Laboratory Report: Antioxidant Efficacy Of Bryophyllum Pinnatum (Katakataka)
Abstract
Antioxidants are substances that utilize oxygen to decrease or lessen harmful damages to the human body, such as cancer. Known substances of antioxidants are vitamin C, vitamin E, and Beta carotene which are utilized for examining the harmful effects of oxidation.
The antioxidant properties of the leaves of Bryophyllum pinnatum (Katakataka) is determined by DPPH free radical-scavenging activity and the Nitric oxide radical-scavenging activity. In the DPPH, its methanolic solution was incubated on the leaves of Katakataka with a temperature of 25ºC. At the end of the incubation period, the optical density of the samples determined at 517nm solution of ascorbic acid was used.
The test had shown that the amount of standard ascorbic acid solution and aqueous extract of Katakataka in EC50 is in ratio of 11. 25 µg/mL: 116. 25 µg/mL. In the nitric oxide activity, 1 mL of sodium nitroprusside in phosphate-buffered saline is mixed with 3. 0 mL of different concentrations of the extracts dissolved in distilled water and incubated at 25°C for 2 hours and 30 minutes and was treated with Griess’ reagent. Based on the treatment done with regards to the nitric oxide radical-scavenging activity of both solutions it showed that the EC50 of standard ascorbic acid solution in total was 15. 5 µg/mL and 90 µg/mL for aqueous extract of Katakataka. In totality both results state and proves that the Katakataka has antioxidant properties. The research can help patients to lessen the effects of cancer and help future researchers on determining the proper medications to reduce damaging effects of oxidation.
Introduction
Antioxidants are compounds that lessens damage through the utilization of oxygen which are caused by free radicals. They could help in decreasing the risk of cancer and its antioxidant properties shows progression to moderate the movement of age-related macular degeneration. Vitamin C, Vitamin E, and Beta Carotene are known substances that are equipped for examining the harming effects of oxidation. These substances are mostly added to food products like vegetable oils and arranged sustenance to keep or defer their decay from the activity of air. The need for an alternative medicine that is more efficient, cheaper, and has an easier access than chemical induced drugs came up to the use of medicinal plants which can help future Filipinos who suffer from oxidation since it is an accessible home remedy for people who cannot afford expensive medicines
Bryophyllum pinnatum (Katakataka) is an herbal plant grown in tropical countries. The plant has constituents that are active which exhibits potent medicinal properties. The goal of the study is to evaluate and examine the capacity of the antioxidant properties of Katakataka. The antioxidant activity was determined in terms of total phenolics content, total antioxidant activity, and reducing power. The results show that the hydroalcoholic extract of Katakataka has exhibited the antioxidant capacity in a significant way. The plant also possesses properties such as antibacterial, antimicrobial, wound healing, antiseptic, astringent etc. The previous study will help future researchers on having additional knowledge about the antioxidant properties of the Katakataka plant. The researchers will focus on the modification of previous studies which concerns the ailment alongside with the utilization of the Katakataka plant. The purpose of the experiment is to determine the antioxidant properties of Katakataka plants and to use its extracts in the treatment of oxidation without the help of other components.
Materials and methods
Plant material
Fresh leaves were gathered from Sta. Maria, Bulacan and authenticated as K. Pinnata (Lam. ) Pers. (Crassulaceae) by the National Museum of the Philippines. The aqueous extract and concentrate of the plant were set up and prepared by utilizing the cool maceration process. The extract was dried in a vacuum evaporator beneath 40º C and stored in air-tight, amber-colored containers at room temperature.
Animals
Healthy, male albino Wistar rats each weighing 150-200 were utilized for this study. The rats were housed in polypropylene cages and kept up under standard conditions (12 h light and dark cycles, at 25±3º C and 35-60% humidity). Standard pelletized feed and tap water were provided.
In vitro antioxidant examination
For all the in vitro antioxidant models specified below, ascorbic acid was utilized as a kind of perspective standard. The concentrations of ascorbic acid were 10, 20, 30, 40, 50 µg/ml and that concentrate were 50, 100, 150, 200, 250 µg/ml.
DPPH free radical-scavenging activity
To determine the antioxidant activity of the leaf extract, a method supported the reduction of a methanolic solution of the colored radical 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) was used. The methanolic solution of DPPH (0. 1 mM, 1 mL) was incubated with 3 mL of various concentrations of the leaf extract ranging from 50 to 250 mg/mL. Incubation was carried out at room temperature (25ºC) for 30 minutes every concentration, the assay was run in triplicate. At the end of the incubation period, the optical density of each sample determined at 517 nm ascorbic acid solution was used as a standard EC50 values (concentration needed to scavenge 50th of the free radicals) for each ascorbic acid and also the leaf extract was determined. The radical scavenging activity of the tested sample was expressed as an inhibition percentage (IP). DPPH Scavenged (%) = (ADPPH – Atest / ADPPH) x 100where ADPPH is that the absorbance of the 0. 1 mL of DPPH solution and Atest is that the absorbance in the presence of the extract or ascorbic acid.
Nitric oxide radical-scavenging activity using the Griess Illosvay reaction
Free radical-scavenging activity was evaluated by knowing the inhibition of the generation of nitric oxide from sodium Nitroprusside. An aqueous solution of sodium nitroprusside at physiological pH spontaneously generates nitric oxide, that interacts with atomic number 8 (oxygen) to provide nitrite ions. The nitrite ions thus produced can be quantified using their reaction with Griess reagent that results in formation of a chromophore, the concentration of that is proportional to that of the generated nitrite ions. Scavengers of gas vie with atomic number 8 resulting in a reduced production of gas. In this assay, 1. 0 mL of sodium nitroprusside (5 mM) in phosphate-buffered saline (PBS) was mixed with 3. 0 mL of various concentrations (50-250 µg/mL) of the extract dissolved in the distilled water. The assay mixture was then incubated at 25°C for 150 minutes. These solutions were treated with Griess' reagent and the optical density of the resultant chromophore determined spectrophotometrically at 546 nm and compared with the absorbance of standard solutions of ascorbic acid simultaneously run in identical assay units. The test was done three times all of which showed that the assay of mixtures was equally run in the absence of the extract or the ascorbic acid that was used.
Results and discussion
Antioxidant activity EC50 of standard ascorbic acid solution EC50 of aqueous extract of K. pinnata DPPH free radical scavenging activity 11. 25 µg/mL 116. 25 µg/mL Nitric oxide radical-scavenging activity 15. 5 µg/mL 90 µg/mL Anti-lipid peroxidation activity 14. 0 µg/mL 125 µg/mL
In vitro antioxidant activity
There are various methods used in estimating the antioxidant properties of K. pinnata, the following methods have proven that K. pinnata contains antioxidant properties hence, the extracts doesn’t come out the same for every method. The results will provide information as to what method is more effective when it comes to the extraction of the substance that contains the said component.
DPPH method
The test had shown that the value of EC50 of standard ascorbic acid solution is 11. 25 µg/mL. On the other hand, for EC50 of aqueous extract of K. pinnata it reached the value of 116. 25 µg/mL.
Nitric oxide radical-scavenging activity
The radical-scavenging activity of both solutions were measured and resulted into 15. 5 µg/mL for EC50 of standard ascorbic acid solution and exactly 90 µg/mL for EC50 of aqueous extract of K. pinnata. Reducing power assay Tests have shown that the growth in the reduction of the effect of Ascorbic acid and extracts from K. pinnata were both proportional and has evidently shown the raise in concentrations.
Anti-lipid peroxidation in liver homogenate
EC50 of the standard Ascorbic acid solution was found to be 14. 0 µg/ml while EC50 for the aqueous extract of the leaves of K. pinnata was found to be 125 µg/ml. The study showed that the aqueous extracts of leaves of Kalanchoe pinnata have in vitro antioxidant activity. Flavonol glycosides like quercetin-3-L-rhamnosido-L-arabinofuranoside, quercetin-3-diarabinoside and kaempferol-3-glucoside, many alkanes C25-C35 (n-hentriacontane, n-triacontane predominating) and alkanols C 26 -C 34 are the different classes of phytochemicals that Kalanchoe pinnata contains. Pentacyclic triterpenoids like α-amyrin, b-amyrin and sterols like sitosterol have likewise been excluded from the non-saponfiable fraction. Moreover, the existence of other phenolic constituents like p -coumaric, ferulic, syringic, caffeic and p -hydroxybenzoic acids and organic acids like isocitric and citric acids has been defined.
Our study results suggest that Kalanchoe pinnata comprises constituents having antioxidant activities, which is equivalent to that of ascorbic acid. Additional investigations by specific fractions of this extract can help to distinguish and recognize antioxidant constituent. Based on the report, it suggests that quercetin has a cleared protective effect on cadmium-induces.
Conclusions and recommendations
After all the tests that have been made the researchers therefore conclude that the nephroprotective effect of the aqueous extract of K. pinnata leaves in gentamicin-induced nephrotoxicity requires antioxidant and oxidant scavenging activities. The data of the study also made the researchers land in few recommendations to the future researchers such as: (1) this research could undertake of finding a way to implement the relationship and make treatments to lower your risk of infections. As stated in the significance of the study and limited by scopes and delimitations. (2) Since, this study was not proven yet to be in the right fit to be applied to humans, it resulted into suggesting in doing further studies that could be recommended and can be used by the people who have suffering from different infections to test if Katakataka extract can be a treatment of different infections. (3) In addition, future studies are recommended to use concentration of a higher range (70%, 80%, 90% and 100%) to see the efficacy and capabilities of the Katakataka with regards to its antioxidant properties. (4) Lastly, the researchers recommend applying the study for both sex in order to expand knowledge about its capabilities and efficacy.
