Mitragyna parvifolia as apotential therapeutic agent for lymphatic filariasis

Abstract

This study investigates the medicinal potential of Mitragyna parvifolia (M. parvifolia) leaves for the management of Lymphatic filariasis (LF). Phytochemical screening of the methanolic leaf extract revealed the presence of alkaloids, terpenoids, phenols, tannins, and flavonoids. The GC–MS analysis identified 24 phytoconstituents, including the major alkaloid “mitraphylline.” Infrared spectroscopy confirmed the presence of various functional groups corresponding to the identified compounds. The extract exhibited significant antibacterial activity against Staphylococcus epidermidis, Bacillus cereus, and Salmonella typhi. In vitro macrofilaricidal screening demonstrated dose-dependent inhibition of worm motility and MTT reduction, indicating its potential as a macrofilaricidal agent. The larvicidal bioassay showed notable effectiveness against Culex quinquifasciatus larvae, with 1% concentration displaying the highest larvicidal activity. Concentration-dependent antioxidant activity was observed using the DPPH assay, with 100 µg/ml showing the highest antioxidant potential. The findings suggest the potential of M. parvifolia leaves for LF management, supporting further research to identify active compounds and elucidate their mechanisms of action. The study highlights the plant’s diverse bioactive compounds, antibacterial and macrofilaricidal activities, larvicidal efficacy, and significant antioxidant properties. Future investigations, including in vivo experiments and clinical trials, are warranted to validate the safety and efficacy of M. parvifolia as a potential therapeutic agent for LF.

Introduction

Lymphatic filariasis (LF), a prevalent neglected tropical disease, poses a significant threat to over 882 million individuals across 44 countries worldwide. To curb the transmission of this parasitic infection, preventive chemotherapy is essential (WHO 2005). LF is caused by filarial nematode worms Wuchereria bancrofti, Brugia malayi and Brugiatimori with mosquitoes being the mode of transmission of the disease. Culex quinquefasciatus, being widespread and capable of breeding in various water sources, is the most common among all filarial vectors and has the greatest impact on the loss of disability-adjusted life years (DALYs) caused by LF (Das and Shenoy 2017). Acute manifestations of lymphatic filariasis (LF) can be triggered by various secondary infections, such as bacterial and fungal infections. Patients with filarial swelling may experience recurrent acute attacks multiple times a year, depending on the presence of precipitating factors. Current evidence supports the presence of bacterial infections, particularly Bacillus cereus, Staphylococcus epidermidis, S. hominis, S. capitis, S. xylosus, S. aureus and Micrococcus spp, in LF patient samples (Olszewski et al. 1997; Pal et al. 2015). The affected limbs often exhibit lesions that facilitate the entry of these infectious agents, contributing to the persistence and progression of lymphedema, ultimately leading to elephantiasis. The prevailing drugs for the treatment of LF in Mass Drug administration (MDA) are Diethylcarbamazine citrate (DEC), Albendazole and Ivermectin which provide only temporary clearance of microfilariae without effectively eliminating all adult worms (Bockarie and Deb 2010). In cases where the worms display insensitivity to a single dose of DEC, administering the drug repeatedly does not result in parasite elimination. Furthermore, treatment with DEC does not appear to reverse the established lymphatic damage in adult worms (Addiss and Dreyer 2000). Providing a vital and endorsed bundle of healthcare measures can alleviate the distress and hinder the progression of disability among individuals affected by diseases resulting from LF. Mitragyna parvifolia is a plant species that belongs to the Mitragyna genus within the Rubiaceae family. It is commonly referred to as Kadam, a highly esteemed medicinal plant species currently facing a critical endangerment crisis (Patel et al. 2020). The botanical resources of this plant have been harnessed for their therapeutic potential in various medical conditions. Its leaves have been employed as a dressing for wounds and ulcers, demonstrating analgesic and anti-inflammatory effects that promote enhanced healing. The fruit juice has shown augmentation properties for lactating mothers, increasing breast milk production and functioning as a lactodepurant (Panwar and Tarafdar 2006). The fresh leaf sap of Mitragyna parvifolia is employed by tribal communities, including the Chenchus, Yerukalas, Yanadis, and Sugalis in Guntur District, Andhra Pradesh, for treating jaundice. In the Tumkur district, Karnataka, India, the stem bark of M. parvifolia is used by local inhabitants to address issues related to biliousness and muscular pains (Choudhary and Jain 2016). While M. parvifolia has been used for centuries in traditional medicine, there is still a lack of scientific research on its safety and efficacy. Some studies have suggested that the plant may have potential health benefits, but more research is needed to fully understand its effects on the body.

Materials and methods

Plant material collection and extract preparation (Vishal and Sanjay 2010)

Fresh leaves of M. parvifolia were collected from the local areas of Madurai. They were washed with distilled water and completely dried in the shade. The dried leaves were finely ground in an electric blender and sieved. About 60 g of dried leaf powder were packed in a filter paper and extracted by continuous hot extraction in a Soxhlet extractor with 250 ml of analytical grade methanol. The extraction procedure was done until the solvent became colourless (about 48 h). The extract was later evaporated in rotary evaporator (Heidolph Hei-Vap Value rotary evaporator system).

Phytochemical screening

Preliminary phytochemical screening was done to qualitatively detect the presence of secondary metabolites.

Test for alkaloids (Evans 2002, Chapter 6):

The filtered extract was treated with Mayer’s reagent (potassium mercuric iodide solution). The yellow precipitate formation would indicate the presence of alkaloids.

The filtered extract was treated with a few drops of Wagner’s reagent (solution of iodine in potassium iodide). Red-brown precipitate formation would indicate the presence of alkaloids.

Test for flavonoids

The extract was treated with a few drops of Sulfuric acid. The formation of orange colour indicates the presence of flavonoids.

Test for terpenoids

0.2 g of the extract when mixed with 2 ml of chloroform and 3 ml of concentrated H₂SO₄ was cautiously added to form a layer. A reddish-brown colouration of the inner face indicates the presence of Terpenoids.

Test for phenols

Extracts were treated with a few drops of 5% ferric chloride solution. The formation of bluish black colour indicates the presence of phenol.

Test for tannins

A small quantity of extract was mixed with water and heated in a water bath. The mixture was filtered, and 0.1% ferric chloride was added to the filtrate. A dark green colour formation indicates the presence of tannins.

Defatting of the leaf extract

The Soxhlet leaf extract (1 g) was subjected to hexane treatment (10 ml) and stirred for 1 h. Afterward, centrifugation was performed at 10,000 rpm for 15 min, discarding the supernatant. The resulting pellet was resuspended in 10 ml of hexane treatment and stirred for an additional hour. This process of hexane treatment and centrifugation was repeated three times. The final pellet was collected and the air-dried extracts were utilized for further studies.

Characterization of M. parvifolia methanolic leaf extract

Gas chromatography-mass spectrometry (GC–MS)

The samples were analyzedusing GCMS-TQ8040NX (SHIMADZU), which was fitted with the capillary column SH-RXi-5SIL Ms (30 mm length X 0.25 mm internal diameter X 0.25 µm thickness). The temperature program was: 100–180 °C at 15 °C min⁻¹, 4 min hold at 180 °C and 180–280 °C at 5 °C min⁻¹ and 10 min hold at 280 °C. The injector temperature was 280 °C The flow rate of carrier gas (helium) was 0.5 ml/min. The sample was derivatized by BSTFA (N,O-bis(trimethylsilyl)trifluoroacetamide) and pyridine. The derivatized sample was further dissolved and diluted in methanol and injected automatically. MS spectra of separated compounds were compared with one from the NIST 2017 mass spectral database. The upper and lower mass limits were set at 40–500 m/z (Tallini et al. 2018).

Fourier transform infrared (FT-IR)

Infrared light from a suitable source pass through a scanning Michelson interferometer and Fourier transformation gives a plot of intensity versus frequency. When the sample is placed, it absorbs specific frequencies, causing their intensities to decrease in the interferogram. The resulting Fourier transform is the infrared absorption spectrum of the sample. The Nicolet 6700 FT-IR spectrometer was used for FT-IR measurements. Fourier transformed infrared spectra weregenerated typically from 4000 to 400 cm⁻¹ representing the molecular fingerprint of the Soxhlet extract to identify various functional groups present in a sample based on the characteristic absorption bands in the infrared spectrum.

Determination of antibacterial activity

The antibacterial activity of the extract against Staphylococcus epidermidis (MTCC 2639), Bacillus cereus and Salmonella typhi, was evaluated by well diffusion method with ampicillin (10 mg/ml) as standard. The stock culture of each bacteria used was subcultured at 37 °C overnight.

Agar well diffusion method

The medium used for this study was the Muller Hinton agar medium. The medium was sterilized by autoclaving at 120 °C. About 30 ml of the molten nutrient agar medium inoculated with respective strains of bacteria was transferred aseptically into each sterilized plate. In each plate, five wells of 5 mm diameter were made using a sterile borer. The test concentrations of 25, 50, 75, and 100 mg/ml and standard were injected into the well. The plates were maintained at room temperature to allow the diffusion of the solutions in to the medium. The petri-dishes used for the antibacterial screen were incubated at 37 °C for 24 h. The diameter of the zone of inhibition surrounding each of the wells was measured and recorded.

Collection of worms and microscopic identification

The Setaria digitata worms were collected in Modified Ringer’s buffer (Singhal et al. 1973) from the peritoneal cavity of cows from the local slaughterhouses in Madurai. The worms were examined both visually and under a Phase contrast microscope to further examine the morphology. The worms were differentiated as male or female based on theirlength and the differences in their cephalic and caudal ends (Sundar and D’Souza 2015).

In vitro macrofilaricidal activity

The motility and the viability of the filarial worm S. digitata against the M. parvifolia leaf extract as reported earlier with a small modification (Mathew et al. 2008). The collected worms were brought to laboratory within 1 h ofculturing and washed with the modified ringer’s buffer thrice to free them from any extraneous materials. The worms transferred to DMEM media supplemented with 10% Fetal Bovine Serum (FBS), penicillin (100U/ml) and streptomycin (100 µg/ml) in a humidified atmosphere of 5% CO₂ at 37 °C were used for the experiment.

Worm motility assay

Dilutions of the leaf extract of M. parvifoliawere made in methanol and the screening was done at concentrations of 0.01, 0.05, 0.1, and 0.5 mg/ml. Control was maintained with medium and methanol alone. Positive control with 10 mg/ml Diethylcarbamazine (DEC) was maintained. One adult female S. digitata worm was introduced into each well of the six well plates containing DMEM supplemented with 10% Fetal Bovine Serum (FBS), penicillin (100 U/ml) and streptomycin (100 µg/ml) in a humidified atmosphere of 5% CO₂ at 37 °C for 48 h. The total volume of the media including the test concentration was 3 ml. Three replicates for each were maintained for both test and control. After the incubation period the number of immobilized worms in each well was counted. Immediately after counting, the worms were washed with fresh medium and transferred to another six well plate containing fresh medium, without the test solution, to find out if any of the immobilized worms regained motility. If the worms did not revive, the condition would be considered irreversible and the concentration lethal.

MTT formazan colorimetric assay for viability of worms

The M. parvifolia leaf extract was further screened for the viability of adult S. digitata through an MTT reduction assay. Adult female worms were used in this assay. After the exposure of these worms to various concentrations of M. parvifolia (0.01, 0.05, 0.1, 0.5 mg/ml) in DMEM at 48 h incubation period the parasites were further incubated for 30 min individually in phosphate buffered saline (pH 7.4) containing MTT (0.25 mg/ml). A control was set up with adult females exposed to methanol but not with test solutions. At the end of the MTT incubation the worms were transferred to spectroscopic grade Dimethyl sulfoxide (DMSO) and allowed to be at room temperature for one hour, with gentle shaking to extract the colour developed. The absorbance of the resulting formazan solution was then determined at 492 nm using a spectrophotometer relative to the DMSO blank. The viability of the worms relative to control worms was estimated as percentage inhibition in formazan formation by using the following equation:

$$\%\text{of}\text{inhibition}=\frac{\left(\text{OD}\text{of}\text{treated}\text{worms}-\text{OD}\text{of}\text{control}\right)}{\text{OD}\text{of}\text{controlworms}}\times 100$$

Larvicidal bioassay

The larvae of Cx. quinquifasciatuswere collected from ICMR-VCRC, Field unit, Madurai and tested for larvicidal activity against M. parvifolia leaf extract (WHO 2005). To determine the activity range, larvae were exposed to a broad range of test concentrations (0.1 ppm–100 ppm/100 ml) across three batches: 0.01%, 0.1%, and 1%. A control with no test material was maintained. The late 3rd or early 4th instar larvae were selected for the assay. Using a strainer and dropper, 10 larvae were transferred to a test vessel containing 100 ml of water and the corresponding test solution for each trial. The test was done in triplicate. Any damaged or unhealthy larvae were removed. To prevent any outside interference, a net was placed over the container and left undisturbed for 24 h. After 24 h, the larvae with no movement even after slight touch with a glass rod areconsidered mortal. The batch with the highest mortality rate was selected and a test with a narrower range of concentration (20, 40, 60, 80, 100 ppm/100 ml) was carried out. Number of dead worms was recorded, and the mortality percentage was determined.

Determination of antioxidant activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method

Blois (1958) described the methodology used to assess the antioxidant activity of M. parvifolia leaf extract by measuring its ability to scavenge free radicals. The 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay was employed with slight modifications. For each assay, a fresh methanolic solution containing 0.2 mM DPPH was prepared. In a 96-well plate, 100 µl of various concentrations of the leaf extract (20, 40, 60, 80 and 100 µg/ml) and ascorbic acid as the standard were added. Subsequently, 100 µl of the DPPH solution was added to each well, followed by a 30-min incubation period in darkness. The absorbance of the samples was then measured at 517 nm. The IC₅₀ value, which represents the concentration of the extract causing 50% inhibition, was determined based on the concentration of the extract and the percentage of inhibition.

The calculation for the percentage inhibition (%) of DPPH radical was as follows:

$$\left[\frac{\left(A_B-A_A\right)}{A_B}\right]\times 100$$

where A_B refers to the absorbance of the DPPH radical plus methanol, and A_A corresponds to the absorbance of the DPPH radical plus the sample extract or standard.

Statistics

All tests were carried out in triplicate. The study reported the findings with statistical analyses as mean ± standard deviation. Statistical analysis was performed using ANOVA (Analysis of Variance) single factor test. Mortality percentage in larvicidal activity was determined through Abbot’s formula and probit analysis, enabling the calculation of LC50 and LC90 values.

Results

Percentage of yield

The leaves of M. parvifolia were extracted by Soxhlet with methanol and later evaporated by rotary evaporator. The percentage yield was calculated using the following equation.

$$\text{Percentage}\text{of}\text{the}\text{yield}=\frac{\text{Amount}\text{of}\text{the}\text{extract}\text{yield}}{\text{Amount}\text{of}\text{the}\text{leaf}\text{powder}}\times 100$$

The result is summarized and shown in Table 1.

Table 1.

Calculated percentage yields from the methanolic extraction of M. parvifolia leaves

Amount of leaf powder (g)	Amount of extract yield (g)	Percentage of extract yield (%)
60	12.4	20

The table details the weight of the initial leaf material, the weight of the extract obtained after the Soxhlet extraction and rotary evaporation process, and the corresponding calculated extraction yield in percentage terms. This data provides insight into the efficiency of the extraction process

Qualitative phytochemical screening

Figure 1 shows the phytochemical screening of the methanolic leaf extract of M. parvifolia revealed a significant presence of alkaloids (a & b), terpenoids (c), phenols (d), tannins (e), and flavonoids (f). The confirmation of various phytoconstituents was achieved through a series of specific phytochemical tests, where distinct colour changes were observed upon treatment with the plant extracts. These results are visually depicted, providing tangible evidence of the presence of these bioactive compounds.

Fig. 1.

Qualitative phytochemical analysis of M. parvifolia methanolic leaf extract, highlighting the significant presence of various phytoconstituents. a and b Confirmation of alkaloids using Wagner’s and Mayer’s reagents, respectively, c Presence of terpenoids, d Presence of phenols, e Presence of tannins, and f Presence of flavonoids. Each part of the figure shows the distinct colour changes observed after treatment with the plant extracts during specific phytochemical tests, visually indicating the presence of these bioactive compounds in the leaf extract

GC–MS analysis

The GC MS chromatogram showed a total of 24 phytoconstituents. The chromatogram is presented in Fig. 2 and the name of the compounds, area, retention time and base m/z are provided in Table 2. The chromatogram indicated the presence of the major alkaloid named mitraphylline (Formosanan-16-Carboxylic Acid) as reported in previous studies.

Fig. 2.

GC–MS chromatogram of the defatted M. parvifolia methanolic leaf extract. The chromatogram displays the identified phytoconstituents, including the major alkaloid mitraphylline (Formosanan-16-Carboxylic Acid), consistent with previous studies. Each peak in the chromatogram represents a different compound found in the extract

Table 2.

Detailed analysis of the phytoconstituents identified by GC–MS in the methanolic leaf extract of M. parvifolia

Peak No	Name of the compound	R. Time	Area	Area%	Similarity	Base M/Z
1	Mesitol	3.218	72,074	0.19	75	135.00
2	Cyclotetrasiloxane	6.533	156,919	0.41	80	281.05
3	Pentasiloxane	6.902	168,934	0.44	96	281.05
4	2,3-Dihydro-Benzofuran	7.370	122,177	0.32	92	120.05
5	2,4-Cresotaldehyde	9.621	205,090	0.54	78	136.05
6	Quinoline	9.690	293,372	0.77	94	158.10
7	Beta.-D-Glucopyranose	10.020	262,828	0.69	81	57.05
8	2,4-Di-Tert-Butylphenol	10.196	844,078	2.22	96	191.15
9	2-Butenedioic Acid (Z)	10.392	324,412	0.85	84	99.05
10	1,3,4,5-Tetrahydroxycyclohexanecarboxylic Acid	11.156	2,275,107	5.97	93	60.00
11	Mome Inositol	11.737	201,539	0.53	86	87.05
12	2(3h)-Benzothiazolone	11.811	272,788	0.72	94	151.00
13	Unidentified	12.063	293,155	0.77	0	75.05
14	Unidentified	13.458	203,045	0.53	0	164.05
15	7,9-Di-Tert-Butyl-1-Oxaspiro (4,5) Deca-6,9-Diene-2,8-Dione	15.883	259,233	0.68	88	205.10
16	2-(1,3-Benzothiazol-2-Ylsulfanyl) Ethanol	16.212	1,080,551	2.84	93	167.00
17	N-Hexadecanoic Acid	16.565	739,070	1.94	95	73.05
18	Ctadecane	16.645	187,108	0.49	87	71.10
19	Sulfur, Mol. (S8)	18.182	462,855	1.21	95	255.75
20	Octadecanoic Acid	20.507	711,397	1.87	91	73.05
21	4-Tert-Butyl-2-(4-Methoxy-Phenyl)-6-P-Tolyl-Pyridine	29.052	327,711	0.86	72	331.25
22	Formosanan-16-Carboxylic Acid	34.339	3,119,561	8.19	81	368.20
23	Formosanan-16-Carboxylic Acid	35.035	24,932,759	65.44	79	223.15
24	Gamma.-Sitosterol	38.481	585,362	1.54	91	414.40

The table includes the names of the compounds, their relative areas, retention times, and base m/z values. The results highlight the presence of the major alkaloid mitraphylline (Formosanan-16-Carboxylic Acid) and other compounds, providing an in-depth understanding of the extract’s phytochemical composition

FT-IR analysis

Infrared spectroscopy was employed to analyse the vibrational frequencies of functional groups present in the methanolic extract of the leaf (Fig. 3). The obtained spectra revealed several characteristic peaks: a peak at 3314 cm⁻¹ indicating the stretching of the –OH group in acids, alcohols, and phenols; a stretching frequency at 2921.96 cm⁻¹ corresponding to C–H stretching in alkanes and aldehydes; a stretching peak at 1698.67 cm⁻¹ corresponding to C=O stretching in amides; a peak at 1615.20 cm⁻¹ representing N–H bend stretching in primary amines; a region at 1515 cm⁻¹ indicative of aromatic C=C bonds; a peak at 1441 cm⁻¹ representing the CH2 bend; and 1197 cm^-1 corresponding to the C–O–C stretching. These observed functional groups, identified through their specific frequencies, signify the presence of alkaloids, phenols, tannins, terpenoids, and flavonoid compounds in the leaf extract.

Fig. 3.

Infrared spectroscopy analysis of the methanolic extract of the leaf, indicating the presence of various functional groups. The spectrum showcases characteristic peaks corresponding to vibrational frequencies of specific functional groups, including: –OH group stretching at 3314 cm⁻¹ (found in acids, alcohols, and phenols); C–H stretching at 2921.96 cm⁻¹ (alkanes and aldehydes); C=O stretching at 1698.67 cm⁻¹ (amides); N–H bend stretching at 1615.20 cm⁻¹ (primary amines); aromatic C=C bonds at 1515 cm⁻¹; CH2 bend at 1441 cm⁻¹; and C–O–C stretching at 1197 cm⁻¹. The identified functional groups suggest the presence of alkaloids, phenols, tannins, terpenoids, and flavonoid compounds in the leaf extract

Antibacterial activity

This study investigated the antimicrobial potential of a crude leaf extract against multiple bacterial strains, including Bacillus cereus (Fig. 4a), Salmonella typhi (Fig. 4b), and Staphylococcus epidermidis (Fig. 4c). The inhibitory effects of the crude methanolic extract were compared to those of the standard antibiotic, ampicillin. Inhibition zones surrounding wells containing varying concentrations of the extract were visually observed and measured. The results summarized in Table 3, provide valuable insights into the extract’s antimicrobial activity against the tested bacterial strains.

Fig. 4.

Visualization of the zones of inhibition caused by various concentrations (25, 50, 75, 100 mg) of a leaf extract on different bacterial strains using the agar well diffusion method. a The effect on Bacillus cereus, b the effect on Salmonella typhi, and c the effect on Staphylococcus epidermidis. Each part of the figure shows a petri dish with the bacterial strain and the inhibition zones around the wells containing different concentrations of the extract. The size of each zone provides a visual representation of the extract’s antimicrobial potency against the corresponding bacterial strain

Table 3.

Comparative assessment of inhibition zones for different bacterial strains—Bacillus cereus, Salmonella typhi, and Staphylococcus epidermidis—responding to various concentrations (25, 50, 75, 100 mg) of a crude leaf extract and the standard antibiotic ampicillin

Conc (mg/ml)	B. cereus (mm)	S. typhi (mm)	S. epidermidis (mm)
25	13	12	9
50	14	13	10
75	14	14	11
100	16	15	13
Ampicillin	17	18	30
−ve control	–	–	–

Each column in the table represents a specific bacterial strain, while each row corresponds to the diameter of the inhibition zone (in mm) observed at a particular extract concentration or with the use of ampicillin. These measurements provide a direct evaluation of the extract’s antimicrobial activity against each tested bacterial strain

Identification of male and female S. digitata worms and its microscopic view

The typical habitat of S. digitata’s filarial nematodes is the peritoneal cavity of cattle. Female worms of S. digitata had an average body length and width measuring 153 mm, while male worms had a length of 82 mm. Phase contrast microscopy was employed to visualize and characterize S. digitata, and female worms were identified based on the presence of a peribuccal crown with a central “helmet” at the cephalic end (Fig. 5a). Additionally, their tail ends terminated in a smooth knob accompanied by oval lateral appendages (Fig. 5b). In contrast, male worms of S. digitata displayed similar cephalic ends to females, but their cuticles exhibited a corrugated appearance (Fig. 5c), distinguishing them from female worms.

Fig. 5.

Phase contrast microscopic images of Setaria digitata worms, highlighting their distinct morphological traits. a The cephalic (head) end of a female S. digitata worm, is characterized by a peribuccal crown with a central “helmet”. b The caudal (tail) end of the same female worm, terminating in a smooth knob. c The caudal end of a male S. digitata worm, distinguished by a cuticle with a corrugated appearance. These identifying features enable differentiation between male and female S. digitata worms

Invitro macrofilaricidal screening

The macrofilaricidal activity of the test material was evaluated through in vitro assays measuring worm motility and MTT reduction. The motility assay revealed complete inhibition of worm movement after 48 h. Screening was performed at different concentrations (0.01, 0.05, 0.1, 0.5 mg/ml) over a 48-h incubation period to determine the dose–response relationship and assess the viability of filarial worms. The findings of the MTT reduction assay were 41.25 ± 0.12, 65.8 ± 0.01, 75.73 ± 0.06, and 92.73 ± 0.03 respectively (P < 0.01) and that of DEC was 81.03 ± 0.015 (Fig. 6). The IC50 value of the extract at 48 h was determined to be 0.027 mg/ml.

Fig. 6.

Bar diagram depicting the macrofilaricidal activity of test concentrations and positive control DEC determined by the MTT assay. The graph illustrates the effect of the extract on Setaria digitata worms, with the X-axis representing the various concentrations of the extract and the Y-axis showing the corresponding inhibition percentage. The error bars represent standard deviations

Larvicidal bioassay

In the initial larvicidal bioassay, the effectiveness of M. parvifolia leaf extract was evaluated against Cx. quinquifasciatuslarvae using three different percentages: 0.01%, 0.1%, and 1%. Each percentage was tested to determine its larvicidal activity. Among the tested percentages, 1% exhibited the highest larvicidal activity, resulting in a significant reduction in larval survival rates. At 0.01% and 0.1%, moderate larvicidal effects were observed, although they were comparatively less pronounced than at 1%. Considering the superior larvicidal activity observed at 1%, this percentage was chosen for further investigation due to its potential as an effective larvicidal agent against Cx. quinquifasciatuslarvae. To investigate the concentration-dependent effects of the plant extract at 1%, a follow-up bioassay was conducted using a narrow range of five concentrations derived from the initial broad range. This narrower range allowed for a more refined examination of the extract’s larvicidal properties. The five concentrations of the plant extract (20 ppm, 40 ppm, 60 ppm, 80 ppm, and 100 ppm) within the selected range were assessed for their larvicidal activity against Cx. quinquifasciatuslarvae. Specifically, 100 ppm exhibited the highest larvicidal activity, followed by 80 ppm, 60 ppm, 40 ppm, and 20 ppm (Fig. 7). The LC50 and LC90 values were 66.5 ppm and 223.87 ppm respectively. The linear regression model was statistically significant (F(1, 3) = 120.13, P < 0.001).

Fig. 7.

Depiction of the dose-dependent larvicidal activity of M. parvifolia leaf extract against Culex quinquefasciatus larvae. The horizontal axis represents the various concentrations of the extract while the vertical axis represents the corresponding larvicidal activity. Each data point corresponds to a specific concentration, and the pattern suggests a relationship between the concentration of the extract and its larvicidal efficacy The error bars represent standard deviations

DPPH radical scavenging assay

The radical scavenging activity of the M. parvifolia leaf extract at different concentrations was depicted by the DPPH assay. Fig shows the percentage of inhibition of the M. parvifolia leaf extract (Fig. 8a) and Ascorbic acid (Fig. 8b). Among the extracts, the 100 mg/ml showed the maximum percentage inhibition (P < 0.01). The IC₅₀ value of the leaf extract was 28.46 µg/ml whereas that of the standard ascorbic acid was 10.02 µg/ml.

Fig. 8.

Evaluation of the radical scavenging activity of various concentrations of M. parvifolia leaf extract. a The effect was measured by the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay, with the horizontal axis indicating extract concentration and the vertical axis displaying the corresponding DPPH radical scavenging activity. b Comparison with the standard antioxidant Ascorbic acid, where the horizontal axis indicates concentration and the vertical axis shows the equivalent radical scavenging activity. Each data point represents a specific concentration, highlighting the relationship between extract/ascorbic acid concentration and their respective antioxidant capacities. The error bars included in the plot represent standard deviations from multiple measurements

Discussion

The present study aimed to conduct a comprehensive analysis of the properties of M. parvifolia leaves and assess their potential as a therapeutic agent for Lymphatic filariasis (LF).

Phytochemical tests conducted on the methanolic leaf extract of M. parvifolia yielded positive results for alkaloids, as confirmed by Mayer’s and Wagner’s tests. Moreover, the extract exhibited positive reactions for flavonoids, phenols, tannins, and terpenoids. These findings indicate that the M. parvifolia leaf extract comprises a diverse range of bioactive compounds with potential pharmacological properties.

In terms of antibacterial activity, the M. parvifolia leaf extract demonstrated significant effectiveness against Staphylococcus epidermidis, Bacillus cereus, and Salmonella typhi, although its potency was not as strong as the positive control, ampicillin.

Regarding macrofilaricidal activity, the methanolic leaf extract of M. parvifolia exhibited promising results, as quantitatively assessed through the MTT reduction assay. The inhibition of formazan formation in a dose-dependent manner indicates its potential as a macrofilaricidal agent. Both the worm motility assay and MTT reduction assay confirmed the macrofilaricidal potential of the extract.

In the context of larvicidal activity, the M. parvifolia leaf extract displayed noteworthy effectiveness against Cx. quinquifasciatuslarvae. The concentration of 1% showed the highest larvicidal activity, suggesting its suitability for further investigation. Notably, the concentration of 100 ppm demonstrated the most potent larvicidal effect, holding significant implications for filariasis prophylaxis, considering Cx. quinquifasciatusas a major disease vector.

To evaluate the antioxidant activity, the DPPH assay was performed at various concentrations of the M. parvifolia leaf extract. The results indicated a concentration-dependent relationship, with 100 µg/ml displaying the highest antioxidant activity. While the extract’s potency may be lower compared to ascorbic acid, it still exhibited significant antioxidant properties. Statistical analysis further supported a robust correlation between the extract’s concentration and its ability to scavenge DPPH radicals.

Our findings align with and extend the results reported by previous researchers. Gupta et al. (2009) assessed the anti-inflammatory and antinociceptive activity of the ethanolic extract of M. parvifolia leaves. Vishal and Sanjay (2010) found significant anthelmintic activity in the methanolic extract of M. parvifolia stem-bark. Ankit et al. (2009) reported no antimicrobial activity against certain bacterial strains, whereas our study demonstrated significant antibacterial activity against different bacterial strains, including Staphylococcus epidermidis, Bacillus cereus, and Salmonella typhi. These variations in bacterial susceptibility could be attributed to the specific bacterial strains selected for evaluation in each study. Our findings highlight the potential of the M. parvifolia extract as a broad-spectrum antimicrobial agent, warranting further investigations into its mechanism of action and the identification of active compounds responsible for its observed activity. Additionally, Sahu et al. (2015) reported significant anthelmintic activity of both ethanolic and aqueous extracts of M. parvifolia leaves against Pheritimaposthuma, particularly at a higher concentration of 50 mg/ml, supporting the potential isolation and development of biologically active components as anthelmintic drugs. Moreover, Kaushik et al. studied the anti-inflammatory activity of ethanolic leaf extract of M. parvifolia and observed significant effects. The extract also showed concentration-dependent antioxidant and free radical scavenging activities, with the highest antioxidant activity observed.

Overall, our study provides valuable insights into the therapeutic potential of M. parvifolia leaves for LF management, presenting its diverse bioactive compounds, antibacterial and macrofilaricidal activities, larvicidal efficacy against disease vectors, and significant antioxidant properties. Further research and exploration of this natural resource may lead to the development of novel treatments and preventive strategies for LF. However, more in-depth investigations are needed to elucidate the exact mechanisms of action and to isolate and identify the active compounds responsible for the observed bioactivities. Additionally, future studies could focus on in vivo experiments and clinical trials to validate the efficacy and safety of M. parvifolia as a potential therapeutic agent for LF.

Author contributions

Jefrillah Jebaseelan was responsible for the conceptualization of the study, performed all methodologies, carried out the formal analysis, led the investigation, and wrote the original draft of the manuscript. Anand Setty Balakrishnan contributed to the conceptualization of the study and provided supervision throughout the research and writing process.Jamespandi Annaraj provided guidance in the characterization studies. Sheerin Banu Sadiq assisted in conducting the antibacterial activity, in-vitro macrofilaricidal assay, and characterization studies.Abi Prakathi Ravikumar assisted in the larvicidal activity component of the research.

Funding

The study was partially funded by RashtriyaUchchatar Shiksha Abhiyan 2.0 (RUSA 2.0) Ref: RUSA/MKU/Internship/2022 and 013/RUSA/MKU/2020-21.

Declarations

Competing interests

The authors have no relevant financial or non-financial interests to disclose.