Antioxidant and Anti Aging Assays of Oryza Sativa Extracts, Vanillin and Coumaric Acid

Ipomoea reniformis Chaos is claimed in Indian traditional medical practice to be useful in the treatment of epilepsy and neurological disorders. In the present study, pretreatment effect of methanolic extract of Ipomoea reniformis on epilepsy and psychosis was evaluated in rodents using standard procedures. Besides evaluating epileptic and behavioral parameters, neurotransmitters such as Gamma-Amino Butyric Acid (GABA) in epilepsy and in psychosis dopamine, noradrenaline and serotonin contents in the rodent brain were estimated. The extract pretreatment reduced maximal electro shock; Isoniazid (INH) and Pentylenetetrazole (PTZ) induced seizures and also significantly inhibited the attenuation of brain GABA levels by INH and PTZ in mice. These results suggested that the observed beneficial effect in epilepsy may be by enhancing the GABAergic system. The test drug also inhibited the apomorphine induced climbing and stereotyped behavior and showed significantly reduced levels of brain dopamine, noradrenaline and serotonin which may be due to blocking of central dopaminergic, noradrenergic and serotonergic pathways or by enhancing the GABAergic system. The results obtained in present study suggest that the title plant possesses antiepileptic and antipsychotic activities in rodents.


Introduction
Ipomoea reniformis (IR) also called as merremia emarginata (Burm. f.) is a procumbent herb belonging to the family convolvulaceae. In India, it is commonly known as Undirkana and Mushakparni. The plant is widely distributed in India, Sri Lanka, Philippines, Malaysia, Tropical Africa and mainly grows in rainy and winter season. In India, it is found in Southern part mainly counting Chennai, and some places of Andhra Pradesh [1]. Traditionally, IR has been used to treat diverse clinical conditions ranging from pain; fever to neurological disorders [2]. IR has been claimed to be useful for inflammation, headache, fever, cough, neuralgia, rheumatism and also in liver and kidney diseases [3]. The powder of leaves is used as a snuff during epileptic seizures. Juice acts as purgative and the root is having diuretic, laxative actions and applied in the disease of the eyes and gums [4].
The plant contains various neuroprotective chemical constituents such as caffeic, p-coumaric, ferulic and sinapic acid esters. Petroleum ether extract contains fats and fixed oil while aqueous extract contains amino acids, tannins (condensed and pseudo tannins) and starch [5]. IR has been reported to possess various pharmacological actions, mainly antidiabetic [6], antiinflammatory [7], nephroprotective [8], antibacterial [9], antioxidant and antimicrobial activity [10]. Further, the principle constituents of IR such as sinapic and ferulic acids have exhibited behavioural and pharmacological

Introduction
Skin aging is the natural process due to photo aging by environmental factors such as chronic UV radiation. The repetitive exposure to UV radiation cause accelerated physical changes in the skin and connective tissue through the formation of lipid peroxides, the cell contents and Reactive Oxygen Species (ROS) 1 . It leads to loss of skin elasticity implicated in formation of wrinkle, uneven pigmentation, brown spots, laxity and leathery appearance, solar elastosis, actinic purpura, precancerous lesions, skin cancer, and melanoma [2][3][4] .
During aging process, collagen, elastin, and hyaluronic acid decrease, that causes loss of strength and flexibility in the skin, resulting in visible wrinkles. It is also related to increasing enzymes activity including Journal of Natural Remedies | ISSN: 2320-3358 www.informaticsjournals.com/index.php/jnr | Vol 16 (3) | July 2016 collagenase, elastase and hyaluronidase. Collagenase is known as an enzyme that plays role in the degradation of collagen. Collagen is the main component with percentage of 70-80 % of the total skin weight, the increasing degradation of collagen is significant in the photo aging process 5,6 . Hyaluronan or hyaluronic acid is one of important components of the tissue matrix substance and has a role in the development, growth, and repair of damaged tissue 6 . Meanwhile, elastin play a role in the maintenance of skin elasticity, but elastase can degrade it 7 . Degradation of the Extracellular Matrix (ECM) has been directly linked to skin aging and is correlated with an increase in activity of certain enzymes involved in skin aging 8,9 . Inhibition of these enzymes is crucial in anti aging prevention 10 . It has been reported that skin aging occurs in the presence of cumulative endogenous damage due to Reactive Oxygen Species (ROS) 11 . ROS are defined as oxygen-containing, highly reactive species. ROS are generated constantly during normal cellular metabolism which is essential for biological functions. Excessive ROS causes oxidative stress and damage of biological molecules 12,13 . Previous studies have investigated that continuous ROS exposure can stimulate skin aging through antioxidant system destruction, wrinkle formation, and melanogenesis 12 . ROS are usually eliminated from the body through antioxidant defense system 14 . Thus, maintaining antioxidant homeostasis is an appropriate strategy to prevent skin aging. Antioxidant properties derived from natural sources have been proposed for aging prevention 15 . Bioactive compounds contained in plants such as isoflavones, anthocyanins, and catechins may have promising antioxidant activity against ROS 16 . Oryza sativa L. is one of the most produced and consumed cereals in the world that contain phenolic compounds, tocopherols, tocotrienols, and g-oryzanol. Phenolic acids were identified in the lignin fraction of rice bran (O. sativa) such as caffeic, chlorogenic, p-coumaric, ferulic, gallicacids, p-hydroxybenzoic, protocatechuic, syringic and vanillin [17][18][19] . Vanillin and coumaric acid have antioxidants activity that can inhibit aging processes 20,21 . In the present study, free radical scavenging activity of O. sativa Extract (OSE) and its compounds, vanillin and coumaric acid ( Figure 1) were evaluated as well as inhibitory activities of collagenase, elastase, and hyaluronidase.

Preparation of O. sativa Extracts
The plants of O. sativa were collected from the plantation in Ciherang, Subang, West Java. The plants were identified by herbarium staff, Department of Biology, School of Life Science and Technology, Bandung Institute of Technology, Bandung, West Java, Indonesia. The grain of O. sativa (600 g) were mashed, extracted using distilled ethanol 70% (2,750 mL) by a maceration method. Every 24 h the ethanol was filtered and the wastes were re-macerated in triplicate. The ethanol filtrate collected was condensed using 50°C rota vapor to obtain OSE. The extract in pasta form (5.64 g) was stored at -20 °C, and used for further assay 22

Qualitative Phytochemical Screening Assay
The phytochemical assay was conducted on O. sativa Extracts (OSE) using modified Farnsworth method to qualitatively identify presence of phenols, steroid/ triterpenoids, saponins, tannins, terpenoids, flavonoids, and alkaloids as listed below 23-25 .

Saponin Identification
Approximately 10 mg of sample was put into the test tube with some water and boiled for 5 min. It was shaken vigorously and saponin content was indicated by persistence of froth on the surface 23-25 .

Terpenoid Identification
Around 10 mg of sample was added into a dropping plate, then vanillin and H 2 SO 4 was added to the sample. Terpenoid presence was indicated by the formation of purple color on the mixture 23-25 .

Flavonoid Identification
About 10 mg of sample was added into a test tube, then Mg [Merck EM105815, USA] and HCl 2N was added to the sample. The mixture sample was heated for 5 to 10 min, then filtered after it was cooled down. Subsequently, amyl alcohol was added into the filtrate. The positive reaction was shown by the formation of red or orange color 23-25 .

Alkaloid Identification
The small amount of sample (10 mg) was introduced into a test tube, then 10% ammonia was added into the sample. After chloroform added to the mixture, two layers of liquid was formed and the bottom layer was collected. HCl 1N was added to the liquid, forming two layers. The upper layer collected and added with 1-2 drops of Draggendorf solution. The presence of yellow colour indicated positive result 23-25 .

2,2-Diphenyl-1-picrylhydrazil (DPPH) Assay
The DPPH assay was conducted using the method from Widowati et al., study 26 . The method is based on the reduction of alcoholic DPPH solution in the presence of a hydrogen-donating antioxidant due to the formation of the non-radical 2,2-diphenyl-1-picrylhydrazine (DPPH-H) 27 . Briefly, 50 µL of various level of samples (50-400 µg/mL for extract and 50-400 µM for compounds in the DMSO) were added to each well in a 96-well micro plate. It was then followed by addition of 200 µl of 2,2-Diphenyl-1-picrylhydrazil (DPPH) [Sigma D9132, USA] solution (0.077 mmol/L in methanol) into the well. The mixture was then incubated in the dark for 30 min at room temperature. Afterward, the absorbance was read using a microplate reader (Multiskan™ GO Microplate Spectrophotometer, Thermo Scientific, Waltham, MA, USA) at 517 nm wave length. The radical scavenging activity was measured using the following formula: % Scavenging = (Ac -As) / Ac x 100 Ac = negative control absorbance (without sample). As = sample absorbance.

Ferric Reducing Antioxidant Power (FRAP) Assay
The

Elastase Inhibitory Activity Assay
Elastase inhibitory activity was measured by modified method of Sigma Aldrich and Thring et al 7   tannins and alkaloids. The result of OSE phytochemical screening can be seen in Table 1. Phytochemical screening of OSE aimed to detect presence of phenols, steroids, saponins, flavonoids, and tannins in OSE. Terpenoids and saponins was detected in high content (+++), phenols and triterpenoids were low content (+), while steroids, flavonoids, tannins and alkaloids were not detected (-) ( Table 1).

2,2-Diphenyl-1-picrylhydrazil (DPPH) Assay
DPPH is a reagent for investigating the free radical scavenging activities of compounds. In the DPPH test, the extracts were able to reduce the stable radical DPPH to the yellow coloured diphenylpicrylhydrazine (DPPH-H) 32,33 . The percentage of DPPH scavenging activity of OSE, vanillin, and coumaric acid can be seen in Figure 2 and the median Inhibitory Concentration (IC 50 ) of samples toward DPPH free radical scavenging activity can be seen in Table 2.  Based on Figure 2, DPPH radical scavenging activity was concentration-dependent manner, in which higher concentration increased DPPH scavenging activity. At the highest concentration (400 µg/mL), OSE has the highest DPPH scavenging activity compared to vanillin and coumaric acid (59.62 ± 5.81%, 23.86 ± 0.57%, and 17.39 ± 0.16%, respectively) ( Figure 2). However, IC 50 value of coumaric acid has the lowest value (255.69 µg/ mL) compared to OSE (314.51 µg/mL) and vanillin (283.76 µg/mL) ( Table 2). These results indicate OSE has low DPPH-scavenging activity among treatments.

ABTS -Reducing Activity Assay
ABTS-reducing activity assay measures the relative ability of antioxidant to scavenge the ABTS generated. The ABTS is generated by reacting a strong oxidizing agent (potassium permanganate/potassium persulfate) with the ABTS salt. Reduction of blue-green ABTS radical colored solution by hydrogen-donating antioxidant is measured by the long wave absorption spectrum 34 . The result ABTS-reducing activity of OSE, vanillin, and coumaric acid based on IC 50 value can be seen in Table 3. The antioxidant activity of OSE, vanillin, and coumaric acid were evaluated by ABTS-reducing activity assay. The IC 50 of ABTS-reducing activity of OSE, vanillin, and coumaric acid can be seen in Table 3. OSE has the lowest ABTS-reducing activity as indicated by highest IC 50 (145.67µg/mL) compared to vanillin (4.96µg/mL) and coumaric acid (1.67µg/mL). The result indicated OSE has weak activity antioxidant compared to these compounds.

Ferric Reducing Antioxidant Power (FRAP) Assay
The Ferric Reducing Antioxidant Power (FRAP) method is based on the reduction of a ferroin analog, the Fe 3+ complex of tripyridyltriazine Fe(TPTZ) 3+ to the intensely blue coloured Fe 2+ complex Fe(TPTZ) 2+ by antioxidants in acidic medium. Absorbance of Fe(II) complex at 593 nm produced by antioxidant reduction of corresponding tripyridyltriazine Fe(III) complex 35 . FRAP activity of OSE, vanillin, and coumaric acid can be seen in Figure 3. The result of the present study showed FRAP activity in concentration-dependent manner, in which higher concentration increased FRAP activity (Figure 3). At the highest concentration (100 µg/mL) OSE has a lowest FRAP with value (21.26±0.21 μM Fe(II)/μg) compared to vanillin (35.05±0.80μM Fe(II)/μg) and coumaric acid (48.52±0.24 μM Fe(II)/μg). That indicates OSE has the lowest antioxidant activity in the FRAP assay compared to vanillin and coumaric acid.

Collagenase Inhibitory Activity
A spectrophotometric method was used to collagenase activity assay, and to detect potential collagenase inhibitor 36 . The collagenase inhibitory activity of OSE, vanillin, and coumaric acid can be seen in Figure 4. Collagenase inhibitory activity of OSE, vanillin, and coumaric acid based on IC 50 value can be seen in Table 4.

Elastase Inhibitory Activity
The elastase inhibitory activity of OSE, vanillin and coumaric acid were measured. The percentation elastase inhibitory activity of OSE, vanillin and coumaric acid can be seen in Figure 5. The IC 50 value an elastase inhibitory activity of OSE, vanillin, and coumaric acid based on IC 50 value can be seen in Table 5. Elastase inhibitory activity of OSE, vanillin, and coumaric acid showed the highest inhibition percentage at the highest concentration (36.82 ± 0.40%, 36.28 ± 0.20%, 29.86 ± 0.45% respectively) ( Figure 5). However, vanillin showed the highest activity in elastase inhibition with IC 50 value 14.46 µg/mL. OSE and coumaric acid have IC 50 value 107.62 µg/mL and 25.38µg/mL, respectively ( Table 5). The result showed that OSE posses low elastase inhibition compared to vanillin and coumaric acid.

Hyaluronidase Inhibitory Activity
Hyaluronidase was assayed by a highly sensitive spectrophotometric method, based on precipitation of HA with cetylpyridinium chloride, which is used for high throughout screening for hyaluronidase inhibitors 37 .
Hyaluronidase inhibitory activity can be used to evaluate anti aging activity of OSE, vanillin, coumaric acid. The percentation hyaluronidase inhibition activity of OSE, vanillin, and coumaric acid can be seen in Figure  6. The IC 50 value for hyaluronidase inhibitory activity of OSE, vanillin, and coumaric acid based on IC 50 value can be seen in Table 6. Based on Figure 6, OSE showed the moderate activity with percentage 36.86±2.28% compared to coumaric acid (48.48±0.40%) and vanillin (20.84±4.78%). Coumaric acid has the lowest IC 50 value (8.22 µg/mL) compared to OSE (203.13µg/mL) and vanillin (45.23µg/mL) ( Table 6). These results show OSE has low hyaluronidase inhibitory activity compared to vanillin and coumaric acid.

4.Discussion
Ayurveda, which means science of long life, is a 5,000-year-old system of Indian medicine (1500-1000 BC) designed to promote good health and longevity  45 . O. sativa methanol and isopropanol extracts exhibited better free radical scavenging activity, which is associated with increasing phenolic compounds that increases antioxidant activity 46 . Coumaric acid has been reported to protect against oxidative stress and play a key role as an antioxidant and anti-inflammatory 47,48 . O. sativa oils such as coumaric acid and vanillin exhibited very good oxidative state with range oxidative stability 5.99-7.40 49 .
OSE has the lowest DPPH scavenging activity compared to vanillin and coumaric acid, this data was not validated with previous study showed that O. sativa extract has rich secondary metabolites, especially alkaloids and phenolic acids that have strong antioxidant activity 50 . Phenolic and flavonoid contents are associated with strong antioxidant activity and especially in terms of DPPH and nitric oxide free radical scavenging 51 53,54 . T. aestivum methanol extract showed good radical scavengers with the inhibition of 12% at 1 mL/ mL concentration 55 . T. aestivum has shown potential anti inflammatory, antioxidant, and anti aging properties 56 .
Collagen, elastin, and hyaluronic acid are the skin main components and have an important role in maintaining its structure and hydration 57 . These enzymes cause repetitive collagen fibers breakdown and responsible for structural defect in dermis and wrinkle development 58 . Collagenase and elastase contribute in production and degradation of these fibers, which is also induced by free radical oxidative stress 59 . In the present study, O. sativa had the lowest activity in collagenase inhibition compared to coumaric acid and vanillin. O. sativa has been studied in anti aging treatment. Watersoluble enzymatic extract from O. sativa served as a protective screen against UV-B radiation on cultivated keratinocytes and in vitro reconstructed skin 60 . In the other study, the extract of O. sativa plants callus improve the human-skin barrier function 61 . Mechanisms of anti aging are via up-regulation of collagen sythesis in normal human dermal fibroblast and down-regulation of matrix metalloproteinase 62 . Vanillin content was also reported to inhibit matrix metalloproteinase-9 expression through Journal of Natural Remedies | ISSN: 2320-3358 www.informaticsjournals.com/index.php/jnr | Vol 16 (3) | July 2016 down-regulation of nuclear factor-kB signaling pathway in human hepatocellular carcinoma cells 63 .
Elastin is a fibrose protein that compose 2-4% of the ECM and involved in the hydration of the skin 64 . Elastase is responsible for increased tissue permeability, inflammation progress and delayed wound healing 65 . Elastase is also the key enzyme that attacks all the major connective tissue matrix protein 66 . In elastase assay, OSE showed the lowest activity compared to coumaric acid and vanillin. Low activity of OSE in the present study might be due to low content of phenol and absence of flavonoids in phytochemical screening. Several studies showed that phenol and flavonoid content of O. sativa possess anti-elastase activity 51,67,68 . However, vanillin has the highest elastase inhibitory activity.
Our present study showed that OSE had the lowest hyaluronidase inhibitory activity compared to CA and vanillin. These results might be correlated with an absence of tannin in OSE. Tannin-rich plants have been reported to inhibit hyaluronidase and elastase release from stimulated neutrophils in vitro 69 . Hyaluronidase (Haases) selectively degrade Hyaluronic Acid (HA), which is a megadalton acidic structural polysaccharide found exclusively in the Extracellular Matrix (ECM). Haase inhibitors are thus potent regulators that maintain HA homeostasis and they might serve as antiinflammatory, anti aging, antimicrobial, anticancer and antitoxin and contraceptive agents 70 . Hyaluronidase degrades hyaluronic acid by lowering its viscosity and increasing the permeability 71 .

Conclusion
Vanillin and coumaric acid have higher antioxidant activity through DPPH scavenging, ABTS-reducing activities and FRAP, and anti-aging through inhibitory activity of collagenase, elastase and hyaluronidase than O. sativa extract.