Phytochemical screening, Antiradical and Anti-inflammatory activity of aqueous bark extract acquired from Acacia nilotica

 

Janani Manoharan1, Nirmal Kumar Chinnakannu2, Babujanarthanam Ranganathan3*

1Department of Biochemistry, Auxilium College (Autonomous),

Gandhi Nagar, Vellore - 632006, Tamil Nadu, India.

2Department of Biochemistry, KMG College of Arts and Science,

Gudiyattam, Vellore -  632602, Tamil Nadu, India.

3Nano and Energy Bioscience Laboratory, Department of Biotechnology,

Thiruvalluvar University, Serkkadu, Vellore - 632115, Tamil Nadu, India.

*Corresponding Author E-mail: babukmg@gmail.com

 

ABSTRACT:

Herbal medicaments are applied in the healing for numerous human illnesses from the sprouting of human civilization. The chemicals existed naturally within the plants displays antiradical features which are the principal agent for the neutralization of reactive oxygen species created within the living entities. Acacia nilotica from the Fabaceae family is a tree with magnificent therapeutic properties. Various sections of this tree are implemented in the traditional healing system. Specifically, the bark are applied to treat numerous health complaints. Here, owned to the medicinal features of bark of A.nilotica in the traditional practices, we prepared the aqueous bark extract from this tree and evaluated the antioxidant (antiradical) and anti-inflammatory activities. Hydroxyl radical scavenging protocol presented dose-based upsurge in the radical inhibition activity of the bark extract with maximum activity at 60 µg/ml and the IC50 value appeared as 43.35µg/ml. ABTS radical scavenging assay executed also displayed dose-lineated upsurge in the radical scavenging property of the tested bark extract with IC50 value as 41.07µg/ml. In both the radical scavenging assays, vitamin C was implemented for reference purpose. Even, the anti-inflammatory nature of the aqueous bark extract was performed applying egg albumin denaturation inhibition protocol. In a dose-lined way, the bark extract presented anti-inflammatory activity with an IC50 calculated value as 73.62µg/ml with diclofenac sodium as reference medicament. Additionally, the phytochemical qualitative scrutiny presented the existence of steroids, saponins, terpenoids, alkaloids, tannins, anthraquinones, carbohydrates, phenols and flavonoids which were chiefly responsible for the pharmacological activities of bark aqueous extract.

 

KEYWORDS: Antiradical, therapeutic, A. nilotica, anti-inflammatory, ABTS.

 

 


INTRODUCTION: 

Plant-based drugs are currently in the limelight as a therapeutic part of human health-related illnesses. As indicated by the World Health Organisation, the maximum percentage (80%) of the human race depends on herbal medicines as a fundamental ingredient in health promotion.

 

Divergent organic plant chemical ingredients offer safety, efficiency and quality1.

 

Both medicinal and medicinal plants were in practice in a variety of cultures from ancient times. Plants with natural phytometabolically active ingredients are potent microbicides. India's diverse traditional life system is related to the usefulness of medical-grade plants.Currently, a vast global experiment is underway on plants by pharmaceutical sectors belonging to the exponential increase in requirements for herbal medicines2. In plants bearing medicament traits are stuffed ubiquitously with the plant metabolite ingredients that include primary metabolites as well as dynamic secondary metabolite ingredients that includes tannins, fatty acids, glycosides as well as their derivatives, flavonoids and alkaloids. All the metabolite ingredients form the core for the innovativeness and newness in the medicinal drug features to frame therapeutic medicaments3.

 

Within the living creatures, free forms of radicals (free radicals) are emerged from the diverse normally occurring consequences of metabolic responses and trigger up the peroxidative reactions in the lipid macromolecules and additionally creating distortion of structure in the other bio-organic macromolecules4. ROS which is an oxygen-derived species in conjugation with other free form of radicals induce human health related pathogenesis that includes hepatic issue, respiratory related issues and carcinoma. The herbal phytophenolic entities are the principal naturally available free radical neutralizers within the human system5. It is evidenced from the experimental outcomes that plant-sourced antioxidants can efficaciously overcome the noxious impression of free radicals and thus save the cells, post-pone the aging process and in addition numerous age-based illnesses6.

 

In the cellular machine of the host, inflammation is an outcome of responsiveness to the re-formation in the homeostasis within the alive tissues as a principal outcome of the wounded cells. An astounding immune functioning is relied on the inflammatory responses which aid to deactivate or lyse the harmful invading organism and noxious chemicals7,8. A well-known plasma protein (albumin) existing in the circulatory system in ubiquitous has straightforward relativity with the response of inflammation. Albumin protein denaturation is stimulated and clearly visualized in rheumatoid arthritis thereby upsurging the impression of inflammation in the patients9,10. Furthermore, long lasting health complaints such as renal issues, heart dysfunction, hypertensive disorders, metabolic disorder (diabetes mellitus) and neuron degeneration are tuned by with inflammation of longer-term11,12. The routinely supplemented non-steroidal anti-inflammatory drugs have already announced their noxious impression on the digestive system. So, the phytophenolic acquired from the plants are the good medicament which blocks the inflammatory-type of reactions by nitric oxide creation suppression, deactivating lipoxygenase as well as cyclooxygenase enzymes13,14.

 

Within the family of Fabaceae, Acacia nilotica (A.nilotica) appears as a medium-featured tree with spherically formed crown look along with densification. The portion of the stem-bark is dark to darkish black in colouration with fissures which are greyish to pinkish. A less good quality of reddish-coloured gum is secreted out from the bark portion of this tree. Bipinnately the leaves are arranged. Globular, golden to yellowish coloured flowers with 1.5cm dimension formed on the peduncles which are usually 2-3cm in length and displayed in axillary as well as whorl type. Astringent properties as well as emetic nature are the characteristics of the bark15. The section of stem-bark of A.niloticaare efficaciously applied in the conventional medicative system for curing human-health complications such as skin issues, haemorrhage, wound-ulcer, dysentery, respiratory complaint (common cold and bronchitis), male reproductive system issues and bleeding piles. Apart from that the other sections of this tree such as root, seed, gum, leaves and pods also have responded well in health complaints as anticancer, hepatic tonic, anti-inflammatory, microbicidal as well as anti-hypertensive agent16.

 

MATERIALS AND METHODS:

Study location and bark sample collection:

The well-formed barks from the A.niloticatree were gathered from Kalapadi Village, Gudiyattam, Vellore district. The bark part was scrapped with the aid of a sterile scrapper in August 2021. The acquired bark material was placed in a sterile container and taken for analysis purpose to the research laboratory.

 

Preparing as well as processing the bark powder:

The protocol acquired for preparing and processing the bark section of A.nilotica has copious impression on the phytophenolic and other ingredient preservation. The soil and the grimy unnecessarily struck on the exterior of the collected bark section were eradicated by applying surplus tap water and additionally rinsed multiple times with the distilled water. The infected and the damages sections of the bark were taken out manually. The entirely mature-dark coloured, completely shade-dried bark were picked and chopped into smaller pieces. The bark pieces were taken into the home mixer and thoroughly blended to achieve finest bark powder. In an air-resistant glass bottle, the acquired bark powdery material was placed and maintained at room environment. For further experimental protocols, the bark powder was applied.

 

Chemical and reagents:

The utilized chemical reagents were procured from the Sigma Aldrich.

 

Extract preparation:

The water (aqueous) extract was made by applying 200 g of powdered bark of A.nilotica and 500ml of water through soxhlation technique. The acquired darkish brown coloured residue was carefully taken and dehydrates in avacuum evaporator to release out the moisture. At last, at 4şC (refrigeration) condition the acquired residue was placed within in a clean-glass vial (3ml capacity) and applied for further experimental protocols.

 

Preliminary qualitative investigation of phytochemicals:

Spotting of active phenolic and other ingredients within the bark of A.nilotica was executed by taking the Soxhlet extracted bark residue as mentioned by the standard phytochemical experimental protocols17. The positive outcomes were provided with plus symbol whereas the negative outcomes were provided with minus symbol.

 

Test to identify alkaloids:

Mayer’s reagent was blended with one ml of bark extract and displaying of cream colouration validate for alkaloids.

 

Test to identify phenols:

Exactly, two ml of the bark extract was stirred with 1 ml of ferric chloride solution. Bluish colouration appearance signifies for phenol.

 

Test to identify flavonoids:

To identify flavonoids presence, 2ml of the bark extract was exposed to few drops of dilute HCL and magnesium ribbon in the test tubes. Reddish to brownish coloured solution formation is indicative for flavonoids presence in the bark extracts.

 

Test to identify steroids:

To trace steroids in the bark extracts, both bark extract and acetic anhydride of 2ml was amalgamated. After exposure to concentrated sulphuric acid of 2ml the reaction solution brings bluish green colouration from violet colour indicative for steroid existence.

 

Test to identify terpenoids:

A reddish-brown colouration on blending chloroform in few drops with concentrated sulphuric acid (2ml) along with equal volume of bark extract display terpenoid presence.

 

Test to identify tannins:

Three drops 1% of gelatine were reacted to 1 ml of bark extract. Whitish coloration justifies for tannins.

 

Test to identify saponins (Froth test):

Exactly, bark (1ml) extract was placed in a sterile test tube and shaken vigorously. Bubble or foam formation validate for saponins.

 

Test to identify anthraquinone:

Bark extract of 1 ml was blended thoroughly with equal proportion of benzene along with ammonia solution (10%). Visualization of reddish colouration on adding ammonia justifies anthraquinone existence.

 

 

Test to identify carbohydrates:

To 3mL of Benedict’s reagent reacted 1ml of bark extract and exposed for boiling for 5 min in a water bath. Alteration of bluish colour to reddish colour attribute for carbohydrates.

 

Antioxidant activity:

a) Assay of hydroxyl radical scavenging18

Two millilitres of bark aqueous extract of diverged dosages (20, 30, 40, 50 and 60µg/ml). EDTA (Ethylene Diamine Tetra Acetic acid) prepared by blending anhydrous FAS (Ferrous Ammonium Sulphate-0.13% prepared in water of 100ml) and 0.26% of EDTA. This solution was further mingled with DMSO (Dimethyl sulfoxide) of 0.85% acquired by diluting in 0.1 M (pH 7.4) of phosphate buffer. Then 0.22% of 0.5ml of vitamin C (Ascorbic acid) was added into the reaction mixtures. The entire reacting solution was maintained at 90şC in a hot water bath for 15 minutes. Termination in the reaction was done by pouring ice-chilled 17.5% of trichloroacetic acid. To all the reaction tubes, Nash reagent of 3 ml was added and the tubes were retained for emergence of coloration for 15minutes at room condition. The yellowish coloured appeared was recorded for its intensity at 412nm through spectrophotometrically. Vitamin C (ascorbic acid) was taken for comparison need. The % reduction in the hydroxyl free radicals was displayed by the below cited formula as

 

% Reduction in hydroxyl radicals = [(Absorbance of control sample- Absorbance of tested sample 0/Absorbance of control sample)] x 100

 

b) ABTS [2,2’-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid] radical scavenging assay19

By mingling equally taken 7mM of ABTS in solution form and 2.4mM of potassium persulphate, preparation of working standard solution was executed. The prepared solution was made to undergo reaction by maintaining the solution in a dark area for 12 hours. For the purpose of this assay, 1ml aqueous bark extract acquired from the A.nilotica was poured into test tubes with diverged ranges (20, 30, 40, 50 and 60µg/ml) and separately blended with one millilitre of prepared working standard solution of ABTS. The whole reaction was executed for 6 minutes and finally at 734nm the intensity was seen spectrophotometrically. Vitamin C (ascorbic acid) was taken as comparison need. The % decrease in the ABTS free radicals in the experimented samples were found out by taking the below-given formula as

 

% Reduction in ABTS radical = [(Absorbance of control sample- Absorbance of tested sample /Absorbance of control sample)] x 100

 

Anti-inflammatory activity:

Egg albumin denaturation assay20

The experimental protocol consisted of 2ml of aqueous extract acquired from the bark of A.nilotica of varying concentration ranges from 25µg/ml to 125µg/ml which was entirely mingled with 2.8ml of phosphate buffered saline (pH 6.4), egg albumin of 2ml acquired from the white part of the egg of hen. The whole reaction content was displayed at 37şC for fifteen minutes and then taken into a water bath holding 70şC for a period of 5 minutes. Diclofenac sodium was applied as a positive standard for comparison (as reference). At 660nm, spectrophotometrically the absorbance was taken and the % Inhibition was provided by executing the below given formula as

% Precipitation inhibition in egg albumin = [(Absorbance of control sample- Absorbance of tested sample/Absorbance of control sample)] x 100

 

RESULTS AND DISCUSSION:

The executed phytochemical testing through the standard protocol application presented dynamic plant phenolic ingredients and the outcomes are clearly displayed as table 1.

 

Table 1: Summarizes the identified phytochemicals through screening protocol in A.nilotica bark extract (aqueous extract).

Plant chemical

Identification test

Outcome

Alkaloids

Mayer’s test

++

Phenols

Ferric chloride test

++

Flavonoids

Shinoda test/ Magnesium hydrochloride reduction test

++

Steroids

Liebermann-Burchard test

++

Terpenoids

Salkowski’s test

+

Tannins

Gelatin test

+++

Saponins

Froth test

++

Anthraquinones

Borntrager’s test

+

Carbohydrates

Benedict’s test

+

Here, (+++) presented: Copiously evidenced; (++) presented: Moderately evidenced; (+) presented: Mildly evidenced

 

Assay of hydroxyl radical scavenging:

Hydroxyl radical which are a type of reactive oxygen species are creator of massive oxidative reactions within the living bodies by distorting the macromolecular structures that include DNA, RNA, proteins, carbohydrates and lipids thereby upsurging the health illnesses21,22. At the studied minimum concentration (20 µg/ml), the % of radical scavenging was 12.13±0.70% which elevated with the concentration increases. At 60 µg/ml, the activity was appeared to be 84.17±0.70%. The IC50 calculated value was 43.35µg/ml for the aqueous bark extract. The standard vitamin C also gave dose-mannered upsurge in the inhibition activity which was 17.01±0.41% at 20µg/ml whereas at 60µg/ml the result was 98.47±0.86%. The IC50measured for ascorbic acid was 37.00µg/ml. The experimental outcomes are provided in the table 2.

 

Table 2: Outlines the outcomes of hydroxyl radical scavenging assay.

Concentration as µg/ml

Aqueous extract from A,niloticabark

(% inhibition)

Vitamin C as reference

20

12.13 ± 0.70

17.01 ± 0.41

30

18.74 ± 0.95

40.38 ± 0.86

40

37.78 ± 0.92

65.69 ± 0.63

50

62.67 ± 0.46

77.17 ± 0.41

60

84.17 ± 0.70

98.47 ± 0.86

 

ABTS [2,2’-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid] radical scavenging assay:

Provoked by chemical means, ABTS free radicals scavenging assay is a most sensitive protocol to screen the antioxidant’s electron or hydrogen giving characteristic. Previous, the authors in their experimental outcomes have strongly communicated the relativity between the degradation in radical ABTS and plant polyphenolic moieties23. In experimented with 20 µg/ml-60 µg/ml concentration ranges, this assay displayed % reduction in ABTS at its maximum of 72.27±0.45% (at 60µg/ml). The IC50 calculated outcome was 41.07µg/ml for the bark aqueous extract (Table. 3). The comparison purpose implemented vitamin C gave an IC50 value of 30.40µg/ml.

 

Table 3: Outlines the outcomes of ABTS radical scavenging assay.

Concentration as µg/ml

Aqueous extract from A,nilotica bark

(% inhibition)

Vitamin C as reference

20

10.60 ± 0.94

18.57 ± 0.85

30

30.30 ±0.69

59.56 ± 0.62

40

50.90 ±0.90

76.36 ± 0.85

50

66.81 ± 0.45

88.11 ± 0.81

60

72.27 ± 0.45

93.85 ± 0.40

 

Egg albumin denaturation assay:

The egg albumin denaturation is a good protocol to notify the anti-inflammatory activity of aqueous bark extract obtained from A.nilotica. At minimum concentration of 25µg/ml, the % of inhibition in albumin denaturation was 16.86±0.53% and at 125µg/ml, the % inhibition achieved was 66.66±0.65% The IC50 measured was 73.62µg/ml for the bark aqueous extract. Here, the IC50 measured for diclofenac sodium was 63.50µg/ml (Table.4).

 

Table 4: Outlines the outcomes of anti-inflammatory activity of aqueous extract of bark from A.nilotica through egg albumin denaturation inhibition.

Concentration as µg/ml

Aqueous extract from A,niloticabark (% inhibition)

Diclofenac sodium as reference

25

16.86 ± 0.53

18.57 ± 0.85

50

25.24 ± 0.75

59.56 ± 0.62

75

42.49 ± 0.66

76.36 ± 0.85

100

54.59 ± 0.60

88.11 ± 0.81

125

66.66 ± 0.65

93.85 ± 0.40

 

CONCLUSION:

In the executed screening of phytometabolites, antioxidant and anti-inflammatory assays of A.nilotica bark aqueous extract it was cleared that the identified alkaloids, tannins, phenol, saponin and other components were chiefly responsive for the antiradical and anti-inflammatory traits exposed by the extract. The aqueous extract acquired from the bark of A.nilotica disclosed efficacious radical inhibition with the ascending dosage of bark extract. Similarly, the anti-inflammatory protocol performed also provided the dose-lineated upsurge in the activity of the extract. So, still more future experimentation has to be executed to unlock the further pharmacological traits of A.nilotica bark aqueous extract.

 

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

 

ACKNOWLEDGMENTS:

The authors are heartfully thankful to the Department of Biotechnology. Thiruvalluvar University, Serkkadu, Vellore for furnishing research need for executing this work.

 

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Received on 19.08.2022            Modified on 24.10.2022

Accepted on 26.11.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(10):4831-4835.

DOI: 10.52711/0974-360X.2023.00783