In Vitro Antioxidant Property and Phytochemical Constituents of Senna alata Leaves Aqueous Extract Collected in Ngaoundéré (Cameroon)

Leaves extract of Senna alata L. are used as indigenous medicine to treat various types of disease like ulcers, stomach, pain and fever. The present study was undertaken to study the phytochemical screening, total phenolic compounds (TPC), total flavonoid compounds (TFC) and in vitro antioxidant of Senna alata leaves’ extract growing in Ngaoundéré town (Cameroon). Respective bioactivities of the phytochemicals were determined. Quantitative analysis of the total phenolic content was determined by using the Folin-Ciocalteu method while total flavonoid was estimated using aluminium trichloride (AlCl3). The antioxidant capacities in the forms of DPPH (2,2-diphenyl-1-picrylhydrazyl) and FRAP (Ferric Reducing Antioxidant Power) were evaluated by spectrophotometric methods. For these purposes, aqueous extract were prepared. Now the experimental screening of phytochemicals showed negative results for the absence of reducing compounds, steroids/triterpenes, and tannins. The results showed that TPC, TFC values were higher: 14.768±0.26 mg GAE per g of extract, 4.32±0.12 mg Ru per g of extract, respectively. The Senna alata Leaves’ extract (SALE) exhibited the best DPPH inhibition concentration 50% (IC50 = 12.05 mg. mL ), and FRAP method (IC50 = 2.79 mg. mL ) compared to that of the positive control, ascorbic acid (IC50 = 17.69 mg/mL); Hence, extract from the leaves of Senna alata contains high secondary metabolites which accounts for its strong antioxidant ability thus justifying its use as natural occurring antioxidants in folkloric medicine.


Introduction
Oxygen is a highly reactive atom that is capable of becoming part of potentially damaging molecules commonly called "free radicals." Living organisms are equipped with a defense system to neutralize free radicals such as superoxide anion radicals (O2¯), hydroxyl radicals (HO•), and non-free-radical species, such as hydrogen peroxide (H2O2) and singlet oxygen ( 1 O2) 1 . If the production of free radicals exceeds the antioxidant capacity of a living system, free radicals are capable of attacking the healthy cells of the body to cause oxidative stress leading to the pathogenesis of several human's degenerative diseases like cancer, inflammation, atherosclerosis, neurological disease, cardiovascular disease and rheumatoid arthritis 2 .
During oxidative stress, free radicals not detoxified by the antioxidant system cause damage to biological molecules contained in cells, including the oxidation of DNA, proteins, lipids, and carbohydrates, but also secondary lesions due to the cytotoxic and mutagenic nature of the released metabolites lipid oxidation 3 . Antioxidants are natural or chemical products which are defined as chemicals which, more specifically, delay the deterioration or discoloration caused by the oxidation and neutralization of free radicals but can also have toxicological effects and suspected carcinogenic potential 5  treatment of ringworm. It is recognizable by its dense and bright orange yellow flowers that are arranged spirally on its rachides, san average height of between 1 and 5 meters and has horizontally spread branches. The inflorescence looks like a yellow candle. It is often cultivated for medicinal purposes, and also as a pantropical ornamental shrub to warm temperate areas.
The fruit is a pod, while the seeds are small and square in shape.
The plant has been shown several pharmacological virtues. Its leaves are used against yellow fever or malaria, and as antiasthmatics, or antidiabetics 6 . The leaves are also specific for the treatment of ringworm and eczema 7 , scabies, athlete's foot 8 , herpes 9 and insect bites 10 . Various extracts and different parts of S. alata have been reported to own many pharmacological activities such as laxative 11 , wound healing 12 , hypoglycaemic 13 , anti-bacterial 14 , analgesic 15 and anti-inflammatory 16 .
In this work, the present study was designed to determine the qualitative screening of phytochemicals, the total phenolic compounds (TPC), total flavonoid compounds (TFC) and to examine the in vitro antioxidant of S. alata leaves extract to ascertain its acclaimed use as natural occurring antioxidants in folkloric medicine.

Collection of plant material
The freshly collected aerial part of the S. alata was gathered in

Preparation of plant extract
The S. alata leaves were prepared according to traditional use.

Ethics statement
For the collection of plant, no specific permits were required for the described field studies. For any locations/activities, no specific permissions were required. The location where the plant was collected was not privately-owned or protected in any way and the field studies did not involve endangered or protected species. This study was approved by the University of Cameroon's institutional review board.

Qualitative phytochemical screening
Preliminary qualitative phytochemical screening for the determination of secondary metabolites was carried out according to standard methods [17][18][19] to detect the presence or absence of bioactive compounds.

Test for flavonoids
In a test tube, introduce 1 mL of extract to be tested, add 1 mL of hydrochloric acid (HCl) and 3 magnesium shavings. The appearance of a red or yellow coloration reveals the presence of flavonoids.

Test for tannins
To 1 mL of extract to be analyzed, add 0.5 mL of a 1% aqueous solution of FeCl3. The presence of tannins is indicated by a greenish or blue-blackish coloration.

Test for Coumarins
Put 5 mL of extract in a tube, add 0.5mL of 10% NH4OH, mix and observe under UV at 366 nm. Intense fluorescence indicates the presence of coumarins.

Test for Alkaloids
The tests are carried out by precipitation reactions with the reagents of Mayer and Wagner.
1 mL of each extract is divided into two equal volumes. One volume is treated with 0.5 mL of Mayer's reagent, the other with 0.5 mL of Wagner's reagent. The appearance of a white or brown precipitate, respectively, reveals the presence of the alkaloids.

Test for saponins: Foam test
In a test tube, introduce 10 mL of extract to be tested and stirred for a few seconds then left to stand for 15 min. A height of persistent foam indicates the presence saponins.

Test for reducing compounds
Put 1 mL of extract in a test tube, add 2 mL of Fehling's liquor (1 mL reagent A and 1 mL reagent B), and incubate the whole for 8 min in a boiling water bath. The appearance of a brick-red precipitate indicates the presence of the reducing compounds.

Total phenolics content determination
For the determination of phenolic compounds present by Folin-Ciocalteu reagent, the method described by Singleton and All measurements were repeated three times and results were expressed as mg gallic acid equivalent (GAE) per grams of extract.

Flavonoid content determination
Colorimetric assay was used to determine the content of flavonoid according to known procedure 21

Ferric reducing antioxidant power (FRAP) activity
The Ferric reducing antioxidant power procedure was followed to

Phytochemical Screening
One of the essential goals of a phytochemical test is the detection of the different families of secondary metabolites existing in the studied part of the plant by qualitative characterization reactions.
The result of phytochemical test is summarized in Table 1

Determination of total phenolic contents
The presence of phenolic compounds identified in the leaves extract of S. alata in the current study could be responsible for its folkloric therapeutic usage for animals. The total phenolic content in the aqueous extract was 14.768 ± 0.26 mg GAE / gram.

Determination of total flavonoid contents
The result of total flavonoid contents of the extract was 4.32 ± 0.12 mg RE / gram of extract. Equation of calibration curve of rutin standard was y = 3.0495×−0.0093, R 2 = 0.9833. The Fig. 2 showed the calibration curve of standard (Rutin). These results are lower than those found by other researchers who revealed that the content of total flavonoids in the methanolic extract of Cassia alata was 24.37 ± 0.25 mg UK / g dry extract 28 .
Flavonoids, for instance, are known to be very potent watersoluble antioxidant. scavenging activity decreased as the concentration of the extract increased.  The production of free radicals is regulated by our body which has developed antioxidant defenses to protect against the potentially destructive effects of free radicals. The present study however showed that the aqueous extract of S. alata is strong antioxidant activity, high total phenolic and total flavonoids content. This extract can be considered as good sources of natural antioxidants for side dishes and medicinal uses. Further in details phytochemical analysis should be done to identify to elucidate the exact bioactive compound which is responsible for the antioxidant action.

Acknowledgements
Nil 6 Authors' contributions JV designed hypothesis, conceived, wrote the paper, analyzed the data and critically reviewed the manuscript. SDS performed the experiments in this study, assisted with analyzing the data, confirmed the results, and contributed to the writing of the article.
All authors reviewed and approved the final manuscript.

Conflicts of interest
The authors declare that there are no conflicts of interest regarding the publication of this paper

Ethics approval and consent to participate
This information is not relevant since our study does involve neither animals nor humans.

Consent for publication
Not applicable since our manuscript does not contain any individual person's data in any form.