INTRODUCTION:

In the very last few decades, there is a tremendous growth in the region of herbal medicine. It is coming popularized in both developing and developed countries due to its natural origin because of its lesser side effects. Herbal remedies provide a lot of drugs for the treatment of internal diseases which are considered to be stubborn and incurable by other system of medicines¹

Inflammation is a protective immune response towards the invasion of any foreign body such as bacteria, viruses, and parasites. Inflammation research is one of the major fields of global healthcare research, due to the side effects of commercially available non-steroidal anti-inflammatory drugs (NSAIDs), such as renal suppression and gastrointestinal problems like ulcers²

Natural products have contributed significantly to the development of modern medicine. Recently traditional medicine worldwide is being re-evaluated by extensive research on different plant species and their active therapeutic principles. The rich wealth of plant kingdom can represent a novel source of newer compounds with significant anti-inflammatory activities. The major merits of herbal medicine seem to be their perceived efficacy, low incidence of serious adverse effects, and low cost³

A.radiata is commonly known in Arabic Nogd Sahrâwi Nogd Lhor⁴ and in Tamazight « Ajjerg; âwjerg » is also called Arabian oxeye⁵, A.radiata is endemic between Algeria and Morocco⁶ It is used in traditional medicine for the treatment of various diseases; gastroenteritis⁷ spasms and colic, arthritis and rheumatoid vaginal discharge, colds, hepatitis, diabetes and stomach ailments lung disease and indigestion⁸, antifungal activity^9,10 antimicrobial activity⁴

The present study was conducted to evaluate the possible in vitro anti-inflammatory effect of A. radiata flowers extract against the denaturation of the protein.

MATERIALS AND METHODS:

Collection of the Plant Materials:

At flowering stage, flowers of A. radiata ware collected during the flowering period from Sahara of Merrara near of Touggourt (33° Northern latitude, longitude of 6° East, altitude: _69 m, under the sea) southeast of Algeria. It was obtained in the month of April 2013. The identification was done on the basis of Quezel and Santa¹¹ by Doctor Halis Youcef researcher in Touggourt’s Scientific and Technical Research Centre for Arid Areas. Plant sample was cleaned, air dried in the shade and well-ventilated place, then grounded and stored in the dark.

Preparation of the extracts of A.radiata Flowers:

Flowers (100g) of A.radiata were extracted at room temperature with MeOH–H₂O (70:30, v/v) for 48h, three times. After filtration, the filtrate was evaporated till dryness (19.34g), recovered with distilled water (60ml) and partitioned successively using chloroform (3 × 35ml) giving 0.278g of Chloroform fraction, ethyl acetate (1 × 35ml) giving 0.149g of Ethyl acetate fraction, and n-butanol (4 × 35ml) giving 1.750g of Butanol fraction. The extracts, also were concentrated under reduced pressure and then re-dissolved with the minimum of methanol or water and stored at 4°C until analysis.

Phytochemical analysis of The extracts of A.radiata Flowers:

The extracts of A.radiata Flowers were subjected to preliminary phytochemical evaluated to determine the presence of alkaloids, flavonoids, glycosides, phenols, terpenoids, sterols, tannins, coumarins, anthraquinones and quinones. According to standard methods⁹

In vitro egg albumin denaturation method:

The reaction mixture (5ml) consisted of 0.2ml of egg albumin from fresh hen's egg, 2.8ml of phosphate buffered saline (PBS, pH 6.4) and 2 ml of varying concentrations of extracts of The extracts of A.radiata Flowers. Was used as the test sample. A similar volume of double distilled water served as the control. Next, the mixture was incubated at 37°C for 15 minutes and then heated at 70°C for 5 minutes. After cooling; the absorbance was measured at 660nm. Diclofenac sodium in the concentrations (0.3125, 0.625,1.25, 2.5, 5 and 10 mg/ml) was used as the reference drug. The percentage inhibition of denaturation which is an index of anti-inflammatory activity was calculated using the following equation:¹²

A_c: absorbance of the control

A_t: absorbance of the test sample

In vitro BSA denaturation method:

The anti-inflammatory activity of the extracts of A.radiata Flowers. was performed by using bovine serum albumin denaturation (BSA) method with some modification^13-15 0.05ml of different concentration of test and reference drugs Diclofenac sodium (0.3125, 0.625,1.25, 2.5, 5 and 10mg/ml) and 0.45 ml of bovine serum albumin (1 % w/v) mixed. The sample was incubated at 37 C° for 20 minutes and then heated at 57C° for 3 minutes. After cooling the samples, add 2.5 ml of phosphate buffer slain (pH 6.4). the absorbance was measured at 660 nm. The percentage inhibition of protein denaturation was calculated as follows:

A_c: absorbance of the control; A_t: absorbance of the test sample

RESULTS AND DISCUSSION:

The extracts of A.radiata Flowers were subjected to qualitative tests for the identification of phytochemical constituents according to standard procedures. The preliminary phytochemical investigation showed the presence of phytoconstutients such as phenols, alkaloids, flavonoids, tannin.

The extracts of A.radiata Flowers was subjected to qualitative analytical tests for the various plant constituents (Table1) the phytochemical analysis have given the presence the flavonoids, glycosides, quinones, coummarine, tannins, sterols and triterpenoids.

*Table 1: Preliminary phytochemical screening of The extracts of A.radiata* Flowers**
Phytochemical test		Extracts of A.radiata
Phytoconstutients	Test	CHCl₃	AcOEt	BuOH	Crude
Alkaloids Glycosides Antraquinones Quinones Phenols Tannins Flavonoids Coumarins Sterols Triterpenoids	Mayer's test Wagner's test Kellarkiliani test Conc. H₂SO₄ test - - Ellagic acid test FeCl₃ test Shinoda's test Alkaline Reagent test - Salkowski test Liberman Burchard test	- - - - - - - - - - + - -	- - + + - - - + + + + + +	- - + + - + - + + + + + +	- - + + - - - + + + + + +
Where, '' +'' indicates positive and '' –'' indicates negative

Table.2 Effect of Diclofenac sodium and extracts of A.radiata Flowers on in vitro heat-induced denaturation of egg albumin protein

Concentrations mg/ml	Standard solution	Chloroform fraction	Ethyl acetate fraction	Butanol fraction	Crude extract
0,3125	23,27±0.05	4,07±0.007	10,00±0.007	12,03±0.06	5,48±0.03
0,625	27,94±0.03	14,98±0.05	14,06±0.01	16,90±0.03	10,75±0.07
1,25	35,31±0.07	24,32±0.05	16,44±0.01	21,64±0.04	13,52±0.09
2,5	53,11±0.02	46,19±0.1	18,15±0.02	36,74±0.1	15,65±0.08
5	79,95±0.05	56,15±0.07	53,69±0.01	64,66±0.08	33,50±0.16
10	89,00±0.05	68,69±0.09	86,71±0.00	77,18±0.02	84,51±0.10
Results are expressed as the mean of 6 values ± standard deviation

Table.3 Effect of Diclofenac sodium and extracts of A.radiata Flowers on in vitro heat-induced denaturation of BSA

Concentrations mg/ml	Standard solution	Chloroform fraction	Ethyl acetate fraction	Butanol fraction	Crude extract
0,3125	38,73±0.021	38,88±0.09	60±0.00	50±0.00	44,44±0.09
0,625	45,41±0.016	38,88±0.09	69,44±0.04	66,6±0.00	62,22±0.03
1,25	52,17±0.037	56,66±0.05	96,57±0.01	90,88±0.02	75,21±0.01
2,5	58,84±0.02	88,62±0.01	98,87±0.01	99,52±0.01	89,41±0.05
5	61,54±0.00	98,38±0.01	99,02±0.008	99,62±0.09	98,44±0.03
10	62,96±0.00	99,40±0.005	99,41±0.005	99,78±0.00	99,11±0.03
Results are expressed as the mean of 6 values ± standard deviatio

Anti-inflammatory activity:

Protein denaturation is a process in which proteins lose their tertiary structure and secondary structure. It is one of the causes of arthritic disease and inflammation Mechanism of denaturation probably involves alteration in electrostatic, hydrogen, hydrophobic and disulphide bonding [16]. In vitro anti-arthritic activity of studied the extracts of A.radiata flowers were evaluated with bovine serum albumin (BSA) denaturation method and denaturation method using egg albumin.

In vitro denaturation of egg albumin:

In the present investigation, the in vitro anti-inflammatory effect of the extracts of A.radiata Flowers was evaluated against denaturation of egg albumin. The results of the extracts of A.radiata Flowers in vitro denaturation of egg albumin of extracts of are summarized in Table 2.

The extracts of A.radiata Flowers had shown have a similar activity inhibition of protein denaturation than standard anti-inflammatory drug Diclofenac sodium, which CHCl₃ extract has less inhibition for them. The maximum inhibition of 89% was observed at 10mg/ml in The extracts of A.radiata Flowers, and the maximum inhibition of Diclofenac sodium has 89.17% at the concentration also 10mg/ml. The minimum inhibition observed at the concentration of 0.3125mg/ml in the CHCl₃ extracts of fruits 4,077% and showed 23,27% in the Diclofenac sodium. This results indicating that the extracts of A.radiata Flowers are a more potent anti-inflammatory agent.

In vitro denaturation of BSA:

In vitro anti-inflammatory activity by bovine serum denaturation method, the results are summarized in table 3

The extracts of A.radiata Flowers had shown a higher inhibition of protein denaturation than standard anti-inflammatory drug Diclofenac sodium.

The maximum inhibition of 99% was observed at 10 mg/ml in all the extracts of- A.radiata Flowers and the maximum inhibition of Diclofenac sodium has 62,96% at the concentration also 10mg/ml. the minimum inhibition observed at the concentration of 0.3125 mg/ml in the Ethyl acetate fraction 20% and showed 38,72% in the Diclofenac sodium. These results indicating that A.radiata Flowers is a more potent anti-inflammatory agent.

Hence, the results of our study reveal that extracts of A. radiata Flowers were capable of controlling the production of auto-antigens and inhibit denaturation of protein especially denaturation of albumin [17]

The inhibition of protein denaturation, albumin denaturation, was studied to establish the mechanism of anti-arthritic activity of A. radiata Flowers. Therefore, our in vitro studies on the extract of A. radiata Flowers demonstrate the significant anti-arthritic activity. Hence, this Nogd Lhor plant can be used as a potent natural anti-arthritic agent. The results show that the extracts of A. radiata Flowers exhibiting anti-arthritic activities might be due to the presence of the secondary metabolites contained in this plant have interesting biological activities (antibacterial, antifungal, anti-inflammatory, anti-cancer, antioxidant ...) as the active principles such as polyphenolic content, triterpenoids, alkaloids, and flavonoids. From the results of the study, it can be concluded that the extract of A. radiata Flowers possessed significant ant-arthritic.

The present study revealed the potential of plant extract in the management of inflammation and arthritis confirming the folk core use of medicinal plants. However, one should try to further figure out extract more as having much better activity in the quest of active candidate or chemical molecule that is mainly responsible for this activity.

CONCLUSION:

In conclusion, the results of this study conclusively show that, the extracts of the extract of A. radiata Flowers. possess potent and dose-dependent anti-inflammatory activity in vitro. This is a novel finding, which scientifically justifies its use as an anti-inflammatory drug in traditional medicine. Possibility also exists for the development of a safe and efficacious anti-inflammatory drug and perhaps anti-arthritic drug.

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Received on 31.10.2019 Modified on 31.12.2019

Asian J. Research Chem. 2020; 13(1): 44-47.

DOI: 10.5958/0974-4150.2020.00010.3