INTRODUCTION:

Amaranthaceae family contains 165 genera and 2040 species¹ most of these plants are green leafy vegetables as a rich in minerals and vitamins, used in diarrhoea, antipyretic and used for ornamental purpose. Generally, these families contain flavonoids, saponins, triterpenoids, steroids, etc. as phytoconstituents². There are various chemical constituents reported like flavonoids, steroids, triterpenoids and alkaloids^3,4. The present work was undertaken to screen with an objective to study the pharmacological and pharmacognostical evaluation for Alternanthera pungens.

Fig 1: Alternanthera pungens

MATERIALS AND METHODS:

Collection of plant material:

Fresh plant leaves of Alternanthera pungens were harvested from Tirunelveli district, Fresh leaves were washed thoroughly 3-4 times with running tap water then finally with sterile water followed by shade drying at room temperature for 15-20 days. Then dried, cut and ground into coarse powder and macerated with ethanol and the extract was dried by rotary vacuum. The dried residue was preserved in airtight container and kept at 4-5°C until further use.

Phytochemical screening:

The extracts were analysed for the presence of phytochemical constituents^5,6 such as phenolic compounds, flavonoids terpenoids, saponins, alkaloids, cardiac glycosides and proteins.

Antimicrobial study:

The antimicrobial properties of Alternanthera pungens were investigated against two-gram negative bacterial strains; Proteus vulgaris, Escherichia coli and two-gram positive bacterial strains; Staphylococcus aureus and Bacillus subtilis. A total of six pathogenic microorganism were obtained from the culture collection of Microbiology Laboratory. The evaluation of antimicrobial activity by cup plate method was performed by the measurement of diameter of zone formed due to constrained growth around the cups, contains different dilutions of test extract⁷.

Anti-inflammatory activity:

Alternanthera pungens extract were suspended into 1% solution of acacia were prepared in two doses 100 and 250mg/kg body weight of each extract and standard (Indomethacin 20mg/kg) by rat paw oedema using Carrageenan^8,9,27 The percentage inhibition of edema was calculated for each group and the results were analysed the data using one-way ANOVA²⁴.

Antioxidant activity:

The antioxidant activity of the ethanolic extract of A. Pungens was measured in vitro by 2, 2 -diphenyl- 1- picrylhydrazyl (DPPH) assay, hydroxy radical scavenging method and superoxide radical scavenging activity.

DPPH free radical scavenging activity:

The free radical scavenging capacity of the ethanolic extract of A. pungens was measured in vitro by 2, 2 -diphenyl- 1- picrylhydrazyl (DPPH) assay^10,11,28. Briefly, a 0.1mm solution of DPPH in methanol was prepared and 1ml of this solution was added to 3 ml of the test, standard and control solution at different concentration (5, 10, and 50μg/ml). The mixtures were shaken vigorously and allowed to stand at room temperature for 30 minutes. Then absorbance was measured at 517nm using a UV-VIS spectrophotometer. Ascorbic acid was used as the reference. The capability of scavenging the DPPH radical was calculated by using the following formula.

Abs (control)-Abs (Test)

DPPH radical scavenging = ------------------------------ X 100

activity Abs control

Hydroxyl radical scavenging activity:

The deoxyribose method¹²was used to determine the hydroxyl radicals trapping capacity of MPE, as per standard method. Here FeCl₃-EDTA-ascorbic acid was used to generate OH radicals, as detailed below. The reaction was carried out in two conditions, in presence of EDTA, (non-site-specific) to determine its OH trapping capacity²⁰ and in absence of EDTA (site specific) to assess its metal chelation property.

Different concentrations (0.1–1000μg/ml) of extract were added to the reaction mixture in a final volume of 1 ml in potassium phosphate buffer (10mM, pH 7.4). This mixture was incubated at 37°C for 1 hr and then mixed with 1 ml of 2.8% TCA (w/v in water) and 1ml of 1% thio barbituric acid (w/v). It was then heated in a boiling water bath for 15 mts, cooled and then absorbance was measured at 532nm.

Abs(control)-Abs (Test)

Hydroxy radical scavenging = --------------------------- X 100

activity Abs control

Superoxide Radical Scavenging Activity:

Superoxide radical scavenging activity was measured by the reduction of NBT according to a previously reported method¹³. The 3ml reaction mixture contains riboflavin (20µg), EDTA (12μm), NBT (0.1mg), NADH (1ml) and various concentrations (5–50μg/ml) of sample solution. After incubation of mixture for 5mts at ambient temperature, the absorbance at 560nm was measured against an appropriate blank to determine the quantity of formazan generated.

RESULTS AND DISCUSSION:

Phytochemical analysis:

The results of phytochemical screening of extracts revealed the presence of phenolic compounds, flavonoids, steroids terpenoids, protein and saponins in the extracts of A. pungens

Table 1: Phytochemical analysis of Alternanthera pungens

S. No.	Name of the test	Observation
1	Steroids	+
2	Triterpenes	+
3	Saponins	+
4	Steroidal saponin	+
5	Glycosides	⁻
6	Alkaloids	⁻
7	Carbohydrates	⁻
8	Flavonoids	+
9	Tannins	⁻
10	Phenols	+
11	Iridoids	⁻
12	Cardiac glycosides	⁻
13	Mucilage	⁻
14	Proteins and amino acid	+

+= Positive ; - = Negative

Antimicrobial activity:

The ethanolic extract of A. pungens was evaluated against antimicrobial activity against two fungal²⁵ and four bacterial strains^,26. The resulted inhibition zones were shown good antibacterial activity. The results of the antibacterial activity (Table 2) reveals that all bacterial strains were more sensitive to A. pungens. The maximum zone of inhibition is achieved with ethanolic extract of A. pungens for Staphylococcus aureus and Escherichia. coli. A. pungens exhibited a moderate activity with Bacillus subtilis.

Anti-inflammatory activity:

The extract was tested at two different doses (100mg/kg and 250mg/kg), the extract exerted considerable inhibitory effect on rat paw swelling after carrageenan administration with more than 50% inhibition for all the two doses. The maximum inhibition (63.85% and 69.84%) was elicited by ethanolic extract of A. pungens for (100 and 250mg/kg). Indomethacin used as reference drug shows a similar inhibitory effect, it was able to exterminate paw edema by 72.35%. The results were analysed by using one-way ANOVA and tabulated in 3 and 4

Table 3: Effect of Ethanolic extract of A. pungens on Carrageenan induced paw edema

Sample	Volume of the paw time intervals (hr)
Sample	1	2	3	4	5	6
Control	4.0694	4.0694	4.0694	3.2674	3.1914	2.4492
(Indomethacin)20mg/kg	3.0395	2.3236	1.7295	0.9231	1.0851	0.6769
Dose-100mg/kg	3.2674	2.512	1.8821	1.7295	1.2459	1.0851
Dose -250mg/kg	2.9635	2.2608	1.7295	1.1655	1.0851	0.7385

Table 4: Percentage Inhibition of A. pungens

Sample	% inhibition of paw volume at various time intervals (hr)
Sample	1	2	3	4	5	6
Indomethacin 20mg/kg	25.31±0.47	42.90±0.45	57.5±0.45	61.95±0.44	71.74±0.45	72.35±0.45
Dose -100 100mg/kg	19.71±0.13	38.27±0.15	53.75±0.56	59.31±0.17	60.96±0.66	63.85±0.67
Dose -250 250mg/kg	27.17±0.14	44.44±0.13	57.5±0.18	64.32±0.19	65.99±0.14	69.84±0.48

Table 6 Scavenging activity of A. pungens by DPPH method

Concentration mcg/ml	Percentage inhibition			Mean % inhibition ±SD
Concentration mcg/ml	Trial 1	Trial 2	Trial 3	Mean % inhibition ±SD
5	2.3751	2.6666	2.6666	2.56±0.168
10	3.3169	3.5645	3.3454	3.40±0.135
15	8.4766	8.3876	8.4768	8.44±0.0514
20	10.4832	11.7768	11.4966	11.25±0.680
25	18.2637	18.5565	18.3647	18.39±0.148
30	26.7813	26.4568	26.3426	26.52±0.227
35	33.7018	33.333	33.7777	33.60±0.237
40	44.5126	44.4557	44.4967	44.48±0.029
45	58.1081	58.5568	58.5668	58.41±0.261
50	61.8755	61.9999	61.8999	61.92±0.065

DISCUSSION:

Invitro Antioxidant activity:

Antioxidant help body immune system to oppose free radicals and protect itself, they do either by scavenging free radicals or defending the dense mechanism¹⁴. The DPPH free radical assay is well reputed for evaluating electron donation potential of crude extracts^15,18. The extent of colour change is directly proportional to concentration and potency of antioxidants.

In our present study the ethanolic extract of A. pungens were performed by DPPH method, hydroxy radical scavenging activity method, superoxide radical scavenging activity method. Percentage DPPH is calculated for test extract and standard. The results are shown in Tab 6. The hydroxy radical scavenging activity was calculated for test extract and standard. The graph was plotted and results were shown in Fig 3. The superoxide radical scavenging activity of methanolic extract of A. pungens and ascorbic acid data were shown in Fig 4.

Invitro Anti-inflammatory activity:

Carrageen brought on paw edema is an approach that may be characterized by using biphasic occasion with the involvement of various inflammatory mediators. Administration of A. pungens extract inhibits irritation which was brought on by way of carrageenan^19,21. It has been said that presence of certain flavonoids posess anti-inflammatory activity by stabilizing lyosomal membrane¹⁶. The outcomes of our present study reveal A. pungens extract exhibit significant activity at all tested dose levels. The results were shown in Table 4.

Antimicrobial activity:

The antimicrobial activity results revealed that all bacterial stains were more sensitive to S. aureus, B. subtilis, P. vulgaris and E. coli. The maximum inhibition is achieved with ethanolic extract of A. pungens for E. coli and S. aureus whereas moderate activity with Bacillus subtilis. The results were shown in Table 2. It has been found that more highly oxidized phenols are more inhibitory to microorganism¹⁷. The phytochemical analysis of A. pungens exhibit the presence of steroid compounds responsible for antibacterial activity.

CONCLUSION:

The results of preliminary phytochemical screening suggest that extracts of Alternanthera pungens are good source of beneficial phytochemicals. The present study provided scientific evidence that extract of A. pungens possess antioxidant, Anti-inflammatory and antimicrobial properties. Our research finding shows good free radical scavenging activity and presence of total flavonoid content. The antimicrobial activity of different extract was performed with statistical analysis were incorporated with the present findings. Therefore A. pungens was explored to evaluate better potential plant as a source for antimicrobial and anti-inflammatory activity.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 22.05.2020 Modified on 15.07.2020

Research J. Pharm. and Tech. 2021; 14(7):3669-3673.

DOI: 10.52711/0974-360X.2021.00634

Micro organism	Standard (Ciprofloxacin 50µg/ml)	T1 (50mg)	T2 (100mg)	T3 (150mg)	T4 (200mg)
S. aureus	24±0.11	12±0.12	14±0.11	14±0.12	14±0.11
B. subtilis	25±0.23	10±0.13	12±0.14	12±0.22	14±0.13
P. vulgaris	24±0.12	12±0.12	12±0.14	13±0.23	14±0.11
E. coli	25±0.22	11±0.11	12±0.14	12±0.16	15±0.16

S. No.	Sample	DPPH radical scavenging activity	Hydroxyl radical scavenging activity	Superoxide radical scavenging activity
1	Alternanthera pungens	42±0.06	8±1.64	44±0.07
2	Ascorbic acid	12±0.05	1±1.18	23±1.04