ANTIOXIDANT PROPERTIES OF METHANOLIC EXTRACT OF BRYPHYLLUM PINNATUM ON ALBINO WISTAR RATS

The result of this study found LD 50 to be above 5000mg/kg. In vitro antioxidant test found the presence of alkaloids, saponins, thiamin, riboflavin etc. Antioxidant activity test result found an increase in GSH, GPx, a decrease in MDA and a stable CAT. In conclusion, we recommend its uses in human, but at a regulated doses because of its efficacy as stated in this study. figure showed that there were no significant different in catalase activity level in the treatment groups that received 400mg/kg and 800mg/kg following Bryophyllumpinnatum administration when compared to the control group. The result showed that the acivities of catalase remained unchanged in control group and the experimental groups following B. pinnatum administration.


Test for the presence of saponins
An estimated 5 ml of extract was shaken vigorously with 5 ml of distilled water in a test tube and warmed. The formation of frothing stable foam) was taken as an indication of the presence of saponins.

Test for the presence of flavonoids
To 2 ml of extract, few drops of lead acetate solution were added. Formation of a yellow precipitate was an indication of the presence of flavonoids.

Test for the presence of tannins
Approximately 2ml of extract was mixed with 2ml distilled water and heated on a water bath. Few drops of ferric chloride (FeCl3) solution were added. Formation of a green coloured precipitate was an indication that tannins were present.
Test for the presence steroids a. Salkowski,s test :-a red color produced in the lower chloroform layer when 2 ml of organic extract was dissolved in 2 ml of chloroform and 2 ml concentrated sulphuric acid was added in it, indicates the presence of steroids. b. Liebermann Burchard test: -development of a greenish color when 2 ml of the organic extract was dissolved in 2 ml of chloroform and treated with concentrated sulphuric acid and acetic acid indicates the presence of steroids.

Detection of Steroids
Two ml of acetic anhydride was added to five mg of the extracts, each with two ml of H2SO4. The colour was changed from violet to blue or green in some samples indicatenthat the presence of steroids.
Test for the presence of terpenoids 2 ml of the organic extract was dissolved in 2ml of CHCl 3 and evaporated to dryness. 2ml of conc. H2SO4 was then added and heated for about 2 minutes. Development of a grayish color indicates the presence of terpenoids

Detection of Terpenoids Salkowski s Test
Five mg of the extract of the leaves, flowers and seeds was mixed with two ml of chloroform and concentrated H2SO4 (3ml)was carefully added to form a layer. An appearance of reddish brown colour in the inner face was indicates that the presence of terpenoids.
Test for the presence alkaloids 3 ml of extract was stirred with 3 ml of 1% HCl on steam bath. 1 ml of mixture was taken separately in two test tubes. Few drops of Dragendorff's reagent were added in one tube and occurrence of orange red precipitated was taken as positive. Two the second tube Mayer's reagent was added and appearance of buff colored precipitate was taken as positive test for presence of alkaloids.

Detection of Alkaloids
Extracts were dissolved individually in dilute hydrochloric acid and filtered. The filtrates were used to test the presence of alkaloids.

Mayer's test:
Filtrates were treated with Mayer's reagent. Formation of a yellow cream precipitate indicates the presence of alkaloids Wagner's test: Filtrates were treated with Wagner's reagent. Formation of brown/ reddish brown precipitate indicates the presence of alkaloids.

Test for the presence glycosides
To about 2 ml of extract with dilute HCl and 2 ml Sodium nitropruside in pyridine and sodium hydroxide solution were added. Formation of pink to blood red color indicates the presence of Cardiac glycosides.

Test for the presence phenols
An estimated 10mg of the extract was dissolved in 5ml of distilled water, then treated with few drops of ferric chloride solution. A dark green or bluish black colour indicated the presence of phenolic compounds.

Nitric oxide inhibition activities of the extracts
Nitric oxide, generated from sodium nitroprusside in aqueous solution at physiological pH, interacts with oxygen to produce nitrite ions which were measured by Griess reaction. The reaction mixture (3 ml) containing sodium nitroppruside (10 mM) in phosphate buffer saline (PBS) and the extract from (50 -800) µg/ml was incubated at 25°C for 150 min. After incubation, 0.5 ml of the reaction mixture was removed and 0.5 ml of Griess reagent (1% (w/v) sulfanilamide, 2% (v/v) H 3 PO 4 and 0.1% (w/v) naphthylethylenediamine hydrochloride) was added. The absorbance of the chromophore formed was measured at 546 nm.

Photometric Assay of 2, 2-Diphenyl-1-Picrylhydrazyl (DPPH)
The free radical scavenging activity of the extract was analyzed by the DPPH Assay using spectrophotometer. The crude extract at concentrations (25, 50, 100, 200 and 400) µg/ml each was mixed with 1 ml of 0.5 mM DPPH (in methanol) in a cuvette. The absorbance at 517 nm was taken after 30 minutes of incubation in the dark at room temperature. The experiment was done in triplicate. The percentage antioxidant activities were calculated as follows. % antioxidant activity (AA) = 100-[{(ABS sample -ABS blank) ×100}/ABS control]. One milliliter of methanol plus 2.0 ml of the test extract was used as the blank while 1.0 ml of the 0.5 mM DPPH solution plus 2.0 ml of methanol was used as the negative control. Ascorbic acid (vitamin C) was used as reference standard.

Ferric Reducing Antioxidant Power
The ferric reducing antioxidant power was carried out as used by Benzie and Strain. The protocol involved is as follows:
FRAP working solution was prepared by mixing solution 1, 2 and 3 in the ratio of ‫,1׃1׃01‬ respectively.

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The working solution was freshly prepared in each test. The aqueous solution of known amount of ascorbic acid was used for calibration.
Assay: Blank FRAP reagent. Sample: FRAP reagent (3 ml) and 100 μl sample solution at concentrations of 50, 100, 200, 400 and 800 µg/ml was mixed and allowed to stand for 4 minutes. Colometric readings were recorded at 593 nm, at 37 0 C. The ascorbic acid standard solution was tested in a parallel process. Calculations were made by a calibration curve.

Experimental Animals
Thirty adult male Wistar albino rats of 12 weeks of age, obtained from the animal house of Abia State University, Nigeria were used for the study. The animals were given standard rodent chow and clean drinking water ad libitum. The animals were kept in a well-ventilated room with a 12 h light/dark cycle at room temperature. All animal experiments were approved by the Animal Research Ethics Committee of the University, in accordance to the guide for care and use of laboratory animals.

Experimental Design
The animals were randomly distributed into three groups of 5 rats per group. The first group served as the control, the second groups were administered 400mg/kg of methanolic extract of B. pinnatum leaf extract by oral gavage daily for 28 days, respectively. The animals in the third group were administered 800mg/kg of ethanolic extract of B. pinnatum daily for 28 days. At the end of extract administration, the rats were sacrificed by cervical dislocation under deep diethyl ether anaesthesia.

Statistical Analysis
The results are presented as mean±SEM for each group. Differences among groups were analysed using one-way analysis of variance (ANOVA) followed by Dunette's multiple comparison test. Data were analysed using SPSS Version 20 and values were considered significant at P<0.05.

Result Of Acute Toxicity Evaluation
No death was recorded within the 24 hours and a further 7 days of the acute toxicity study, even at the highest dose of 5000 mg/kg body weight. The animals instead had normal disposition and were emotionally stable and all survived the period of the study.       The bar chart shows glutathione activity level is significantly increased in the treatment groups that received 400mg/kg and 800mg/kg following Bryophyllumpinnatum administration when compared to the control group. This result showed that Bryophyllumpinnatum extract increases glutathione levels in cells and organ, and it does so in a dose dependent manner as shown in fig1.0.

Discussion And Conclusion:-Acute Toxicity Testing
Acute toxicity describes any adverse effect that occurs within 24 hours of administering any agent or substance. To be described as acute toxicity the effects should be noticed within 14 days of giving any substance.
Animals in the phase one testing were observed to have normal physical activity as those in the control group, while animals in the phase two testing had normal activity, but were always clustering together and calm. Our study concluded that the LD 50 of methanolic extract of B. pinnatum were above 5000mg/kg, reason being that no death occurred across the group within 24hrs of the toxicity testing and 14 days after the testing there was no fatality across the groups.

Phytochemical properties of b. Pinnatum
The phytochemical analysis was done using standard method as described by Sofowora (1990) and Evans (1989). The analysis reveals the presence of Alkaloids (11.70 mg/100g), Flavonoids (4.49 mg/100g), and phenols (5.13 mg/100g) in high quantity as shown in table 6. These phytochemicals helps to reduce oxidative stress and fight against free radicals. This is in agreement with the study done by Nwali

Exogenous Defense Mechanism
To maintain homeostasis in the redox system and protect the body against ROS Photometric assay of 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenge activities was measured using spectrophotometer. The asorbance of methanol extract of B. pinnatum was measured at 517nm after 30 minutes of incubation (Ijioma et al,. 2019). In the current study we found that DPPH scavenging activities of methanolic extract of B. pinnatum exhibited a concentration-dependent relationship as seen in table 9 of results.
Glutathione is an antioxidant produced in cells. GSH is capable of preventing damage to important cellular components caused by reactive oxygen species (ROS) such as free radicals and lipid peroxide (White et al., 2003). In our study, glutathione increases in a dose dependent manner (p>0.05) across the groups. This suggests that B. pinnatum increased GHS in the tissues tested for GHS, by fending off ROS and free radicals in the organs.
Glutathione peroxidase (GPx), is a cytosolic enzyme that catalyzes the reduction of hydrogen peroxide to water and lipid peroxide to alcohols. Research have shown that low GPx in serum maybe a contributing factor to vitiligo (Zedan et al., 2015), diabetes and multiple schlerosis (Socha et al., 2014). GPx also increases across the groups in a dose-dependent manner in this current study.

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Catalase together with GPx decomposes hydrogen peroxide to water and oxygen. It also protect cells and tissues against oxidative stress. No, significant (p>0.05) change were observed for the CAT between the control groups and the experimental groups.
Malondialdehyde is a natural occurring compound and it is a maker for oxidative stress. It results from lipid peroxidation of polyunsaturated fatty acids (Nair et al., 2008). High levels of MDA may depict tissue damage in an animal, such as abnormal spermatozoa, osteoarthritis (Collodel et al., 2015). In our study, there was a significant decrease in the levels of MDA inferring that Bryophyllumpinnatum can reduce damage caused by high MDA levels in cells and tissues. This study is in agreement with Chioma et al., (2017).

Conclusion:-
The phytochemical properties of B. pinnatum shows it contains phenols, flavonoids, riboflavin and other important vitamins.
The scavenging activity of Bryophyllumpinnutum on Niric oxide, FRAP and DPPH has shown it can fight free radicals. It has also shown to decrease MDA levels in the animals administered it and increases GHS in the groupsadministeredB. pinnatum.