INTRODUCTION:

Even now, natural resources maintain a significant role in drug research; in the previous thirty years, natural products have been the source of up to fifty percentage of authorized medications¹. Medical practice has used herbal medicines for thousands of years. They are still used today, particularly as a valuable and easily has accessible form of healthcare². The flowering parts of the plant are boiled and consumed by rural people who

need to increase their haemoglobin levels. Hygrophila auriculata it is a highly effective treatment for kidney stones; researchers have sought to demonstrate the plant's efficacy through scientific testing. The plant contains alkaloids, flavonoids, glycosides, tannins, quinone, and saponin, which comprise the majority of phytochemicals. Literature reviews identified a number of important pharmacological effects.

The process of developing new drugs is intricate, varied, innovative, and tightly regulated.^3.A different research proposed that the phytochemical composition of Hygrophila auriculata, including flavonoids and polyphenolic chemicals, was responsible for the plant's hepatoprotective properties⁴ In the present review, we highlight the ethnobotanical and traditional uses, phytochemical analysis, and pharmacological studies of Hygrophila auriculata⁵ Antioxidants are naturally produced by plants containing beneficial phytochemicals ⁶. Their selection to analyze a methanol flower extract of Hygrophila auriculata using GC-MS and Based on the literature, its anti-oxidant and anti-microbial activities was investigated.

MATERIALS AND METHODS:

Hydrophila auriculata flowers were collected from vadakku kanjankollai Village, Cuddalore District, Tamil Nadu, Dr. P. Mani, the director of the herbarium and centre for molecular systematics, identified and authenticated the flower. In Kumbakonam, Tamil Nadu, a voucher specimen (0821/22) has been deposited in the herbarium at the Annai College of Arts and Sciences. India

Extraction:

A mixture of 95% methanol, acetone, ethyl acetate, and chloroform (Sigma Aldrich Co., India) was used for extraction. The initial stage of the experimental work involves first isolating chemical compounds from the chosen plant and then identifying those isolated compounds. Hygrophila auriculata (3kg) dried and powdered flowers were exhaustively extracted with 95% methanol (MeOH, 2.5 litres x 3) at room temperature. Under reduced pressure, methanol extract was dried to yield 110g. In the fractions, Acetone (15g), CHCl3 (36.0 g), EtOAc (12.5g) MeOH (17.4g) were collected. By using FT-IR and GC-MS spectroscopic techniques, the structure of a compound isolated from a methanolic flower extract of Hygrophila auriculata was elucidated.

Method of antimicrobial activity discs:

The antibacterial activity of lemongrass oil was investigated by cultivating Staphylococcus aureus, Bacillus subtillis, Sarcina lutea and E. coli, Pseudomonas aeruginosa, and Candida albicans on agar plates with varying amounts of oil applied using the Agar Well Method. The plates were incubated for 24 hours at 37°C. The organism treated with the test substance was observed to compare its growth with that of the control.⁷

TEST ORGANISMS:

For the microbiological work, the pure bacteria cultures stored in the microbiology lab were utilized. The test organisms were maintained in nutrient agar medium. In this investigation, Staphylococcus aureus, Bacillus subtillis, Sarcina lutea and E. coli, Pseudomonas aeruginosa and Candida albicans were used as test organisms. The culture media for this experiment was either potato dextrose agar or nutrient agar. In pre-sterilized Petri dishes (25ml), the molten nutrient agar, also known as potato dextrose agar, was distributed and permitted to cool. Previously, the test bacteria were suspended in distilled water and evenly incubated on these agar plates. It was permitted for the plates to solidify. After the agar had set, 6 mm-diameter holes or wells were bored into it using a flamed cork borer. For each plate, three wells were formed. Plant extract was poured into one hole at a 100% concentration and another well at a 50% concentration. The last hole was filled with solvent as a control. For 24hours, the Petri plates were cultured for microorganisms at 37°C. Petri plates and discs containing individual doses of plant extracts as well as control and standard (novobiocin, chloramphenicol) discs were made. Following the incubation period, each well or cup's inhibitory zone's diameter was measured, and the results were recorded. Each experiment began with the preparation of a well by filling it with nothing but solvent as a negative control. Following that, the outcomes were recorded while comparing the methanolic floral extract to the standard, control, and methanol settings. In millimeters, the results were expressed.⁸

ANTI-OXIDANT STUDIES:

DPPH scavenging assay:

Associates used spectrophotometry to carry out the radical scavenging experiment⁹. The technique examined the absorbance at 517nm, which indicates how well the DPPH free radical can be scavenged. 90.25 mM of 2,2-Diphenyl-1-picryl hydrazyl (DPPH) were employed in methanol under dim illumination. The produced HECOL was tested using the usual methodology with minor changes for the DPPH (1,1-Diphenyl2-picryl hydrazyl) assay^10,11

Procedure:

A methanolic solution of DPPH (90.25mm) was combined with a methanolic extract of Hydrophila auriculata (250–1500g), and the mixture was then increased in volume with methanolic DPPH to 1.0mL. The absorbance at 517nm was found using a UV-visible Spectrophotometer from Systronics after 20minutes. Ascorbic acid served as a benchmark for comparison in the market. To calculate the percentage (%) by which DPPH inhibited free radicals, the following equation was utilised.

Scavenging of H₂O₂

The approach was used to assess Hydrophila auriculata capacity to scavenge H₂O₂. A 40mm H₂O₂ solution in phosphate buffer was created (pH 7.4). In a spectrophotometer, the absorbance at 230nm was used to estimate the H₂O₂ concentration (SL 159, UV-visible spec, E. coli, India). A H₂O₂ solution was supplemented with extracts (200, 400, 600, 800, and 1000g). The following equation was used to determine how well Hydrophila auriculata scavenged H₂O₂ in comparison to the standard.

Compound – (1) 4-methoxy-3-(methoxymethyl) Phenol − MP 67 ^oC; UV: 223-325 nm; IR (KBr): νmax 3575, 3010, 2850, 1590,1622,1608,1272, 1052, 850, and 698; M ⁺ m/e: 166.4815; ¹H NMR: 7.02, 6.93, 3.61-3.42, 4.59. ¹³C-NMR: δ156.7, 118.7 12,7.12, 152.3, 111.13, 115.28,51.62, 72.85, 55.85.

Compound (2) (E)-N'-(5-bromo-2-methoxybenzylide4-methoxy benzohydrazide – MP 65 ^oC; UV: 273 - 378 nm; IR (KBr): νmax 3285, 3080, 3030, 3025, 3017, 2985, 1666, 1620, 1575, 1525, 685, M⁺ m/e: 362.1281, ¹H NMR: 7.63, 6.95, 6.78, 8.20, 7.16, 6.85,3.82, 12.1: ¹³C NMR: 55.23, 57.20, 159.3,112.8, 133.1, 126.1, 141.7, 162.8, 128.7, 131.3, 117.4, 161.3.

RESULT AND DISCUSSION:

The current work presents the isolation, antimicrobial, and anti-oxidant properties of Compound 1 and compound 2. A significant peak was visible in the GC/MS data at the retention time of 11.38. Peaks at m/z 150.40 and 120.32 in the compound 1 mass spectrum are indicative of the ions that the molecule produces ¹². The following elemental analysis results, anal, were used to validate this molecular formula. O - 28.59 (calcd, 28.65) %; C - 62.27 (calcd, 63.14); H - 7.11 (calcd, 7.14). The peak areas of absorption at 223–325 nm in the UV–VIS spectra. A phenolic group is present, as evidenced by the existence of IR absorptions at 3575, 1622, 1608, and 1272cm1¹³. The band at 1052 cm1 was identified as the source of the symmetric stretching between the -CH3 and -OCH3 groups. Compound 1 signal pattern at 7.02, 6.93 which was ascribed to H2, H5, and H6 and revealed the presence of aromatic rings that are 1, 3, and 4 trisubstituted. ¹⁴

The signals for substituted carbons in the¹³C-NMR spectra at 156.7, 127.12, and 152.3. Further validated the tri-substituted aromatic ring. When compared to C-1, C-3 and C-4 move downfield, indicating that they include oxygen functions. The existence of groups that donate electrons, such as (-OCH3) at C-7 caused a strong signal at 3.61 and 55.85ppm. The ¹H NMR signals at 4.59 and 3.42, respectively, with 2 and 3 proton intensities. Following a discussion of the compound 1 spectrum evidence, 1 was identified as 4-methoxy-3-(methoxymethyl) phenol.¹⁵

compound 2 was obtained. The peak of the molecular ions at m/z 362.1281. The following elemental analysis results, anal, were used to validate this molecular formula. C -52.81 (calculated as 52.92); H -4.15 (4.12); N -7.69 (7.76); Br -21.99 (22.0); and O -13.12 (4.12; calculated as 13.24) %.¹⁶

The C=N and -O=C-N- stretching frequencies in the 1620–1666 cm1 range were observed by the two azomethine groups and the NH bands were detected to absorb in the area of 3285cm1 in the IR spectra. This fell within the range that had been mentioned for the same class of Schiff base ligands.Additionally, the IR spectra revealed the stretching bands Ar-H in the area of 2985, C=C on the aromatic ring at 1575, and C-Br at 685¹⁷.

The 1H NMR spectra displayed one pattern of ABX-type signals that emerged at 7.63, 6.95, 6.85, and 7.16, respectively. Additionally, methoxy protons were detected in the 3.82ppm range, and the 13C NMR spectra revealed signals at 55.2 and 57.20, respectively, the production of Schiff bases was also supported by the 13C NMR data. C=O and C=N have been credited for two peaks at 162.8 and 141.7. carbons respectively¹⁸.

Antimicrobial Activity:

Plant-based antimicrobial activity is beneficial in the treatment of a number of illnesses. The other interactions to inactivate microbial adhesions¹⁹. The disc diffusion technique was used in this study to assess the methnoal flower extract. Anti-microbial activity using a variety of clinically significant strains at doses of 50μl/disc, 100μl/disc and 150μl by disc diffusion method. Staphylococcus aureus, Bacillus subtillis, Sarcina lutea gram positive group and E. coli, Pseudomonas aeruginosa and Candida albicans gram negative group. were the microbes used for the study. The table below lists the methnoal floral extract of Hygrophila auriculata's antibacterial properties. Hygrophila auriculata methanol flower extract was shown to be the most efficient antibacterial agent. For Staphylococcus aureus, Bacillus subtillis, Sarcina lutea and E. coli, Pseudomonas aeruginosa and Candida albicans, respectively. The ethnoal flower extract exhibited a mean zone of inhibition between 35.0 and 25.0mm.

The commercial antibiotic produced one between 1.0 and 27.0 mm. Based on the results, In comparison to the commercial antibiotic, the methanoal extract at 200μg demonstrated the highest zone of inhibition against all tested bacteria. Furthermore, it was more potent against S. aureus and E. coli, exhibiting no growth in both species. In comparison to the other studied microorganisms, S. aureus showed a small reduction in the inhibitory zone at 200μg. Nevertheless, there was no action seen by the methanol extract against C. albicans.

Table:1 Anti microbial effects of methanol extract drug determined by agar diffusion method. (zone of inhibition in mm)

Microorganisms	50 µl	100 µl	200 µl	Standard-1 novobiocin (50µg)	Standard-2 chloramphenicol (50µg)
S. aureus	15	21	27	32	-
B. subtillis	6	13	18	35	-
S.lutea	7	14	18	-	26
E.Coli	8	16	23	-	27
P.aeruginosa	1	4	9	30	-
C. albicans	-	-	-	-	25

Antioxidant activity:

One of the nitrogen atoms in the stable free radical known as DPPH has an unpaired valence electron. One of the more often used antioxidant tests is the scavenging of DPPH free radicals. is a decolorization assay that does so by tracking their absorbance with a spectrophotometer at a wavelength of 517nm²⁰. Flower extract to find out how well it scavenges free radicals. The 1,1-diphenyl-2-picrylhydrazine free radical is reduced by the floral extract of H. auriculata to the yellow 1,1-diphenyl-2-picrylhydrazine, and with increasing extract content, the potential to reduce increases. At a concentration of 120g/mL, the scavenging activity was 66.20.19%. The IC50 was compared to the IC50 of traditional ascorbic acid, which was 173.56g/mL.

Hydrogen peroxide scavenging assay:

According to Figure 1 (P 0.05), the scavenging activity of several H. auriculata extracts on hydrogen peroxide was concentration-dependent (50-250g/mL). Strong H₂O₂scavenging activity was shown by the methanol:water extract (IC 50: 147.62g/mL). In contrast, water extract had an IC 50 of 133.72g/mL. (Table 1). In the natural world, H₂O₂ may be found in small amounts in the air, water, human body, plants, microbes, and food²¹

Many of the enzymes needed by cells and microbes to carry out regular metabolic reactions are also inhibited by them. It is so claimed that flavonoids inhibit the ability of several bacteria and viruses to spread and multiply within cells ^22,23

H₂O₂ASSAY OF PLANT EXTRACT

Test	Concentration of plant extract (mg/ml)	% of Inhibition	Ascorbic acid
H₂O₂ assay	50	30.0	34.0
	100	43.7	44.0
	150	52.6	52.0
	200	63.0	61.4
	250	63.0	71.0
IC₅₀ VALUE		147.62	133.72

DPPH SCAVENGING ACTIVITY OF PLANT EXTRACT

Test	Concentration of plant extract (mg/ml)	% of Inhibition	Ascorbic acid
DPPH assay	50	27.2	34.3
	100	31.5	42.4
	150	46.2	51.1
	200	55.1	63.2
	250	65.1	71.6
IC₅₀ Value		173.56	135.65

Fig: Hygrophila auriculata flower extract GC-MS chromatogram

CONCLUSION:

Utilizing GC-MS and FT-IR, the current work defined the phytochemical profile of the Hydrophila auriculata flower extract. The concentrations of different components that were eluted as a function of retention time are displayed on the chromatogram. Flower extracts could have produced many biologically active constituents in the present study. The flower extracts from Hydrophila auriculata were more efficient in their anti-microbial and antioxidant activities, according to experimental research.

FUNDING:

This work is not supported by any further funding.

CONFLICT OF INTEREST:

There is no conflict of interest, according to the authors.

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Received on 03.01.2024 Revised on 09.05.2024

Accepted on 12.07.2024 Published on 20.01.2025

Available online from January 27, 2025

Research J. Pharmacy and Technology. 2025;18(1):39-43.

DOI: 10.52711/0974-360X.2025.00006

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