INTRODUCTION:

Infectious diseases have become a critical health problem in most developing countries, including Indonesia¹. The causative agents of infectious diseases are pathogenic microorganisms, such as fungi, bacteria, viruses, or parasites². Infectious diseases constitute the second most prominent contributor to global mortality, accounting for a staggering 14.9 million fatalities, which is over a quarter of all deaths³.

Infectious diseases acquired by patients receiving treatment at the hospital are called nosocomial infections, which increase the mortality rate of hospitalized patients. Air in the emergency room of the Abepura General Hospital, Jayapura, Indonesia showed the presence of Escherichia coli (12%) and Staphylococcus aureus (11%), which can cause nosocomial infections⁴. E. coli causes urinary tract infections, diarrhea, and septic meningitis. S. aureus causes several skin diseases, acute respiratory infections (ARI), and urinary tract infections⁵.

Antibiotics are widely used in infectious diseases treatment mostly caused by bacterial pathogens. Nevertheless, the use of inappropriate antibiotics leads to bacterial resistance⁶. Therefore, exploring plants with antibacterial properties could be an alternative to overcome antimicrobial resistance in Indonesia⁷. People believe that natural ingredients have relatively fewer side effects and are more affordable than synthetic drugs⁸. To increase its effect, the synergistic mechanism of substances with antibacterial and antioxidant activities will provide benefits for medication. Antioxidants against inflammation caused by resistant strains of bacteria⁹.

Bawang Dayak, scientifically identified as Eleutherine americana (Aubl.) Merr., is a botanical species that exhibits promising potential in the field of antibacterial and antioxidant research. The origin of this plant may be traced to the region of tropical America; nonetheless, it has gained significant popularity and is currently extensively grown in several regions such as South Africa, Thailand, South China (specifically Hainan Island), and Indonesia (specifically Kalimantan Island)¹⁰. Empirically, the Dayak tribe (in Kalimantan, Indonesia) and people in other areas use its bulb to increase breastmilk production, and treat hypertension, diabetes, stroke, breast cancer, sexual disorder¹¹, coronary disorder¹², and wound¹³. The plant part of E. americana used as food or herbal remedies is the bulb, whereas its leaves are useless¹⁴. E. americana leaves from Pontianak, West Kalimantan, was known to contain alkaloids, flavonoids, saponins, phenols, triterpenoids, and steroids as antibacterial agents¹⁵. Additionally, another study revealed that 70% of ethanolic extract of E. americana bulb contained flavonoid of 34%¹⁴. Previous studies have shown antibacterial^16–22and antioxidant^11,20,23activities of its bulb. Furthermore, E. americana has been reported to contain antibacterial and antioxidant agents, such as naphthalene^24,25, anthraquinone^26–29, naphthoquinones^30,31, and stigmasterol^32–34.

The study aimed to assess the antibacterial and antioxidant activities, total phenolic content, total flavonoid content, and phytochemical screening of ethanolic extract from E. americana leaves. The plants used in this study originated from different cultivation sites: West Kotawaringin (Central Kalimantan), Malang (East Java), and Probolinggo (East Java). A previous study reported that different plant cultivation sites affected the metabolite profiles of Justicia gendarussa. Plants of different origins have various environmental factors, such as altitude, temperature, rainfall, climate, and soil³⁵. Various factors have the potential to qualitatively and quantitatively impact the primary and secondary metabolites of plants, hence leading to variations in their efficacy and safety^36–39.

MATERIALS AND METHODS:

Materials:

Eleutherine americana (Aubl.) Merr. leaves were collected from Madurejo Village, South Arut, West Kotawaringin, Central Kalimantan; Batu, Malang, East Java; and Klaseman Village, Gending, Probolinggo, East Java. The plant was determined at the UPT Laboratory of Materia Medica Batu (voucher number of 074/463A/102.7/2019). The culture of Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 33591 were acquired from Microbiology and Biotechnology Laboratory, Faculty of Pharmacy, University of Jember. Nutrient Agar (NA), Muller Hinton Agar (MHA), acetone, ethanol, Na₂CO₃, Folin-Ciocalteu reagent, and AlCl₃.5H₂O were provided by Merck (Darmstadt, Germany). Dimethyl sulfoxide (DMSO), DPPH, gallic acid, quercetin, BaCl₂, and H₂SO₄were obtained from Sigma-Aldrich (St. Louis, MO, USA). Blank disks and gentamicin disks were obtained from Oxoid (Cheshire, UK). Distilled water was provided by Brataco (Tangerang, Indonesia).

Extracts preparation:

The leaves of E. americana underwent a complete washing process using running water, subsequent drainage, and aeration for a duration of three days. Following this, the leaves were dried at a temperature of 50 °C. The leaves were ground into a fine powder and extracted using maceration method for three days by soaking the powder in 96% ethanol (1:10) and stirring several times a day. The macerate was separated, filtered using a Buchner funnel, and evaporated at 50 °C using a rotary evaporator (Heidolph Laborota 4000).

Inoculum preparation:

E. coli and S. aureus were cultured using colonies in NA media under aseptic conditions. The incubation time was 18-24 hours at 37 °C. Four or five colonies in NA media were added 10 mL of 0.9% NaCl, then homogenized using a vortex to obtain bacterial suspension as inoculum.

Antibacterial assay:

The disc diffusion method was utilized to conduct an antibacterial assay based on previous studies with minor modifications^40–42. The samples were ethanolic extracts of E. americana leaves at doses of 1, 5, 10, 20, and 40%. The bacterial suspension was flattened using a spreader to MHA media in a petri dish. Disc paper with the samples was placed on the surface of MHA media. A positive control consisting of a gentamicin disk with a mass of 10 µg was positioned at the center of the petri dish. Additionally, a negative control containing 10% DMSO was placed at the periphery of the positive control, aligned parallel to the samples located on the disk. The petri dishes have been covered and subjected to incubation for a duration of 18 hours at a temperature of 37°C. The measurement of the inhibitory zone's diameter was conducted using callipers.

Antioxidant assay:

A quantity of 0.8 mL of the DPPH solution (0.004% w/v) was introduced into 0.2 mL of the sample and allowed to incubate for a duration of 30 minutes under conditions of darkness. The measurement of antioxidant activity was conducted by assessing the reduction in the intensity of the purple colour ofthe DPPH solution using a UV-Vis spectrophotometer (Hitachi U1800) at 515 nm and calculated using equation 1^43–45.

A_control – A_sample

% DPPH inhibition = ------------------ x 100% ……(1)

A_control

The half maximal inhibitory concentration (IC₅₀) value was used to express antioxidant activity as an effective concentration for 50% reduction of DPPH free radicals⁴⁶.

Total phenolic content determination:

A 0.5 mL aliquot of a sample solution with a concentration of 2% w/v was subjected to a reaction with 5 mL of Folin Ciocalteu reagent in a volumetric ratio of 1:10 v/v and allowed to incubate for a duration of 5 minutes. A 4 mL of sodium carbonate was introduced into the combined solution, agitated for a duration of 15 seconds, and subsequently allowed to stand undisturbed for a period of 50 minutes. The absorbance was measured using a UV-Vis spectrophotometer at 743 nm. In order to determine the overall phenolic content of the samples, a calibration curve was plotted using gallic acid as the standard. The range of gallic acid concentrations used in the curve ranged from 60.00 to 270.00 µg/mL. Total phenolic content is expressed in gallic acid equivalent (GAE) in 1 g of extract⁴⁷.

Total flavonoid content determination:

A volume of 1 mL of a sample (2% w/v) was subjected to a reaction with 1 mL of AlCl₃ reagent with a concentration of 2% v/v. The resulting mixture was allowed to rest for a duration of 45 minutes. The sample absorbance was determined using a UV-Vis spectrophotometer at 437 nm. A calibration curve was constructed by plotting the concentrations of quercetin, which ranged from 4.00 to 8.00 µg/mL, in order to determine total flavonoid content. The quantification of total flavonoid content is expressed as quercetin equivalent (QE) in 1 g of extract^47,48.

Phytochemical screening:

The assay was conducted based on previous studies with slight modifications^49–52.

a) Flavonoids assay:

Three milliliters of n-hexane were mixed with 0.3 g of extract and agitated until the extract became colorless or pale. The residual substance was mixed with ethanol and filtered.

Bate-Smith and Metcalf test: A concentrated solution of hydrochloric acid with a volume of 0.5 mL was introduced into the filtrate. The mixture was then heated for 2-3 minutes, during which the alteration in color was carefully monitored. The presence of leucoanthocyanins was indicated by the red or purple color.

Wilstarter test: A volume of 0.5 mL of concentrated HCl and four pieces of magnesium were added to the filtrate, then a color change was observed. A half mL of distilled water and 1 mL of butanol were introduced to the mixture, and the discoloration was observed. The red orange color indicated the flavones content. The color change to pale red showed the presence of flavonols. Additionaly, a dark red color change indicated the existence of flavonones.

TLC: The filtrate was applied as spots onto a silica gel plate. The composition of the mobile phase consisted of a mixture of butanol, acetic acid, and water in a ratio of 4:1:5, and the staining agent was ammonia vapor. An intense yellow stain indicated the existence of flavonoids.

b) Alkaloids assay:

To 0.3 g of extract, 5 mL of HCl 2 N was added, heated and stirring for 2-3 min. A quantity of 0.3 g of NaCl was introduced into the mixture, agitated, and filtered. Subsequently, 5 mL of HCl 2N was introduced to the filtrate.

Mayer’s test: Mayer’s reagent was introduced to the mixture. The yellowish-white precipitate showed the existence of alkaloids.

Wagner’s test: Wagner’s reagent was added to the mixture. The brown precipitate indicated the existence of alkaloids.

TLC: The filtrate was added with 28% NH₄OH solution to result in an alkaline condition, then 5 mL of chloroform was added and filtered. The filtrate underwent evaporation, resulting in the formation of a residue that was subsequently dissolved in methanol. The solution was then spotted onto a silica gel plate. Ethyl acetate: methanol: water (9:2:2) was the mobile phase composition. The staining reagent was Dragendorff. The presence of alkaloids was indicated by the orange color.

c) Saponins assay:

Foam test: In this experiment, a mass of 0.3 g of extract was mixed with 10 mL of distilled water. The mixture was then agitated for a duration of 30 seconds. The existence of saponins was subsequently assessed by observing the foam formation.

d) Terpenoids/steroids assay:

A solution was prepared by dissolving 0.3 g of extract in 15 mL of ethanol.

Liebermann-Burchard test: Three drops of acetic acid and one drop of concentrated H₂SO₄were added to a 5 mL solution. The mixture was then gently agitated, and observed for the discoloration. The existence of steroidal saponins was indicated by a bluish green color, whereas the presence of steroidal triterpenoids was shown by a purplish red color. Additionally, the presence of saturated saponins was confirmed by a light yellow color.

Salkowski test: One or two milliliters of concentrated H₂SO₄ was added to a solution with a volume of 5 mL. The red ring appearance indicated the presence of unsaturated steroids.

TLC: The solution was spotted onto a silica gel plate. The mobile phase utilized in this study consisted of n-hexane and ethyl acetate (4:1) with anisaldehyde sulfuric acid as the staining reagent. A red-purple or purple color confirmed the presence of terpenoids or steroids.

e) Tannins assay:

A quantity of 0.3 g of extract was immersed in 10 mL hot distilled water, stirred, and cooled. Five milliliters of 10% NaCl and gelatin were then added to the mixture. The observation of a white precipitate suggests the existence of tannins.

f) Polyphenols assay:

A volume of 10 mL of heated distilled water was introduced to 0.3 g of extract, agitated, and subsequently cooled. The mixture was subjected to the addition of three to four drops of a 10% NaCl solution, which was subsequently followed by agitation and filtration.

FeCl₃ test: Several drops of FeCl₃ were mixed into a 4 mL filtrate. The blackish-green color showed the existence of polyphenols.

TLC: The filtrate was spotted onto a silica gel plate. The mobile phase was chloroform: ethyl acetate (1:9), and the staining reagent was FeCl₃. The black color indicated the presence of polyphenols.

Data analysis:

All procedures were performed in triplicate. The data were presented as the mean value ± the standard deviation and analysed using a one-way analysis of variance (ANOVA). The Least Significant Difference (LSD) approach was used to assess the presence of statistically significant differences. The statistical analysis results revealed a significant difference within the samples (α=0.05), as indicated by the p-value being less than 0.05.

RESULT AND DISCUSSION:

Extraction yield:

The extraction yields of E. americana leaves from different cultivation sites were 13.45% for EW, 16.61% for EM, and 14.85% for EP. The results showed that different numbers of metabolites were dissolved in the same solvent. The different cultivation sites of E. americana may cause different chemical compounds that affect its biological activities^53–55.

Determination of antibacterial activity:

Gentamicin disk as a positive control is an aminoglycoside inhibiting the growth of both gram-negative and gram-positive bacteria⁵⁶. The diameter of the inhibitory zone was in accordance with E. coli and S. aureus requirements of the Clinical and Laboratory Standards Institute (CLSI), i.e. 19-26 mm and 19-27 mm, respectively⁵⁷. According to the data shown in Table 1, it was known that gentamicin exhibited larger inhibition zones compared to all doses of E. americana.

The antibacterial capacity of E. americana leaves extract against S. aureus was higher than E. coli for all administered doses. The results indicated that the extract is more effective as an antibacterial agent against positive gram bacteria. S. aureus exhibited greater sensitivity in comparison to E. coli because of the structure differences of bacterial cell wall. E. coli possessed a cell wall enveloped by an outer membrane consisted of phospholipids, lipopolysaccharides, and proteins. The composition of the cell wall of S. aureus is characterized by a small number of lipids, thick peptidoglycan, and teichoic acid⁵⁸.

E. americana extract at a dose of 1% exhibited the lowest activity, which may be related to the lowest concentration of antibacterial compounds. Otherwise, the extract dose of 40% had the highest antibacterial capacity against E. coli and S. aureus. Nevertheless, the activity of 40% dose remained lower in comparison to gentamicin. It is possible that an increase in extract dose resulted in antibacterial activity enhancement to obtain similar capacity like gentamicin. The degree of bacterial growth inhibition increases in direct proportion to the concentration of the antibacterial substance. This is consistent with the findings of the research, which indicated that as the extract concentration increased, so did the diameter of the inhibition zone.

From Table 1, it was observed that the antibacterial activity of EP40 (EP at a dose of 40%) against E. coli was not significantly different compared to EM40 (EM at a dose of 40%) and EW40 (EW at a dose of 40%), although EM40 showed the largest inhibition zone of 9.70±0.20 mm. Meanwhile, the order of the antibacterial activity assessment of the ethanolic extract of E. americana leaves against S. aureus was as follows: EW40< EM40< EP40. In this study, EP40 exhibited the highest capacity against S. aureus with inhibition zone of 12.25±0.17 mm. Different antibacterial activity among the samples is possible because plant extract from different cultivation sites contained distinct chemical compounds⁵⁹. The previous study reported that naphthalene as an active chemical constituent of E. americana bulb exhibited inhibitory effects on the growth of S. aureus at a dose of 0.0125% and E. coli at a dose of 0.025%⁶⁰. The inhibitory zone of E. americana bulb extract with a concentration of 40% against S. aureus was measured to be 14.00 mm, whereas against E. coli was slightly smaller at 13.85 mm⁶¹.

Table 1: Inhibition zone of E. americana leaves extract from different cultivation sites against E. coli and S. aureus

Groups	Inhibition zone (mm)
Groups	*E. coli*	*S. aureus*
EW1	0.00±0.00^a	6.40±0.32^a
EW5	6.53±0.23^b	7.04±0.13^b
EW10	7.47±0.34^c	7.62±0.41^c
EW20	8.36±0.23^d	8.47±0.15^d
EW40	9.04±0.41^e	9.26±0.14^e
EM1	6.68±0.10^b	7.00±0.22^b
EM5	7.59±0.03^c	7.75±0.42^c
EM10	8.36±0.04^d	8.67±0.25^d
EM20	9.28±0.13^e	9.33±0,22^e
EM40	9.70±0.20^f	10.15±0,09^f
EP1	0.00±0.00^a	0.00±0.00^g
EP5	6.77±0.09^b	8.31±0.42^cd
EP10	7.91±0.22^cd	9.42±0.23^e
EP20	8.49±0.15^d^e	11.25±0.18^h
EP40	9.60±0.34^e^f	12.25±0.17ⁱ
Positive control (gentamicin)	23.44±0.66^g	24.13±0.66^j
Negative control (10% DMSO)	0.00±0.00^a	0.00±0.00^g

The data were presented as the mean values of three samples ± the standard deviation (SD). Significant differences among the superscript letters within the same column were observed based on the Least Significant Difference (LSD) test at a significance level of p<0.05. EW1: E. americana from West Waringin at a dose of 1% (b/v); EW5: E. americana from West Waringin at a dose of 5% (b/v); EW10: E. americana from West Waringin at a dose of 10% (b/v); EW20: E. americana from West Waringin at a dose of 20% (b/v); EW40: E. americana from West Waringin at a dose of 40% (b/v); EM1: E. americana from Malang at a dose of 1% (b/v); EM5: E. americana from Malang at a dose of 5% (b/v); EM10: E. americana from Malang at a dose of 10% (b/v); EM20: E. americana from Malang at a dose of 20% (b/v); EM40: E. americana from Malang at a dose of 40% (b/v); EP1: E. americana from Probolinggo at a dose of 1% (b/v); EP5: E. americana from Probolinggo at a dose of 5% (b/v); EP10: E. americana from Probolinggo at a dose of 10% (b/v); EP20: E. americana from Probolinggo at a dose of 20% (b/v); EP40: E. americana from Probolinggo at a dose of 40% (b/v).

Determination of antioxidant activity:

The assessment of antioxidant activity for E. americana leaves extract was conducted using the DPPH method and analyzed utilizing a UV-Vis spectrophotometer at 515 nm. The mechanism for DPPH scavenging as free radicals by antioxidants is through hydrogen atoms (protons) donation to free radicals⁴⁶. The non-radical form of DPPH exhibits a gradual reduction in its purple color after extract addition, which is indicated by a decrease in DPPH absorption. The result for antioxidant activity of E. americana leaves extract is presented in Table 2. The research findings indicated that EP exhibited the most significant antioxidant activity, as evidenced by its IC₅₀ value of 32.83±3.51 µg/mL. Antioxidant activity of E. americana leaves ethanolic extract was ordered as follows: EP > EM > EW. This may be related to the different growth locations for all samples⁶². A prior study showed that 70% ethanolic extract of E. americana leaves from Kubu Raya, West Kalimantan showed comparable antioxidant capacity with IC₅₀ value of 31.97437 µg/mL⁶³. Several mechanisms of action of antioxidant compounds as antibacterial agents are inhibiting oxidation at several points depriving cells of energy, destruction of membrane structure causing cell nutrients leakage, and blocking binding sites of gyrase, ceasing DNA coiling and retarding cell growth⁹.

Table 2: Antioxidant activity, TPC and TFC of E. americana leaves extract from different cultivation sites

Groups	IC₅₀ (µg/mL)	TPC (mg GAE/g extract)	TFC (mg QE/g extract)
EW	83.78±1.54^a	84.99±0.40^a	0.45±0.01^a
EM	66.94±1.37^b	99.88±0.29^b	0.56±0.01^b
EP	32.83±3.51^c	129.40±0.19^c	0.61±0.01^c
Gallic acid	11.09±0.73^d	-	-

Determination of total phenolic content:

Phenolic compounds play an essential role as antioxidants through its ability to decrease the permeability of the outer membrane, induce leakage of cytoplasmic contents, and inhibit the synthesis of nucleic acids. Polyphenols interact with non-specific forces, including hydrogen bonding and the hydrophobic effect, as well as lipophilic forces, such as the formation of covalent bonds. These interactions result in the disruption of cell envelope transport proteins, microbial adhesion, and enzymes. Another mechanism of antibacterial activity for phenolic compounds is iron chelation, which is important for most bacteria survival. Additionally, these compounds cause cell wall rupture, enhance cytoplasmic membrane permeability, and release of lipopolysaccharides⁹. The total phenolic content of E. americana extract was shown in Table 2, calculated based on gallic acid standard curve (Figure 1).

The extract of EP had the highest total phenolic content of 129.40±0.19 mg GAE/g extract. EW exhibited the lowest total phenolic content, measuring at 0.61±0.01 mg GAE/g extract. The research findings implied that the ethanolic extract obtained from E. americana leaves, which were cultivated in various locations, exhibited variations in their total phenolic content. The results obtained were consistent with the findings published by Noriega et al.⁶⁴, who observed variations in the total phenolic content and antioxidant capacity of white strawberries (Fragaria chiloensis) from different geographical regions.

Figure 1: Calibration curve for total phenolic content (A) and total flavonoid content (B) determination (n=3)

Determination of total flavonoid content:

Besides phenolic compounds, flavonoids possess antioxidant properties and exhibit antibacterial effects. These compounds act through deterioration functions of a bacterial membrane, including altering the fluidity of the cytoplasmic membrane and inhibiting the formation of the cell wall and cell membrane. They interfere with the synthesis of nucleic acids and impair respiratory metabolism⁹. The total flavonoid content of E. americana extract was calculated based on a quercetin standard curve (Figure 1), as shown in Table 2. EP exhibited the highest total flavonoid contentin comparison to other extracts (0.61±0.01 mg QE/g extract). EW had the lowest total flavonoid content of 0.45±0.01 mg QE/g extract. This result was in accordance with previous study, which has demonstrated that several onion varieties planted in different locations with distinct environmental conditions exhibited different antioxidant activity, total phenolic and antioxidant content⁶⁵.

Phytochemical screening:

The present investigation comprised the application of phytochemical screening to determine secondary metabolites content in ethanolic extract of E. americana using the tube test and TLC methods. Previous studies reported that 70% ethanolic extract of E. americana leaves from Pontianak, West Kalimantan had alkaloids, flavonoids, saponins, phenolics, triterpenoids, and steroids content^14,66. The research exhibited that EW, EM, and EP were found to contain alkaloids, terpenoids, flavonoids, polyphenols, and tannins (Table 3). The chemical compounds were suggested to promote the antibacterial activity of the extracts.

The secondary metabolites have different mechanisms of antibacterial activity. Alkaloids inhibit topoisomerase enzymes and DNA interchelators. Alkaloids also inhibit bond formation to DNA, interrupting protein and nucleic acid synthesis. Hence, this leads to metabolism disruption and cell lysis⁶⁷. Tannins are involved in protein binding by coagulating the protoplasm of bacterial cells. Tannins are also involved in the disruption of bacterial cell membranes and the formation of metal ion complex bonds, resulting in toxicity⁶⁸. Polyphenols have the ability to inhibit the activity of hydrophilic enzymes, including proteases and carbohydrolases. They can also disrupt the functioning of adhesins and non-specific interactions involving carbohydrates, leading to their inactivation⁶⁹. Terpenoids react with transmembrane proteins, specifically porins, located on the outer membrane of bacterial cell wall, thereby reducing the permeability of bacterial cell wall⁷⁰.

Table 3: Phytochemical screening of E. americana leaves extract from different cultivation sites against E. coli and S. aureus

Phytochemical compounds	Reagent/Test	EW	EM	EP
Alkaloids	Mayer/tube test	+	+	+
	Wagner/tube test	+	+	+
	Dragendorff/TLC	+	+	+
Saponins	Foam test/tube test	-	-	-
Terpenoids/ steroids	Liberman-Burchard/tube test	+	+	+
	Salkowski/tube test	+	+	+
	Anisaldehyde sulfuric acid/TLC	+	+	+
Flavonoids	Bate-Smith and Metcalf/tube test	+	+	+
	Wilstater/tube test	+	+	+
	Ammonia/TLC	+	+	+
Polyphenols	FeCl₃/tube test	+	+	+
Polyphenols	FeCl₃/TLC	+	+	+
Tannins	Gelatin/tube test	+	+	+

(+): Positive result; (-): Negative result; EW: E. americana from West Waringin; EM: E. americana from Malang; EP: E. americana from Probolinggo.

CONCLUSION:

The study showed that EW, EM, and EP had been proven to inhibit E. coli and S. aureus growth. EP had higher antioxidant activity, total phenolic and total flavonoid content than EW and EM. Different growing sites of the plant may cause the different results. The activity of E. americana leaves extract was expected related to its contents of alkaloids, terpenoids, flavonoids, polyphenols, and tannins.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors acknowledge thanks to Institute for Research and Community Service (LP2M) University of Jember for supporting this research through Hibah Reworking Skripsi/Tesis under grant number of 6326/UN25.3.1/LT/2022.

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Received on 14.11.2023 Modified on 09.02.2024

Research J. Pharm. and Tech 2024; 17(11):5597-5604.

DOI: 10.52711/0974-360X.2024.00854