela produce significant quantities of guava. The guava industry provides a variety of processed products: beverages, syrup, ice cream, jams, jellies, cheese, toffee, juice, wine, and dehydrated and canned products (Adsule RN et al., 1995). It is popularly known as guava and has been used traditionally as a medicinal plant throughout the world for a number of ailments. There are two most common varieties of guava: the red (P. guajava var. pomifera) and the white (P. guajava var. pyrifera) Brazil is among the world’s top producers of guava and most of the country’s production is destined for the food industry to produce candies, juices, jams and frozen pulp. Guava is a good source of minerals like iron, calcium, and phosphorus as well as many vitamins like ascorbic acid, pantothenic acid, vitamin A, and niacin (Paull and Goo, 1983). It is also reported to be rich in antioxidants like phenolics and carotene (Luximon-Ramma et al., 2003). Psidium guajava is inexpensive, easily available and widely popular fruit. The variety difference, degree of ripeness in guava can affect its nutritional and non-nutrient bioactive. It is rich in ascorbic acid, dietary fibers and phenolic content (Jimenez-Escrig et al., 2001). They are rich source of natural antioxidants mainly due to their phenolic constituents. Recent study also confirmed substantially higher amount of phenolic compounds and ascorbic acids in the peel than in pulp for most of the fruits. Phenolic compounds play an important role as a protector for inner material from insects and microorganisms. They also contributed for the colour and appearance of the fruits. With an increased pressure on finding alternative ways to utilise food waste together with scarce information on individual bioactive phenolic compounds and huge research interest on antioxidant activity are expected to widen the opportunities for commercial exploitation. The better varieties are sweet while others may be astringent. On average, the fruit contains 74–87% moisture, 13–26% dry matter, 0.5–1% ash, 0.4–0.7% fat and 0.8–1.5% protein (Chin and Yong, 1980). It is rich in ascorbic acid (vitamin C), at levels far higher than most imported and local fruits. The fruit, in particular the pink flesh cultivar, has a fair amount of vitamin A (beta-carotene). The international guava market is dominated mainly by white guava, while red guava dominates the Brazilian market.Guava (Psidium guajava L.) also known locally as jambu batu, is grown commercially and in many home gardens in Malaysia.Brazilian guava has several carotenoids such as phytofluene, b-carotene, bcryptoxanthin,g-carotene, lycopene, rubixanthin, cryptoflavin, lutein and neochrome (Mercadante et al., 1999).(Setiawan et al., 2001) reported that Indonesian guava is an excellent source of provitamin a carotenoids.

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Major biochemical changes occurring during ripening are:  Seed maturation (in pollinated fruits)  Peel colour change  Abscission (detachment of fruits from parent plant)  A dramatic rise in respiration rate  Increased ethylene production  Increased peel permeability  Softening of pulp tissue  Decrease in starch: sugar ratio  Production of volatiles (flavor and smell) Fruit ripening is a highly coordinated, genetically programmed and irreversible process which involves a series of physiological, biochemical, and organoleptic changes allowing for the development of an edible ripe fruit (Prasanna et al., 2007). The typical course of fruit development involves expansion, sweetening and increasing pigmentation (Coombe, 1976). The appearance, texture and taste of the fruit are all of high importance. These properties involve attaining a suitable composition of sugars, organic acids, amino acids and carotenoids. Citrus is a widely grown fruit crops, which exhibits non-climacteric ripening behaviour. Its fruit contains a juicy pulp made of vesicles within segments (Iglesias et al., 2007). Citrus fruit provides a convenient vehicle to study gene regulation during nonclimacteric fruit development and ripening. Citrus fruits accumulate most of their sucrose in the juice cells (Soule et al., 1986). As the major acid present in citrus fruit, citric acid contributes > 90% of the total organic acid content. MATERIAL & METHOD For Total Phenolic Content The total Phenolic content in Lemon(Citrus limon) and Guava(Psidium guajava) were determined by the method of Malik, C.P. and Singh,1980 by using Gallic acid as standard. Chemicals (1) 80% Ethanol. (2) Folin Ciocalteau Reagent. (3) 20% Sodium carbonate. (4) Gallic acid as Standard. Requirements Test tubes, beakers-100ml, 250 ml, 500ml, glass rod, spatula, filter paper, muslin cloth, pestle and mortar, petridishes, oven, weighing machine, vacuum evaporator, UV-spectrophotometer, centrifuge machine. Method 1. The samples of Lemon(Citrus limon) and Guava(Psidium guajava) were weighed exactly 0.5g. 2. The samples were grinded with a pestle and mortar in 10 times volume of 80% ethanol.

3. The homogenate was centrifuged at 10,000 rpm for 20 min. The supernatant was separated and collected. 4. Then we re- extracted the residue with five times the value of 80% ethanol and again centrifuged it. The supernatant was again pooled. 5. Then evaporated the supernatant to dryness by keeping it in an open petriplates. 6. Dissolved the residue in 5ml of distilled water. 7. Then pipette out different amounts of dissolved residue from 0.2 to 1.8 ml into different test tubes. 8. Then the volume in each test tube was made to 3 ml by adding distilled water into them. 9. Then we added 0.5 ml of Folin- ciocalteau reagent in all test tubes. 10. After three min., 2 ml of 20% Sodium carbonate solution was added to each test tube. 11. Mixed thoroughly and test tubes were placed in boiling water for exactly one min, cooled and measured absorbance at 650 nm against a blank reagent. 12. Standard curve was prepared using different concentrations of Gallic acid. Preparation of standard Gallic acid curve To prepare a standard curve, 0.5mL aliquots of 0.2, 0.6, 1.0, 1.4 and 1.8μg/mL methanolic gallic acid solutions were mixed with 2.5 mL Folin–Ciocalteu reagent (diluted ten-fold) and 2.5 mL (75 g/L) sodium carbonate. After incubation at 25°C for 30 min, the quantitative phenolic estimation was performed at 650nm against reagent blank by UV Spectrophotometer. The standard curve was constructed by putting the value of absorbance vs. concentration. For Total Protein determination The soluble protein in various fruits was determined by the method of Lowery et al 1951 using Bovin Serum Albumin as standard protein. This method is based on the protein reacts with folin ciocalteau’s reagent to give a coloured complex. The colour so formed was due to the reaction of alkaline copper with the protein and reduction of Phosphomolybdate by tyrosine and tryptophan residues present in protein. Chemicals Reagent A: 2% sodium carbonate in 0.1N NaOH (2gm sodium carbonate + 100ml distilled water + 0.4gm NaOH). Reagent B1: 1% Copper sulphate in distilled water, stored at 4˚C (0.1gm CuSO4+10ml H2O). Reagent B2: 2% sodium potassium tartarate in distilled water, stored at 4˚C (.2gm C4H4KNaO6.4H20+10ml distilled water). Reagent B: B1+B2 in 1:1 ratio, prepared freshly at time of use. Reagent C: Reagent A+ Reagent B in 50:1 ratio always prepared freshly at time of use.

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Reagent D: Folin- Ciocalteau’s reagent (2N) and distilled water in 1:1 ratio. It was also prepared fresh. Tris-Cl buffer: Prepared 0.5 M buffer and adjusted the Ph at 7.5. Requirements Test tubes, beaker, pestle and mortar, pipette, distilled water, centrifuge machine, UVspectrophotometer.

including the blank. Mixed it well and allowed it to stand for 10 min. 4. Then added 0.5 ml of reagent D. Mixed it well and incubated at room temperature in the dark for 30 min. Blue colour developed. 5. Took the readings with UV-spectrophotometer at 750 nm.

Extraction method The sample of Lemon were weighed 500mg and were grinned separately with pestle and mortar in 10 ml of buffer. Centrifuged and used the supernatant for protein estimation.

Preparation of standard BSA curve Different concentrations such as 0.2, 0.4, 0.6, 0.8 and 1 ml were taken in 0.5 ml distilled water from stock solutions of BSA (100mg/ml) and the above procedure was followed for protein determination. A curve was plotted between A 750 and different concentrations of BSA solution.

Estimation of Proteins 1. Pipette out 0.2, 0.4, 0.6, 0.8 and 1 ml of the obtained supernatant into series of test tube. 2. The volume in all test tubes was made 1 ml. Another test tube with 1 ml of distilled water served as blank. 3. Now added 5 ml of the reagent C to each test tube

RESULT The present study instigates the amount of total phenolic content and protein content in Lemon and Guava. Gallic acid was used as standard for phenolics. Phenolic compounds are very important constituents because their hydroxyl group confer scavenging ability.

Table 1. Phenols in Lemon were determined at λ of 650nm where we obtained maximum amount of phenol in Lemon at 0.631nm λ which is 0.98µg/ml in concentration. The total phenolic contents are expressed as mg of gallic acid equivalent (GAE) per gm of extract. Total Phenol in Lemon Absorbance (λmax=650nm) Concentration obtained (µg/ml) 0.362 0.55 0.429 0.66 0.486 0.74 0.548 0.83 0.631 0.98 Table 2. Phenols in Guava were maximum at λ of 0.948nm where the concentration of phenol in Guava was 1.45µg/ml, where as the minimum concentration was obtained at λ of 0.543 which was 0.82µg/ml. Total Phenol in Guava Absorbance (λmax=650nm) Concentration obtained(µg/ml) 0.543 0.82 0.615 0.94 0.740 1.11 0.834 1.28 0.948 1.45 Table 3. The standard curve for Protein was obtained at λ 750nm. The curve was constructed by putting the value of absorbance vs. concentration. The standard curve equation: y=1.7729x+0.0419, R 2=0.9977, where y is absorbance at 750 nm and x is total protein content obtained. Standard calibration curve for Protein Concentration(µg/ml) Absorbance(λmax=750nm) 0 0 0.2 0.45 0.4 0.75 0.6 1.1 0.8 1.47 1 1.8

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Table 4. Total protein in Lemon was obtained at an absorbance of 750 nm. The maximum amount of protein in Lemon was obtained at an absorbance of 0.384 nm where the concentration was 0.20µg/ml. Total Protein in Lemon Absorbance(λmax=750nm) Concentration obtained (µg/ml) 0.068 0.02 0.165 0.09 0.192 0.12 0.283 0.15 0.384 0.20 Table 5. Total protein in Guava was maximum at an absorbance of 1.823nm, where concentration of protein was 1.02µg/ml. The minimum amount of protein which was 0.26µg/ml was obtained at an absorbance of 0.500nm. Total Protein in Guava Absorbance(λmax=750nm) Concentration obtained(µg/ml) 0.500 0.26 0.815 0.44 1.210 0.66 1.531 0.84 1.823 1.02 Fig 1. Standard curve for Gallic acid

Fig. 2 Total Phenolic content in Lemon

Fig 3. Total Phenolic content in Guava

Fig 4. Standard curve for BSA (Bovin Serum Albumin).

Fig. 5 Total Protein in Lemon

Fig. 6 Total Protein in Guava

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DISCUSSION In our recent experiments we estimated the total phenolic content and total protein in two fruits, Psidium guajava and Citrus limon by using Folin-ciocalteau’s method and Lowry method respectively. We determined that Phenolic content in Guava is higher than Lemon. This is due to the fact that Psidium guajava has very high level of Antioxidants and the high level of Antioxidant contributes to the high level of Phenolic content in Guava. Phenolic compounds are very important plant constituents because their hydroxyl groups confer scavenging ability (Hatano T, Edamatsu R,1989). Plant materials rich in phenolics are increasingly being used in the food industry because they retard oxidative degradation of lipids and improve the quality and nutritional value of food (Pan YK et al., 2008). Antioxidant activity of extracts is strongly dependent on the solvent due to the different antioxidant potentials of compounds with different polarity (Kähkönen MP et al., 1999). In this research, amongst the two extracts, guava exhibited the high total phenolic content of phenolic compound. For Total Phenolic content Total phenolic content in guava and lemon was determined by the method of Malik, C.P. and Singh, 1980 by using Gallic acid as standard in which Folin Ciocalteau reagent was used. Standard of Gallic acid was used to identify peaks by UV spectra to build calibration curves for quantification of total phenolic content. The phenolic content were quantified at 650nm, the quantity was determined by the calibration curve. The total phenolic content in guava and lemon were determined by standard calibration curve of gallic acid which were different at different absorbance of UV spectra. The phenolic content of guava and lemon were increased with increase in absorbance. Lemon(Citrus limon) The total phenolic content in lemon was determined by equation obtained from the standard curve for gallic acid which was y= 0.6632x -0.011, R2=0.9969 and in this equation y was absorbance and x was concentration obtained. The minimum concentration of phenolic content in lemon (0.55µg/ml ) was determined at absorbance 0.362 nm and maximum concentration of phenolic content in lemon (0.98 µg/ml) was determined at absorbance 0.631nm.It was observed that the concentration was increased with absorbance. Guava (Psidium guajava) According to our standard equation: y= 0.6632x 0.011, R2=0.9969, for Gallic acid, the maximum amount of phenol in Guava was obtained at an absorbance of 0.948nm where the content of phenol was 1.45µg/ml. And the minimum content of phenol was 0.82 µg/ml at 0.543nm. Thus by studying the obtained data we can

conclude that as the concentration of phenolic content increases with increase in the absorbance. Total Protein content Total protein content in guava and lemon was determined by the method of Lowry et al; 1951 by using BSA (Bovin Serum Albumin).BSA was used to identify peaks by UV spectra to build calibration curves for quantification. The protein content were quantified at 750nm, the quantity was determined by the calibration curve. This method was based on the protein reacts with Folin reagent to give a coloured complex. The colour so formed was due to the reaction of alkaline copper with the protein and reduction of Phosphomolybdate by tyrosine and tryptophan residues present in protein. Lemon (Citrus limon) The total protein content in lemon was determined by Lowry method for protein estimation. Total protein was determined by the equation obtained by the standard curve of BSA (Bovine Serum Albumin) which was y=1.772x +0.041 where y was the absorbance and x was the concentration. The total protein content was also increased with increase in absorbance and was observed that the minimum concentration (0.02 µg/ml) was obtained at absorbance 0.068nm and the maximum concentration (0.20 µg/ml) was obtained at absorbance 0.384nm.As compared to guava lemon has low protein content. Guava (Psidium guajava) Guava has more protein content as compared to lemon which was determined by Lowry method. According to standard equation: y=1.7729 xs + 0.0419, R2= 0.9977, the minimum protein concentration (0.26 µg/ml) was obtained at absorbance 0.500nm and the maximum concentration (1.02 µg/ml) was obtained at absorbance 1.823nm and the concentration increased with increase in absorbance. It was observed in study that guava has higher protein content than lemon. DISCUSSION AND CONCLUSION In this study the total phenolic content and total protein content was determined by using gallic acid and BSA (Bovin Serum Albumin) as standard. The total phenolic and protein was determined in guava and lemon. Phenolic compounds, including flavonoids, anthocyanins and tannins are the main group of antioxidant phytochemicals and have deeply value due to their biological and free radical scavenging activities. It was noticed that the highest values of antioxidant activity were found in fruits with a high phenolic compound concentration confirming the results reported by that fruits characterized by a low phenolic compound concentration expressed a low antioxidant activity. Phenolic compounds mainly responsible factor for the high antioxidant activity . Fruits are rich with antioxidants that help in lowering

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incidence of degenerative diseases such as cancer, arthritis, arteriosclerosis, heart disease, inflammation, brain dysfunction and acceleration of the ageing process. Besides the protection that phenolic compounds provide to plants, they may also have beneficial preventive effects on animals their intake and the prevention of certain major chronic diseases, such as cardiovascular disease or certain kinds of cancer. Standard of Gallic acid was used to identify peaks by UV spectra to build calibration curves for quantification of total phenolic content.The phenolic content were quantified at 650nm,the quantity was determined by the calibration curve. Phenolic compounds

mainly responsible factor for the high antioxidant activity in guava. A strong positive correlation was found between antioxidant activity and phenolic compounds. It was observed that guava has more protein than lemon. For protein determination BSA (Bovine Serum Albumin) was used as standard. The total protein content was determined by the Lowry method of protein estimation in which reagent Follin was used which give a coloured complex. Peel of guava has more protein as compared to lemon and most of the protein present in the form of enzymes, as protein suddenly starts degrading when ripening of fruit starts.

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