antioxidant defense system. Consequently, in this 2.2 Extraction and antioxidant activity analysis study aim to evaluate the potential of extraction and Fresh and dry stem of O. cochenillifera was antioxidant activity of fresh and dry extracted by digestion method. Antioxidant activity cochenillifera. was determined by DPPH+ test method [9]. Solution

preparation process following: weighing solution of EVALUATION OF EXTRACTION AND ANTIOXIDANT 2. MATERIALS AND METHODS 2,2-diphenyl-1-picrylhyclrazyl (DPPH) to 0.0083 g, ACTIVITY OF FRESH AND DRY OPUNTIA COCHENILLIFERA then place into the beaker. 2.1 Sample preparations

1 Opuntia cochenillifera (Fig. 1) was collected from the Natchaya Lamnoi , ABSTRACT: Nowadays, research on phytochemicals and their Maejo University campus, Chiang Mai, Thailand. Rameshprabu Ramaraj2, effects on human health has been increased. In particular, research has 1 Sawitree Tipnee focused on the search for antioxidants, hypoglycemic agents, and 1, Yuwalee Unpaprom * anticancer agents from vegetable, fruit, tea, spice and medical herbs.

1 Opuntia cochenillifera is flowering belonging to the Cactaceae Program in Biotechnology, Faculty family. Traditionally, the O. cochenillifera have been used for both of Science, Maejo University, Chiang medicinal and edible purposes used at tropical/subtropical regions. Mai, Thailand Therefore, the objectives of this study were to examine the extraction

2 School of Renewable Energy, Maejo yield and antioxidant properties of stem of O. cochenillifera. Fresh and University, Chiang Mai dry opuntia stems were extracted by digestion method using ethanol 1 School of Renewable Energy, Maejo 50% and 95% with 4hrs extraction duration. Extraction from the fresh stem using ethanol 50% was achieved high yield (i.e. 2.9897g) University, Chiang Mai 50290, Thailand. compared to others. Finally, the extracts were investigated for the Fig. 3 Sample preparation for microplate reader antioxidant activity using DPPH. Consequently, this study results show * Corresponding author e-mail: that the tested O. cochenillifera could be considered as a natural Then dissolved with 99.9% ethanol and adjusted [email protected] ; antioxidant source. to 100 ml volume in a volumetric flask DPPH solution [email protected] Keyword: Opuntia cochenillifera, Extraction, Antioxidant activity, was diluted with 10 ml DPPH pipette, dissolved with Natural antioxidant 99.9% ethanol in 100 ml volume volumetric flasks, and then sample analyzing by microplate reader

procedure presented in Fig. 3 and 4. Although in some countries different parts of the plant 1. INTRODUCTION Fig.1 Opuntia cochenillifera Phytochemicals are found in abundance at are utilized in the food and cosmetic industry [4]. different levels in many . It is important to O. cochenillifera contains flat stems, curved top. The The plants were washed and known amount was measure the total phenolic compounds correctly in base is round, spiny, thorny and plant is green color, dehydrated. The 20-grams sample was cut into small such medicinal plants, the better to assess their when the tree is brown, a single, small, succulent can pieces (Fig. 2) and then stored at − 20 °C until antioxidant capacity. There is ample evidence that be seen only the new branch. The leaves will fall out, extraction. Each 20 grams of fresh and dry material reactive oxygen species generated in the human body single flowers, reddish orange, a single round effect, were blended using electrical blender and then mixed can cause oxidative damage associated with many soft texture, early on, and then reddish orange, scales well with 50 ml of 95% ethanol. The extracts were degenerative diseases such as atherosclerosis, like leaves around the fruit. The physiological role of moved to a flask, subsequently 150 ml of 95% ethanol coronary heart diseases, aging, and cancer [1]. Due to the plant mucilage is to regulate the cellular water was added, and the mixture was kept in water bath at Fig. 4 Sample analyzing by microplate reader this troubling correlation, there is currently an upsurge content during prolonged drought and to regulate the 50oC temperature for 4 hrs digestion. The sample was of interest in phytochemicals as potential new sources calcium fluxes of the plant [6]. O. cochenillifera manually shacked. The extracts were prepared from all the of natural antioxidants. Natural antioxidants have cladodes also represent a source of phytochemicals, gained considerable interest in recent years for their such as phenolics acids and flavonoids. It has been processed products for determination of % DPPH role in preventing the auto oxidation of fats, oils and found that fruit juice contains a rich variety of natural radical scavenging assay. The scavenging effect for fat containing food products. The goal is to use them antioxidants, many phenol compounds, ascorbic acid, DPPH radical was monitored according to the in foods and pharmaceutical preparations to replace betalains, betacyanins, and a flavonoid fraction that procedure described in Seneviratne et al. [9], the synthetic antioxidants [2]. Most antioxidants isolated consists mainly of rutin and isorhamnetin derivatives absorbance at 517 nm was measured by Microplate from plants are polyphenols. However, reliable [7]. Reader. scientific information on the antioxidant properties of A number of studies have revealed a positive various wild plants is still rather scarce, particularly correlation between a diet rich in plant-based foods 2.3 Statistical analyses for those plants that are less widely used due to their and reduced risk of diseases associated with oxidative The data obtained was expressed as mean ± endemic nature. stress such as cancer and cardiovascular and standard deviation (SD). The statistical analyses were Opuntia cochenillifera was freshly consumed neurodegenerative diseases. Opuntia fruit, and stem performed using one-way analysis of variance and traditionally used in folk medicine for health extract reportedly protected erythrocytes against lipid (ANOVA), IBM SPSS statistical package version 22.0 benefits by the local people [3]. According to previous oxidation induced in vitro by ethanol [8]. The (IBM Corp., New York, USA). The statistical significances were reached when p < 0.05. reports, family Cactaceae is reported to contain about normalization of scavenging activity by prickly pear 130 genera and nearly 1500 all well adapted to arid juice supplement could be due to the natural Fig. 2 Sample preparations (A-F) lands and to a diversity of climates and are naturalized antioxidants, which could modulate the intrinsic in several areas all over the world, including Thailand. imbalance between oxidant species and the

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372 antioxidant defense system. Consequently, in this 2.2 Extraction and antioxidant activity analysis study aim to evaluate the potential of extraction and Fresh and dry stem of O. cochenillifera was antioxidant activity of fresh and dry Opuntia extracted by digestion method. Antioxidant activity cochenillifera. was determined by DPPH+ test method [9]. Solution preparation process following: weighing solution of 2. MATERIALS AND METHODS 2,2-diphenyl-1-picrylhyclrazyl (DPPH) to 0.0083 g, then place into the beaker. 2.1 Sample preparations Opuntia cochenillifera (Fig. 1) was collected from the Maejo University campus, Chiang Mai, Thailand.

Fig. 3 Sample preparation for microplate reader

Then dissolved with 99.9% ethanol and adjusted to 100 ml volume in a volumetric flask DPPH solution was diluted with 10 ml DPPH pipette, dissolved with 99.9% ethanol in 100 ml volume volumetric flasks, and then sample analyzing by microplate reader procedure presented in Fig. 3 and 4. Fig.1 Opuntia cochenillifera

The plants were washed and known amount was dehydrated. The 20-grams sample was cut into small pieces (Fig. 2) and then stored at − 20 °C until extraction. Each 20 grams of fresh and dry material were blended using electrical blender and then mixed well with 50 ml of 95% ethanol. The extracts were moved to a flask, subsequently 150 ml of 95% ethanol was added, and the mixture was kept in water bath at Fig. 4 Sample analyzing by microplate reader o 50 C temperature for 4 hrs digestion. The sample was manually shacked. The extracts were prepared from all the processed products for determination of % DPPH radical scavenging assay. The scavenging effect for DPPH radical was monitored according to the procedure described in Seneviratne et al. [9], the absorbance at 517 nm was measured by Microplate Reader.

2.3 Statistical analyses The data obtained was expressed as mean ± standard deviation (SD). The statistical analyses were performed using one-way analysis of variance (ANOVA), IBM SPSS statistical package version 22.0 (IBM Corp., New York, USA). The statistical significances were reached when p < 0.05.

Fig. 2 Sample preparations (A-F)

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3. RESULTS AND DISCUSSION of antioxidants are very important to good health, because if free radicals are left unchallenged, they can 3.1 Characteristics Opuntia cochenillifera and cause a wide range of illnesses and chronic diseases. extraction yield So antioxidants are supposed to beneficially interfere The O . cochenillifera is an introduced with diseases-related oxidative stress, however, the ornamental plant in several countries. It is a tropical interplay of endogenous and exogenous antioxidants and subtropical plant able to grow in arid climates, with the overall redox system is far from clear [9,14]. growing in Thailand as well. It has recently received Therefore, in this study antioxidant assays were an ever increasing attention from researchers carried out to determine the antioxidant potentials of worldwide for its multivalent pharmaceutical and the extracts on fresh and dry stem of O. cochenillifera. cosmetical potential [10]. The stem/ cladodes ware The DPPH is a stable free radical with a mainly rich in carbohydrate containing polymers, maximum absorbance at 517 nm it can readily known as mucilage, made up of several sugar residues undergo scavenging by an antioxidant, and gets such as arabinose, galactose, rhamnose, xylose and converted in to 1,1-diphenyl-2-picrylhydrazine [12]. galacturonic acid. It has been previously reported that The degree of discoloration indicates the scavenging polysaccharides from Opuntia plants can be used as potentials of the antioxidant extract. In the present mucoprotective agents due to their capability to form study, the free radical scavenging potentials of the a molecular network and to retain huge amount of fresh and dry O. cochenillifera extracts a were tested. water. Table 1 Antioxidant activity of DPPH radical Scientific classification: scavenging assay and inhibition Kingdom: Plantae Sample DPPH Inhibition (%) Clade: Angiosperms Positive 0.3202±0.0149 - Clade: Negative 0.0682±0.0079 - Order: Fresh sample 1 0.2436±0.0081 3.3201±3.2104 Family: Cactaceae Dry sample 2 0.1909±0.0054 24.2593± 2.1540 Genus: Opuntia Species: O. cochenillifera The concentration of extract necessary to decrease the initial concentration of DPPH by 50% Several researchers have studied this whole (IC50) under the specified experimental condition was reaction and found out that we could extract two water calculated and the results of antioxidant activity of soluble polymers which were the mucilage and pectin DPPH radical scavenging assay were shown in [11,12]. Table 1. Among the extracts, dry sample of The mucilage is the result of the polymerization O. cochenillifera showed significantly higher DPPH of more monosaccharides whose majority is scavenging activity and inhibition higher than fresh associated with uronic acids. This complex sample. hydrocolloid which is naturally abundant in certain plant seeds and plants is a viscous substance 4. CONCLUSIONS responsible for retaining water [13,14]. The mucilage In the present study, antioxidant activities of gums were used in food and medicine because of their ethanolic extracts of fresh and dry stem of functional properties such as gelling, thickening and O. cochenillifera were investigated. All the extracts emulsifying and their ability to prevent and treat were found to possess radical scavenging and certain diseases. The two types extraction process antioxidant activities, as determined by scavenging (Fresh and dry stem) with digestion method. The effect on the DPPH free radical and inhibition obtained results (Table 1) showed that the extraction properties. Extraction by digestion method was yield of fresh stem was 0.2805 g and dry stem was carried. This study results suggested that dry sample 1.6320 g. The fresh stem extraction yield was lower could be proper chance for extraction yield and than the dry stem yield. In most cases, extraction inhibitory activities. yields obtained from dry material were higher than the yields from fresh material. This is not surprising 5. REFFERANCE considering the lower content of water in dry plant [1] Uttara B. Singh A.V. Zamboni P. Mahajan R. material. Oxidative Stress and Neurodegenerative Diseases: A Review of Upstream and Downstream 3.2 Antioxidant activity Antioxidant Therapeutic Options. Current Antioxidants come up frequently in discussions Neuropharmacology, 2009; 7(1): 65-74. about good health and preventing diseases. These [2] Taghvaei M. Jafari S.M. Application and stability powerful substances, which mostly come from the of natural antioxidants in edible oils in order to fresh fruits and vegetables we eat, prohibit, the substitute synthetic additives. Journal of Food oxidation of other molecules in the body. The benefits Science and Technology, 2015; 52(3): 1272-1282.

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3. RESULTS AND DISCUSSION of antioxidants are very important to good health, [3] du Toit A. de W it M . Osthoff G . H ugo A. [9] Mangge H. Antioxidants, inflammation and because if free radicals are left unchallenged, they can Antioxidant properties of fresh and processed cardiovascular disease. World Journal of 3.1 Characteristics Opuntia cochenillifera and cause a wide range of illnesses and chronic diseases. cactus pear cladodes from selected Opuntia ficus Cardiology, 2014; 6: 462-477. extraction yield So antioxidants are supposed to beneficially interfere indica and O. robusta cultivars. South African [10] Coetzer G.M. Fouché H.J. Fruit yield and quality of The O . cochenillifera is an introduced with diseases-related oxidative stress, however, the Journal of Botany, 2018; 118, 44-51. cactus pear (Opuntia spp.) cultivars in the Central ornamental plant in several countries. It is a tropical interplay of endogenous and exogenous antioxidants [4] Lekameera R. Vijayabaskar P. Somasundaram S.T. Free State, South Africa. Acta Horticulturae, 2015; and subtropical plant able to grow in arid climates, with the overall redox system is far from clear [9,14]. Evaluating antioxidant property of brown alga 89-96. growing in Thailand as well. It has recently received Therefore, in this study antioxidant assays were Colpomenia sinuosa (Derb. Et sol). African Journal [11] Joubert E. Processing of the fruit of five prickly an ever increasing attention from researchers carried out to determine the antioxidant potentials of of Food Science, 2008; 2: 126-130. pear cultivars grown in South Africa. International Journal of Food Science and Technology, 1993; 28: worldwide for its multivalent pharmaceutical and the extracts on fresh and dry stem of O. cochenillifera. [5] Oktay M. Gûlcin İ. Kûfrevioglu Ö.İ. Determination of in vitro antioxidant activity of fennel 377-387 cosmetical potential [10]. The stem/ cladodes ware The DPPH is a stable free radical with a (Foeniculum vulgare) seed extracts. Leben Wissen [12] Sowndhararajan K. Kang S.C. Free radical mainly rich in carbohydrate containing polymers, maximum absorbance at 517 nm it can readily Technology, 2003; 36: 263-271 scavenging activity from different extracts of leaves known as mucilage, made up of several sugar residues undergo scavenging by an antioxidant, and gets [6] Osuna-Martínez U, Reyes-Esparza J, Rodríguez- of Bauhinia vahlii Wight & Arn. Saudi Journal of such as arabinose, galactose, rhamnose, xylose and converted in to 1,1-diphenyl-2-picrylhydrazine [12]. Fragoso L (2014) Cactus (Opuntia ficus-indica): A Biological Sciences, 2013; 20(4): 319-325. galacturonic acid. It has been previously reported that The degree of discoloration indicates the scavenging Review on its Antioxidants Properties and Potential [13] Medina-Torres L. Vernon-Carter E.J. Gallegos- polysaccharides from Opuntia plants can be used as potentials of the antioxidant extract. In the present Pharmacological Use in Chronic Diseases. Natural Infante J.A. Rocha-Guzman N.E. Herrera-Valencia mucoprotective agents due to their capability to form study, the free radical scavenging potentials of the Products Chemistry and Research, 2:153. E.E. Calderas F. Jiménez-Alvarado R. Study of the a molecular network and to retain huge amount of fresh and dry O. cochenillifera extracts a were tested. [7] Morales P. Ramírez-Moreno E. Sanchez-Mata M. antioxidant properties of extracts obtained from water. de C. Carvalho A.M. Ferreira, I.C.F.R. Nutritional nopal cactus (Opuntia ficusindica) cladodes after Table 1 Antioxidant activity of DPPH radical and antioxidant properties of pulp and seeds of two convective drying. Journal of the Science of Food Scientific classification: scavenging assay and inhibition xoconostle cultivars (Opuntia joconostle F.A.C. and Agriculture, 2011; 91: 1001-1005. Kingdom: Plantae Sample DPPH Inhibition (%) Weber ex Diguet and Opuntia matudae Scheinvar) [14] Liguori G. Inglese P. Sortino G. Inglese G. Dry Clade: Angiosperms Positive 0.3202±0.0149 - of high consumption in Mexico. Food Research matter accumulation and seasonal partitioning in Clade: Eudicots Negative 0.0682±0.0079 - International, 2012; 46:279-285. mature Opuntia ficusindica (L.) Order: Caryophyllales Fresh sample 1 0.2436±0.0081 3.3201±3.2104 [8] Guevara J.C. Yahia E.M. Brito de la Fuente E. Mill. fruiting trees. Italian Journal of Agronomy, 2014; 9: 44-47. Family: Cactaceae Dry sample 2 0.1909±0.0054 24.2593± 2.1540 Modified atmosphere packaging of prickly pear cactus stems (Opuntia spp.). LWT - Food Science Genus: Opuntia and Technology, 2001; 34: 445-451. Species: O. cochenillifera The concentration of extract necessary to

decrease the initial concentration of DPPH by 50%

Several researchers have studied this whole (IC50) under the specified experimental condition was Under graduate student Dr. Rameshprabu Ramaraj, reaction and found out that we could extract two water calculated and the results of antioxidant activity of soluble polymers which were the mucilage and pectin Program in Biotechnology, M.Eng., M.Sc., M.Phil., DPPH radical scavenging assay were shown in Maejo University. Ph.D., Lecturer, School of [11,12]. Table 1. Among the extracts, dry sample of The mucilage is the result of the polymerization Renewable Energy, Maejo O. cochenillifera showed significantly higher DPPH University, of more monosaccharides whose majority is scavenging activity and inhibition higher than fresh Professional in scientific and associated with uronic acids. This complex sample. engineering field including hydrocolloid which is naturally abundant in certain biology (animal, plant & plant seeds and cactus plants is a viscous substance Natchaya Lamnoi Rameshprabu 4. CONCLUSIONS Ramaraj microbes), sustainable responsible for retaining water [13,14]. The mucilage In the present study, antioxidant activities of resource engineering, gums were used in food and medicine because of their ethanolic extracts of fresh and dry stem of environment and ecological functional properties such as gelling, thickening and O. cochenillifera were investigated. All the extracts engineering, and biofuels. emulsifying and their ability to prevent and treat were found to possess radical scavenging and certain diseases. The two types extraction process antioxidant activities, as determined by scavenging Scientist at Program in Lecturer, Program in (Fresh and dry stem) with digestion method. The effect on the DPPH free radical and inhibition Biotechnology, Maejo Biotechnology, Maejo obtained results (Table 1) showed that the extraction properties. Extraction by digestion method was University, Chiang Mai. University. yield of fresh stem was 0.2805 g and dry stem was carried. This study results suggested that dry sample Professional in plant Research Interests: plant 1.6320 g. The fresh stem extraction yield was lower could be proper chance for extraction yield and extraction and biotechnology, plant than the dry stem yield. In most cases, extraction inhibitory activities. phytochemical analysis. physiology and biochemistry, and yields obtained from dry material were higher than the sustainable fuels/bioenergy. yields from fresh material. This is not surprising 5. REFFERANCE Sawitree Tipnee Yuwalee Unpaprom considering the lower content of water in dry plant [1] Uttara B. Singh A.V. Zamboni P. Mahajan R. material. Oxidative Stress and Neurodegenerative Diseases: A Review of Upstream and Downstream 3.2 Antioxidant activity Antioxidant Therapeutic Options. Current Antioxidants come up frequently in discussions Neuropharmacology, 2009; 7(1): 65-74. about good health and preventing diseases. These [2] Taghvaei M. Jafari S.M. Application and stability powerful substances, which mostly come from the of natural antioxidants in edible oils in order to fresh fruits and vegetables we eat, prohibit, the substitute synthetic additives. Journal of Food oxidation of other molecules in the body. The benefits Science and Technology, 2015; 52(3): 1272-1282.

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