Phytochemical Analysis, OHR-LCMS Assisted Metabolite Profiling, and Antifungal Activity of Natural Products from the Medicinal Plant Tragia Plukentii A.R. sm as Antitinea Agents

doi:10.21203/rs.3.rs-4953347/v1

Background

Tinea, a fungal infection caused by dermatophytes, affects approximately 25% of the global population, and Trichophyton rubrum is the main causative agent. Although these infections usually appear as superficial skin issues, they can become serious in diabetic or immunocompromised individuals. Despite the traditional antifungal reputation of Tragia plukentii, scientific studies on its phytoconstituent profile via OHR-LCMS analysis and its efficacy against T. rubrum are lacking. This study aimed to investigate the antifungal activity of Tragia plukentii against T. rubrum and to assess its potential as an alternative treatment for Tinea pedis.

Methods

Tragia plukentii plants were authenticated, and healthy leaves were collected and shade-dried for 10‒15 days. The dried leaves were ground into a fine powder and extracted via the Soxhlet method with solvents of various polarities. The physical and chemical properties of the leaf powder were assessed, and the extracts were analyzed for bioactive phytocompounds via OHR-LCMS. The antifungal activity of the extracts against Trichophyton rubrum (ATCC28188) was evaluated using the cell diffusion method, and the efficacy of the extracts was compared with that of the standard drug terbinafine by measuring inhibition zones.

Results

This study identified 92 and 29 bioactive phytoconstituents in the positive and negative ionization modes of the OHR-LCMS spectrum, respectively, including alkaloids, flavonoids, phytosterols, glycosides, and terpenoids, in glacial acetic acid extract for the first time. The key compounds identified included NP-001787, quercetin, methyl hippuric acid, and xanthohumol, which were validated using mzCloud and the Spider Search Database. Among the six extracts tested, the glacial acetic acid extract showed highly potent antifungal activity with a notably larger inhibition zone of 45 mm, surpassing the standard drug terbinafine (29 mm), and the water (11 mm), cyclohexane (11 mm), and methanol (10 mm) extracts exhibited moderate antifungal activity, whereas the other extracts showed weaker activity.

Conclusions

This study revealed the significant antifungal potential of Tragia plukentii extracts, particularly the glacial acetic acid extract, which exhibited a 45 mm inhibition zone against Trichophyton rubrum, outperforming the standard antifungal drug terbinafine (29 mm). This study also highlighted the importance of OHR-LCMS in generating a detailed phytochemical profile crucial for identifying bioactive compounds.

The trial registration number (TRN):

‘Clinical trial number: not applicable.’

Antifungal activity

OHR-LCMS analysis

Phytoconstituent profiling

Tragia plukentii A.R.sm.

Tinea pedis

This study aimed to develop an effective alternative therapy for tinea dermatitis, a condition current medications can fully treat.[1], Tragia plukentii, which is traditionally known for its antifungal properties, was evaluated for its effectiveness against the fungal strain Trichophyton rubrum (ATCC28188). [2] Tinea, a common fungal infection caused by dermatophytes that affect the skin, is found worldwide. The infection appears in specific body regions, including the trunk, neck, arms, and legs. Different names describe dermatophyte infections in other areas, such as the scalp (Tinea capitis), face (Tinea faciei), hands (Tinea manuum), groin (Tinea cruris), and feet (Tinea pedis). Symptoms included swollen red patches, dry scaly rashes, red rings (ringworms), itching, hair loss, and an itchy scalp. [3, 4] The development of Tinea capitis involves various dermatophytes, classified as anthropophilic, zoophilic, or geophilic. Although geophilic dermatophyte species are widespread, some anthropophilic and zoophilic fungi may be restricted to specific geographic areas on the basis of their host range. In the USA, Tinea capitis caused by geophilic organisms is uncommon, generally leading to an increased inflammatory response when these species infect humans. Conversely, anthropophilic species were associated with fewer inflammatory conditions. [5, 6] In India, approximately 57.3 million people (4.1% of the population) are estimated to be affected by serious fungal diseases. The prevalence figures (in millions) for various conditions include 24.3 for recurrent vulvovaginal candidiasis, 2.0 for allergic bronchopulmonary aspergillosis, 25.0 for Tinea capitis among school-aged children, 1.36 for severe asthma with fungal sensitization, 1.74 for chronic pulmonary aspergillosis, and 1.52 for chronic fungal rhinosinusitis. Additionally, the annual incidence rates of Pneumocystis pneumonia are 58,400; invasive aspergillosis accounts for 250,900 cases; mucormycosis accounts for 195,000 cases; esophageal candidiasis in HIV patients accounts for 266,600 cases; candidemia accounts for 188,000 cases; fungal keratitis accounts for 1,017,100 cases; and cryptococcal meningitis accounts for 11,500 cases. [7] Over the last 50 years, the epidemiology of Tinea capitis in the Americas has undergone significant changes. In the United States, Trichophyton tonsurans has emerged as the leading cause, surpassing Microsporum audouinii. Once uncommon, T. tonsurans now represents 90% of cases in Brooklyn and has spread throughout the nation, partly due to immigration. This fungus affects both children and adults, with a notably higher incidence among women. Research in cities such as New York, Chicago, and the San Francisco Bay Area has shown a significant increase in T. tonsurans infection over time. For example, the prevalence of T. tonsurans Tinea capitis in the San Francisco Bay area increased from 60% in the 1970s to 91% by the early 1990s. In Canada, T. tonsurans has also become the primary causative agent, especially in metropolitan locations. Moreover, between 1979 and 1989, M. canis and T. mentagrophytes were more prevalent in Puerto Rico. In Brazil, M. canis continues to be the main causative agent, followed by T. tonsurans, whereas in Argentina, M. canis is the most common causative agent among children under 11 years of age. The increasing prevalence of T. tonsurans as a major cause of Tinea capitis highlights the need for updated diagnostic and treatment strategies. [8, 9] Dermatophytosis, caused by the filamentous fungus Trichophyton rubrum, affects approximately 25% of people worldwide. These infections thrive on keratin in human and animal skin, hair, and nails and are categorized on the basis of their environment. These bacteria produce virulence enzymes that facilitate infection. [10] Tragia plukentii A.R.sm., a member of the Euphorbiaceae familyily, is found in tropical regions of India, Africa, Nigeria, Zimbabwe, and Sri Lanka.[11] In India, these individuals are located in states such as Andhra Pradesh, Goa, Gujarat, Karnataka, Madhya Pradesh, Maharashtra, Rajasthan, Tamil Nadu, Uttar Pradesh, and West Bengal. Tragia is a large botanical family encompassing 168 species that are found primarily in tropical and subtropical regions across Africa, America, and Asia. India hosts seven distinct species within this genus, with two species found in Uttar Pradesh. [12] Tragia plukentii A.R. Sm. is recognized by several synonyms, such as Croton status L, Tragia cannabina L.F., Tragia cannabina var. hastate and Tragia tripartita Beille. [13, 14] It is commonly referred to as Khaj Kholi or Chur Churi. Taxonomically, it falls under the Eukaryote kingdom Plantae, phylum Magnoliophyta, class Equisetopsida C. Agardh, and order Malpighiales Juss. ex Bercht. and J. Presl, the genus Tragia, and the species Tragia plukentii A.R. Smith. [15] A comprehensive literature review revealed that there are currently no scientific reports on the antifungal activity of Tragia plukentii against Trichophyton rubrum. Therefore, our study aimed to investigate the antifungal activity of Tragia plukentii and its potential use in plant-based products. OHR-LCMS analysis of glacial acetic acid leaf extracts from Tragia plukentii revealed novel bioactive compounds with high precision and sensitivity. This technique integrates liquid chromatography‒tandem mass spectrometry (LC‒MS) with other chromatographic methods, providing accurate identification and quantification of compounds, even at trace levels. The rapid analysis and separation capabilities of these materials are ideal for intricate phytochemical profiling. By offering detailed insights into the chemical composition and identifying less-studied compounds, OHR-LCMS enables comprehensive profiling and cataloging of bioactive constituents.[16] correlating these compounds with the observed antifungal activity can reveal key contributors to the therapeutic effects, guiding future research and development.[17]

2.1 Chemicals and reagents

Analytical-grade solvents were used for the extraction of Tragia plukentii plants, which were procured from SD Fine Chemicals Limited (SDFCL, Mumbai, India). The reagents employed for phytochemical screening of the extracts involved conducting chemical tests for various compounds, including carbohydrates, saponins, glycosides, amino acids, proteins, steroids, reducing sugars, alkaloids, flavonoids, phenols, tannins, and terpenoids. These reagents were obtained from Fisher Scientific (India Pvt., Ltd.). Additionally, the solvents used for high-resolution chromatography–mass spectrometry (HR-LCMS) were of spectroscopic grade and procured from SD Fine Chemicals Limited (SDFCL, Mumbai, India).

2.2 Collection of plant material

Fresh healthy and naturally grown Tragia plukentii A.R. sm leaves were dark green and were collected for research purposes from local places in Ukhalad, Parbhani, Maharashtra. Figure 1 shows an image of the Tragia plukentii A. R. sm plant and the location where the leaves were collected. Plant botanical identification was performed by Dr. B. S. Surwase, Head of Department, Department of Botany, School Of Life Sciences SRTM University Nanded, who confirmed that the selected plant was Tragia plukentii A.R. sm. An authentication certificate of the plant was issued and is provided in additional file 1. A voucher specimen is preserved in the Department of Pharmacognosy, School of Pharmacy, S.R.T. M. University Nanded, Maharashtra, India. The plant material was collected and placed in sterile plastic bags for later processing in the laboratory. [18, 19].

2.2.1 Drying and powdering of Tragia plukentii leaves

Mature, healthy Tragia plukentii plants free of disease or pests were selected for drying. [19] Leaves were cleaned with water to remove dirt or soil and then shade-dried for 10–15 days to remove moisture. [20] Dry leaves are brittle and can be easily crushed. The drying process continued until the desired dryness was achieved. The dried leaves were ground into powder using an electric grinder and passed through a fine mesh sieve to remove coarse particles. The powdered Tragia plukentii leaves were stored in airtight containers. [21]

2.2.2 Preparation of extracts

Tragia plukentii leaf powder was extracted using the Soxhlet extraction method, with methanol, glacial acetic acid, benzene, ethyl acetate, cyclohexane, and water used as solvents. For Soxhlet extraction, 50 g of powdered material was wrapped in muslin thimble and placed in a round bottom flask. The flask was then connected to a Soxhlet apparatus and extracted with 400 mL of solvents ranging from low to high polarities. Extraction was carried out using a continuous hot percolation process until the solvent was colorless. The solvent was evaporated using a rotary evaporator, and the concentrated extract was dried in a desiccator. The dried extract was stored in an airtight container until subsequent use. Chemical analyses were performed to determine the presence of several bioactive phytoconstituents in the extracts as well as their physical qualities. [22]

2.3 Equipment and software

A Soxhlet apparatus was used for the extraction. The whole extraction assembly included a stand, a water pump, etc.[22] The samples were examined using Orbitrap high resolution liquid chromatography mass spectrometry (OHR–LCMS) at the Sophisticated Analytical Instrumentation Facility (SAIF),IIT Bombay. The Thermo Scientific Data Acquisition and Q Exactive Plus Biopharma platforms were used for OHR-LCMS analysis. Thermo Scientific Xcalibur software version 4.2.28.14 was used for the analysis. Compound Discoverer 3.2 SP1 was used for data processing. The column sections consisted of Hypersil Gold 150 × 2.1 mm, 1.9 microns (Thermo Scientific) Solvent A comprised 0.1% formic acid dissolved in Milli-Q water, while Solvent B was acetonitrile. The mzCloud and ChemSpider software programs were used in the OHR-LCMS analysis for phytocompounds identification. [23]

2.4 Fungal strains

Antifungal activity was assessed using Trichophyton rubrum (ATCC28188) fungal strain cultures preserved at the Infinite Biotech Laboratory in Sangali, Maharashtra, India. For inoculum preparation, fungal strains were subcultured on nutrient agar (HiMedia) and incubated at 37°C overnight.

2.5 Morphological study of plant leaves

The plant is a creeping or climbing shrub that occasionally grows erectly up to 1 meterm tall, with branches that soften with age. Its leaves are palmate, three-lobed, and sometimes bilobed with a suppressed lobe, measuring 3 to 10 cm long and 1.5 to 7 cm wide. The leaves have a paper-like texture, with sparse hairs on the underside and irregular sawtooth edges on the central lobe. Lateral lobes 0.5–4 cm long and 0.3–1.5 cm wide were measured. The petiole is 3 to 20 mm in length. The terminal racemes are 2 to 5 cm long, with male flowers or 1 to 3 female flowers at the base. Male flowers have a pedicel length of 1 mm, a broadly ovate calyx, and rectangular anthers. The female flowers had a 1 mm peduncle, a calyx with stiff hairs, a 2 mm diameter ovary, and a 3 mm column with three leaves. The fruit is compressed, 3 to 5 mm long, 6 to 8 mm wide, and three-lobed, with a linear or rectangular sepal and 4 to 10 lobes bearing brown hair, containing three spherical seeds. [12]

2.6 Physicochemical study of powdered drug

The physical and chemical properties of Tragia plukentii leaf powder, such as loss on drying, foaming index, ash content, and moisture content, were assessed using standardized methods described in the literature [24, 25], as these parameters are crucial in determining the quality and integrity of the crude drug powder. The loss on drying (LOD) measurement reveals the capacity of the material to prevent the growth of bacteria, fungi, and yeast, whereas the percentage of ash content is a sign of purity. Furthermore, determination of the physicochemical properties of the extract confirmed the existence of both polar and nonpolar molecules. The results of the physicochemical analysis of the powdered drugs are presented in Table 2.

2.7 Physical characterization of the plant extracts

The Soxhlet extraction technique was employed to obtain extracts of Tragia plukentii leaf powder using glacial acetic acid, cyclohexane, methanol, benzene, ethyl acetate, and water. Physicochemical characterization, such as determination of the color, appearance, and yield of these extracts, was carried out according to the standard methods mentioned in the literature [26] to determine their quality and effectiveness. The observations and results obtained are presented in Table 3.

2.8 Preliminary phytochemical screening of plant extracts

An extensive phytochemical investigation was conducted on all the extracts following established protocols. [26, 27] The extracts were examined for the presence of carbohydrates, saponins, glycosides, amino acids, proteins, steroids, reducing sugars, alkaloids, flavonoids, phenols, tannins, and terpenoids, and the results are shown in Table 4. [28, 29]

2.9 Orbitrap high resolution-liquid chromatography mass spectrometry (OHR-LCMS) analysis

Metabolite analysis was conducted using Orbitrap high resolution liquid chromatography mass spectrometry (OHR–LCMS). Figure 2 shows the OHR-LCMS chromatograms in positive and negative ionization modes for the glacial acetic acid extract of T. plukentii plants. The OHR-LCMS instrument used was Software Version 2.8–280502/2.8.1.2806. The model is a Q Exactive Plus, and the instrument is an Exactive Series slot. The Hardware ID is 901B0EBB4135. Thermo Scientific Data Acquisition and Q Exactive Plus Biopharma were used for the OHR-LCMS investigation. Before injection, all the samples were filtered through nylon membrane filters with pore dimensions of 0.2 µm. A hypersil gold column with dimensions of 150 × 2.1 mm and a particle size of 1.9 microns (originating from Thermo Scientific) was used for chromatographic separation in conjunction with a solvent gradient setup involving (a) 10% water containing 0.1% formic acid and (b) acetonitrile containing 10% water and 0.1% formic acid. [30, 31] The gradient profile was as follows: 2–20 minutes (A) 95% (B) 5%, 20–25 minutes (A) 5%, (B) 95%, and 26–30 minutes (A) 95%, (B) 5%. The flow rate during this process was maintained at 0.2 ml/minute, and the pressure was held steady at 1,200 bar. Mass spectra were acquired by electro spraying in both positive and negative modes. The capillary voltage, source cone voltage, and extraction cone voltages were set at 3.25 kV, 30 V, and 4 V, respectively, for both the positive and negative modes. Nitrogen served as the desolvation gas and was supplied at a rate of 900 l/hr. The source and desolvation temperatures were maintained at 120°C and 550°C, respectively. Mass spectra were obtained within the m/z range of 100–1200 with a mass resolution of 22,000 full-width at half maximum (FWHM). [32, 33]

2.9.1 Data processing and identification

The raw data were preprocessed using Thermo Scientific Xcalibur software, with default parameters for peak alignment, extraction, normalization, deconvolution, and chemical identification. Data processing was performed using Compound Discoverer 3.2 SP1. Untargeted data analysis using the mzCloud spectrum database and the Spider search database revealed 131 and 32 chemical features in T. plukentii leaf extracts in ESI + and ESI modes, respectively. The precise mass, retention time, mass error, isotopic similarity, and peak intensity were used to distinguish these features. [34, 35]

2.9.2 Building the bioactive metabolite database

To identify the phytoconstituents in the glacial acetic acid extract of T. plukentii, a range of online databases, such as Metlin, the Human Metabolome Database (HMDB), ChemSpider, CHEMEBI, and ChEMBL, were used to gather structural and spectral information on the metabolites. These resources enabled the identification of metabolites in the T. plukentii plant extract, and the data were obtained in “.sdf” and “.mol” file formats. Each file was meticulously examined for precision before being combined into a single .sdf” file for additional analysis. [36]. The bioactive phytocompounds identified based on OHR-LCMS analysis are presented in Tables 5 and 6 for the positive and negative ionization modes, respectively.

2.10 Determination of the antifungal activity of T. plukentii plant leaf extracts against Trichophyton rubrum

The antifungal activity of the six extracts of T. plukentii leaves was evaluated against the Trichophyton rubrum strain ATCC28188 using the well diffusion method. The procedure for determining antifungal activity is described below. The fungi were cultured in a laboratory, and a small sample was extracted and dispersed on a Petri dish containing a nutrient medium named Saubroad agar. Subsequently, tiny holes were made in the agar, and varying concentrations of the test compound and standard antifungal drug (terbinafine) were added to the holes. The Petri dishes were incubated at 37°C. for 24 hours. The diameter of the clear region surrounding each hole (zone of inhibition) was precisely measured to evaluate the potential of each compound to prevent fungal growth. The experiment was replicated three times to ensure the reliability of the results. [37]. The antifungal activities of T. plukentii leaf extracts against Trichophyton rubrum are shown in Table 7 and Figs. 3 and 4.

3.1 Morphological study of plant leaves

Results

Morphological study of T. plukentii plant leaves with three lobes growing from the same point, rarely without lobes, or with only two lobes, as one of the side lobes is missing. The leaves are between 3 and 10 cm long and between 1.5 and 7 cm wide. They are papery and have sparse, stiff hairs or longer, softer hairs on the veins. The undersides of the leaves are hairless or nearly hairless. Three veins were observed at the base of the leaf. The middle lobe is long and narrow, resembling a rectangle or an upside-down oval. It is between 1.5 and 8 cm long, between 0.5 and 2 cm wide, and has irregular teeth-like notches along the edges. The side lobes are between 0.5 and 4 cm long and between 0.3 and 1.5 cm wide. The veins that branch from the main vein at the center of the leaf enter the side lobes. There are between three and seven lateral veins on each side of the middle lobe, which mostly curve along the leaf margin. The petioles or leaf stalks are between 3 and 20 mm long. The morphological characteristics of Tragia plukentii AR sm leaves are shown in Table 1.

Table 1

Morphological characteristics of *Tragia plukentii* A.R sm leaves
Sr.No.	Morphological character	Observation
1	Color	Darkish Green
2	Odor	Characteristic
3	Taste	Pungent and Bitter
4	Size	3 to 10 cm long and 1.5to 7 cm wide
5	Shape	Rectangle or an upside-down oval Shape

3.2 Physicochemical study of powdered drug

Results

To evaluate the quality and purity of the powdered drug derived from Tragia plukentii leaves, various physicochemical parameters, including the total ash content, moisture content, alcohol soluble extractive value, swelling index, and foaming index, were analyzed. The results revealed a total ash content of 4.60% w/w, moisture content (loss on drying, LOD) of 1.21% w/w, alcohol soluble extractive value of 9.12% w/w, swelling index of 1.5 ml, and foaming index of 142.85 fu/cm. The results of the physicochemical analysis of Tragia plukentii AR sm leaf powder are shown in Table 2.

Table 2

Determination of the physicochemical parameters of *Tragia plukentii* leaf powder
Sr. No.	Parameter	Value
1	Total ash value	4.60% w/w
2	Moisture content(LOD)	1.21% w/w
3	Alcohol Soluble Extractive value	9.12% w/w
4	Swelling Index	1.5 ml
5	Foaming Index	142.85 fu/cm

3.3 Physical characteristics of the plant extracts

Results

A literature review suggested six solvents for extraction on the basis of their polarity indices, methanol, glacial acetic acid, cyclohexane, ethyl acetate, benzene, and water, all of which were used to extract T. plukentii leaf powder using the Soxhlet extraction method. The extraction revealed notable variations in yield and physical properties depending on the solvent used. The glacial acetic acid extract produced the highest yield (18.2% w/w) and had a light yellowish, sticky texture, indicating its effectiveness over other solvents. The methanol extract yielded 16.4% w/w and had a dark yellowish sticky appearance. The aqueous extract yielded 9.8% w/w with a dark radish color and sticky consistency, indicating lower efficiency but distinct characteristics. The benzene extract yielded a yield of 6.2% w/w and had a light green, sticky texture. The ethyl acetate and cyclohexane extracts gave yields of 4.2% w/w and 4% w/w, respectively, with dark green and greenish sticky appearances. Table 3 presents the physical properties of the various Tragia plukentii leaf extracts.

Table 3

Physical characteristics of different extracts of *Tragia plukentii* A.R sm leaves.
Sr.No.	Name of Extract	Characteristics	Yield (% w/w)
1	Methanol	Dark yellowish sticky extract	16.4%
2	Glacial acetic acid	Light yellowish sticky extract	18.2%
3	Cyclohexane	Greenish sticky extract	4%
4	Ethyl acetate	Dark green sticky extract	4.2%
5	Benzene	Light green sticky extract	6.2%
6	Aqueous	Brown sticky extract	9.8%

3.4 Preliminary phytochemical screening

Results: Primary phytochemical studies of six different leaf extracts of Tragia plukentii (methanol, glacial acetic acid, cyclohexane, ethyl acetate, water, and benzene) included a comprehensive set of tests: Mayer’s reagent, iodine, picric acid, Hager’s reagent, Molisch, Benedict, Fehling’s, alkaline reagent, ferric chloride, lead acetate, ammonia, concentrated sulfuric acid, Borntrager, Legal, Keller Kiliani, iodine solution, Salkowski’s, acetic anhydride, ninhydrin, and Millon tests. Positive test results indicated the presence of a wide range of phytochemicals, especially alkaloids, reducing sugars, flavonoids, phenols, phytosterols, proteins, amino acids, carbohydrates, and terpenoids. These results indicate that Tragia plukentii leaf extract is rich in bioactive compounds with potential therapeutic effects, as detailed in Table 4.

3.5 Metabolite profiling

Results

As shown in Fig. 2, a chromatogram obtained via Orbitrap high-resolution liquid chromatography‒mass spectrometry analysis identified 121 bioactive chemicals with a base peak of 1.29. This analysis provided precise information on the chemical structure, molecular formula, molecular weight, and retention time of each metabolite. Tables 5 and 6 present the results. Furthermore, metabolite profiling analysis revealed unique metabolite abundance patterns in the samples.

3.6 Antifungal activity

Results

Biological evaluation of antifungal activity against Trichophyton rubrum ATCC28188 was performed, and six different solvent extracts were evaluated and compared with the standard drug terbinafine, which has a zone of inhibition of 29 mm. The glacial acetic acid extract had the highest antifungal activity, with an inhibition zone of 45 mm, which significantly surpassed that of terbinafine. Both the water and cyclohexane extracts exhibited moderate activity, with zones of inhibition of 11 mm each, while the methanol extract also showed moderate activity at 10 mm. In contrast, the ethyl acetate and benzene extracts presented the lowest antifungal activity, with zones of inhibition of 8 mm and 7 mm, respectively. These results highlight the remarkable efficacy of the glacial acetic acid extract and suggest its potential as a potent antifungal agent while also emphasizing the different degrees of antifungal activity among the different extracts. The results are presented in Table 7 and Figs. 3 and 4.

Table 4

Phytochemical screening of different extracts of *Tragia Plukentii*
Name of Test	Solvent Extracts1
Name of Test	E1	E2	E3	E4	E5	E6
1. Test for Alkaloids
a) Mayer’s Reagent test	-ve	-ve	-ve	-ve	-ve	-ve
b) Iodine test	-ve	-ve	-ve	-ve	-ve	-ve
c) Picric Acid Test	-ve	-ve	+ve	-ve	-ve	-ve
d) Hager’s reagent test	-ve	-ve	-ve	+ve	-ve	+ve
2. Test for Carbohydrates
a) Molish’s test	-ve	+ve	-ve	-ve	+ve	+ve
b) Benedict’s test	+ve	+ve	-ve	+ve	+ve	+ve
3. Test for Reducing Sugars
a) Felling’s test	+ve	-ve	+ve	+ve	-ve	+ve
b) Benedict’s test	+ve	+ve	+ve	+ve	+ve	-ve
4 Test for Flavonoids
a) Alkaline reagent test	+ve	+ve	+ve	+ve	+ve	+ve
b) Ferric chloride test	+ve	-ve	-ve	-ve	-ve	+ve
c) Lead acetate test	+ve	+ve	+ve	+ve	+ve	-ve
d) Ammonia test	+ve	+ve	+ve	+ve	+ve	-ve
e) Conc. sulfuric acid test	-ve	-ve	-ve	-ve	-ve	+ve
5. Test for Glycosides
a) Borntrager Test	-ve	-ve	-ve	+ve	-ve	-ve
b) Legal’s Test	+ve	-ve	+ve	-ve	-ve	-ve
c) Keller-kiliani Test	-ve	-ve	+ve	+ve	-ve	-ve
6. Test for Tannins/Phenols
a) Ferric chloride test	+ve	+ve	-ve	-ve	+ve	+ve
b) Iodine solution	+ve	+ve	-ve	-ve	+ve	+ve
7. Test for Phytosterols
a) Salkowski’s Test	-ve	-ve	+ve	-ve	-ve	-ve
b) Acetic anhydride Test	+ve	-ve	+ve	-ve	-ve	-ve
8. Test for Proteins
a) Ninhydrin test	+ve	-ve	-ve	-ve	-ve	+ve
b) Millon’s test	+ve	-ve	-ve	-ve	-ve	+ve
c) Xanthoproteic test	+ve	-ve	-ve	-ve	-ve	+ve
9. Test for Amino Acids a) Ninhydrin Test	+ve	-ve	+ve	-ve	-ve	-ve
10. Test for Terpenoids a) Salkowski Test	-ve	-ve	+ve	+ve	-ve	-ve
Note: 1. The names of the solvent extracts E1 to E6 are listed below. E1 – Methanolic extract.

E2 – ethyl acetate extract.

E3 – Glacial acetic acid extract. E4 – Benzene extract.

E5 – Water extract.

E6 – Cyclohexane extract.

Table 5 Bioactive compounds identified in the glacial acetic acid extract of Tragia plukentii by OHR-LCMS analysis (positive ionization mode)

Phytoconstituents	RT1	MF2	MW3	Phytoconstituents	RT1	MF2	MW3
NP-001787	18.32	C15H12O8	320.0532	L(-)-Pipecolinic acid5	2.52	C6H11NO2	129.0790
Maltol	5.13	C6H6O3	126.0317	NP-016582	22.26	C20H35NO	305.2719
Corilagin	8.88	C27H22O18	634.0806	Quercetin	8.56	C15H10O7	302.0427
Indole-3-acetic acid	2.67	C10H9NO2	175.0633	3-(2-Hydroxyethyl)indole	3.08	C10H11NO	161.0841
Salsolinol	7.37	C10H13NO2	179.0946	Caprolactam	32.6	C6H11NO	113.0841
Sulfamethazine	15.17	C12H14N4O2	278.0837	NP-019150	8.74	C17H28O12	424.1581
Acetylcholine	1.31	C7H15NO2	145.1103	Robinetin	1.31	C7H15NO2	145.1103
NP-015145	2.25	C14H13NO2	227.0946	4-Hydroxy-6 methyl-2-pyrone	11.35	C6H6O3	126.0317
2-Hydroxyphenylalani ne	1.32	C9H11NO3	181.0739	5-Methoxyindoleacetic acid	8.56	C11H11NO3	205.0739
9-Oxo-10(E),12(E) octadecadienoic acid	19.85	C18H30O3	294.2195	Pyridoxal	1.73	C8H9NO3	167.0582
Trigonelline	1.25	C7H7NO2	137.0477	Arachidonoyl amide	21.25	C20H33NO	303.2562
DL-Carnitine	2.23	C7H15NO3	161.1052	4-Aminobenzoic acid	4.25	C7H15NO3	161.1052
Methyl 2-furoate	20.20	C6H6O3	126.0317	Oleamide	22.96	C18H35NO	281.2719
NP-020273	17.25	C17H26O5	310.1780	NP-004713	19.31	C15H24O2	236.1776
NP-020521	21.94	C18H32O3	296.2351	Biotin	8.84	C10H16N2O3S	176.0685
DL-Tryptophan	8.84	C11H12N2O2	204.0899	9-Oxo-ODE	24.25	C18H30O3	294.2195
Spectinomycin	25.25	C14 H24 N2 O7	332.1584	Methylhippuric acid	10.10	C10H11NO3	193.0739
Octyl hydrogen phthalate	20.03	C16H22O4	278.1518	6-Gingerol	17.25	C17H26O4	294.1831
(±)13-HpODE	19.49	C18H32O4	312.2301	Luotonin A	1.79	C18H11N3O	285.0902
NP-008095	12.35	C15H24O3	252.1725	L-Tyrosine	15.32	C9H11NO3	181.0739
Perillartine	8.69	C10H15NO	165.1154	12-Oxo phytodienoic acid	18.86	C18H28O3	292.2038
N-(2,4 Dimethylphenyl) formamide	1.33	C9H11NO	149.0841	Laudanosine	12.32	C21H27NO4	149.0841
NP-005289	21.78	C33H34N4O5	566.2529	NP-014517	11.34	C14H18O9	330.0951
Nicotinamide	1.74	C6H6N2O	122.0480	3-Succinoylpyridine	1.75	C9H9NO3	122.0480
3-Succinoylpyridine	1.75	C9H9NO3	179.0582	Mono(2-ethylhexyl) phthalate	12.25	C16H22O4	278.1518
N-(2 hydroxyphenyl) acetamide	9.77	C8H9NO2	151.0633	Palmitic Acid	15.96	C16H32O2	256.2402
5-Aminovaleric acid	14.25	C5H11NO2	117.0790	Valine	14.35	C5H11NO2	117.0790
L-Pyroglutamic acid	1.75	C5H7NO3	129.0426	3-Hydroxypicolinic acid	10.25	C6H5NO3	139.0269
NP-013538	7.95	C12H16O8	288.0845	Adenine	1.73	C5H5N5	135.0545
19-Nortestosterone	10.25	C18H26O2	274.1933	NP-002322	24.25	C18H32O4	312.2301
KLK	22.24	C18H37N5O4	387.2846	Nicotinic acid	20.24	C6H5NO2	123.0320
NP-010770	20.33	C33H40N2O9	608.2734	NP-021018	16.41	C12H18O4	226.1205
Stearoyl Ethanolamide	24.42	C20H41NO2	327.3137	2-(3,4dihydroxyphenyl)acetamide	22.351	C8H9NO3	167.0582
Guvacoline	3.31	C7H11NO2	141.0790	Xanthohumol	16.10	C21H22O5	354.1467
tetranor-12(R)-HETE	16.25	C16 H26 O3	266.1882	6-Hydroxynicotinic acid	14.25	C6 H5 N O3	139.0269
Tetranor-12(S) HETE	15.38	C16 H26 O3	266.1882	NP-020403	15.35	C17 H2O O3	272.1412
NP-004036	14.25	C12H22O4	230.1518	NP-020020	15.35	C7H12O5	176.0685
Sphingosine (d18:1)	15.35	C18H37NO2	299.2824	NP-022454	15.25	C12H16O3	208.1099
Thromboxane B15	26.35	C20H36O6	372.2512	Noroxymorphone	18.35	C16H17NO4	287.1158
Coumarin	10.35	C9H6O2	146.0368	2-Hydroxycinnamic acid	10.25	C9H8O3	164.0473
2-Arachidonoyl glycerol	15.01	C23H38O4	378.2770	Palmitoleic Acid	15.36	C16H30O2	254.2246
Isovanillic acid	11.25	C8H8O4	168.0423	4-oxododecanedioic acid	15.36	C12H20O5	244.1311

1 Retention time.

2 Molecular formula.

3 Molecular weight.

Table 6 Bioactive compounds identified in the glacial acetic acid extract of Tragia plukentii by OHR-LCMS analysis (negative ionization molecule)

Phytoconstituents	RT1	MF2	MW3	Phytoconstituents	RT1	MF2	MW3
NP-009092	17.513	C19H22O3	298.1569	Gallic acid	2.18	C7H6O5	170.0215
D-(-)-Quinic acid	1.27	C7H12O6	192.0634	NP-012534	17.66	C15H24O5	284.1624
Aflatoxin G1	11.25	C17H12O7	328.0583	Carminic acid	13.25	C22H20O13	266.1518
19(R)-hydroxy PGE	15.25	C20H36O6	372.2512	Rutin	9.94	C27H30O16	610.1534
Phloroglucinol	11.35	C6H6O3	126.0317	isoquercitrin	15.12	C21H20O12	464.0955
NP-004596	12.25	C32H38O21	758.1906	NP-021293	15.25	C30H48O4	472.3553
NP-015929	15.25	C22H18O11	458.0849	EGCG	15.25	C18H32O4	458.0849
Phenyloxan-3-yl-5-THB OEA	17.25	C20H39NO2	325.2981	NP-018609	14.35	C26H36O12	382.1839
13,14-dihydro PGE1	15.35	C20H36O5	356.2563	NP-004400	14.35	C26H36O12	540.2207
Esculin	12.35	C15H16O9	340.0794	NP-020236	12.35	C23H24O9	444.1420
NP-004022	14.25	C23H22O7	410.1366	NP-005128	15.35	C28H38O13	582.2312
8-iso PGE1	7.35	C22H39N O5	397.2828	NP-007735	18.25	C18H24O12	432.1268

1 Retention time.

2 Molecular formula.

3 Molecular weight.

Table 7 Antifungal activity of T. plukentii leaf extracts against Trichophyton rubrum

4.1 Morphological study of plant leaves

Discussion

The Tragia plukentii plant is dark green and has a distinct pungent and bitter odor. The reported leaves have a distinct three-lobed morphology, with varied degrees of lobation caused by occasional absence or diminution of the side lobes. They measure 3 to 10 cm in length and 1.5 to 7 cm in breadth. These papery leaves have sparse, stiff hairs or longer, softer hairs, which are predominantly found on the

veins, whereas the underside is typically hairless. The base of the leaf has three prominent veins, and the middle lobe is strikingly elongated and thin, with irregular tooth-like grooves along its edge. The smaller side lobes contain branching veins that join the main central vein. With three to seven lateral veins that curve along the edges and petioles, ranging from 3 to 20 mm, these characteristics generate a unique leaf morphology that is critical for recognizing species and studying their adaptations.

The morphological characteristics of Tragia plukentii are distinct and provide valuable insights into its identification and ecological adaptations. Detailed morphological features, including the unique three-lobed shape, vein patterns, and hair distribution, are crucial for the accurate identification of Tragia plukentii. Additionally, the adaptations of plants, such as sparse, stiff hairs and specific vein structures, may play a role in their ecological interactions and survival. Understanding these characteristics enhances our ability to study the taxonomic and ecological significance of this species.

4.2 Physicochemical study of powdered drug

Discussion

Analysis of the physicochemical properties of Tragia plukentii plant powder is crucial for evaluating the quality and purity of the raw materials used in herbal medicine and pharmacognosy. The leaves of Tragia plukentii had a total ash content of 4.60%, which is within the standard range of 1–10% typically observed in plant materials, indicating a moderate mineral content. This implies that the plant material is pure; however, confirming that there are no significant contaminants from the inorganic substances is crucial. Although a total ash value of 4.60% is generally deemed suitable for various plant materials, it is important to assess it against specific benchmarks for Tragia plukentii to ensure its integrity and quality. The moisture content, also known as the loss on drying (LOD), determines the quantity of water in Tragia plukentii powder. A value of 1.21% indicated that the material was dry. This low moisture level implies that all the water evaporated, resulting in greater stability by reducing the possibility of microbial development and degradation, which is required for pharmaceutical and phytochemical preparations. It also promotes a longer shelf-life and helps prevent fungal or bacterial contamination in plant extracts and powders. The alcohol-soluble extractive value of Tragia plukentii powder indicates the presence of phytochemicals such as flavonoids, alkaloids, glycosides, tannins, and essential oils, suggesting potential therapeutic or biological activities. This efficiency makes it relevant for developing herbal medicines or supplements. The swelling index of Tragia plukentii powder (1.5 mL) indicates its hydration capacity when immersed in water, which is crucial for understanding its behavior in aqueous environments. This index affects the disintegration and release of the active ingredients in pharmaceutical formulations, particularly tablets and capsules. A value of 1.5 ml ensures proper formulation disintegration and effective ingredient dissolution for optimal therapeutic action. The Foaming Index measures the ability of a substance to form a foam when mixed with water. A moderate value of 142.85 fu/cm indicates moderate foam formation and stable properties, which are crucial for pharmaceuticals, cosmetics, and food products. This finding highlights the surface-active properties of T. plukentii, including its ability to lower surface tension and maintain foam stability. In summary, assessing these physicochemical properties is crucial to guarantee the quality, safety, and effectiveness of raw medicinal substances. This process plays a significant role in advancing standardized herbal medicines and promoting their integration into contemporary healthcare practices.

Physicochemical analysis of Tragia plukentii powder revealed several key attributes that underscore its potential for use in herbal medicine and pharmacognosy. The total ash content of 4.60% falls within the acceptable range for plant materials, indicating moderate mineral content and suggesting the purity of the material if there are no significant contaminants. The low moisture content (1.21%) contributed to the stability of the powder by reducing microbial growth and degradation risks and increasing shelf-life. The alcohol soluble extractive value indicates the presence of various phytochemicals, such as flavonoids, alkaloids, glycosides, tannins, and essential oils, which may possess therapeutic or biological activities. The swelling index (1.5 ml) and moderate foaming index (142.85 fu/cm) highlight the hydration capacity and surface active properties of the plant powder, which are critical for pharmaceutical formulations. Overall, this study highlighted the importance of evaluating these physicochemical properties to ensure the quality, safety, and efficacy of Tragia plukentii as a raw medicinal substance. This comprehensive assessment facilitates the development of standardized herbal medicines and supports their integration into modern healthcare systems.

4.3 Physical characteristics of the plant extracts

Discussion

Several solvents used in the extraction of Tragia plukentii leaf powder, including cyclohexane, methanol, benzene, glacial acetic acid, ethyl acetate, and aqueous solutions, enable the selective separation of diverse chemical classes of bioactive phytoconstituents. Specific components were extracted from each solvent fraction according to their polarity, solubility, and chemical properties. The glacial acetic acid extract of Tragia plukentii was light yellowish and sticky, accounting for 18.2% w/w of the plant material, yielding the highest extract quantity. The methanol extract, characterized by its dark yellowish color and sticky texture, constituted approximately 16.4% w/w, making it the second highest yield. The benzene extract is light green and sticky, making up 6.2% w/w, whereas the ethyl acetate extract is dark green and sticky, comprising approximately 4.2% w/w of the plant material. The cyclohexane extract, which has a greenish, sticky texture, constitutes approximately 4% (w/w) of the plant material used. The unique color and consistency of each extract reflect the efficiency of the specific solvent for extracting different phytochemicals, with glacial acetic acid and methanol yielding higher quantities than benzene, ethyl acetate, and cyclohexane. Variations in extract characteristics highlight the diversity in phytochemical composition, which influences the potential antifungal activity and other therapeutic applications of these extracts. For example, the glacial acetic acid extract showed the highest antifungal activity, whereas the other extracts demonstrated moderate to low activity. This study demonstrated that the choice of solvent significantly affected the extraction yield and phytochemical composition of Tragia plukentii leaf powder. Among the chosen solvents, glacial acetic acid and methanol were the most effective at extracting bioactive compounds, with glacial acetic acid yielding the highest quantity (18.2% w/w), followed by methanol (16.4% w/w). The distinct colors and stickiness of the extracts obtained from different solvents suggest that various chemical profiles are influenced by solvent polarity and solubility characteristics. Notably, the glacial acetic acid extract exhibited the highest antifungal activity, indicating its superior ability to extract potent antifungal agents compared with other solvents. These findings revealed the importance of solvent selection in optimizing the extraction process for therapeutic applications, highlighting the potential of glacial acetic acid for increased antifungal activity. Further research should explore the specific phytochemicals responsible for these effects and their mechanisms of action.

4.4 Preliminary phytochemical screening

Discussion

Alkaloid detection in various extracts was assessed using several tests. Mayer’s reagent test and iodine test yielded negative results for all extracts, suggesting that alkaloids might not be present in significant amounts in these extracts. Conversely, the picric acid test indicated the presence of alkaloids only in the glacial acetic acid extract, whereas Hager’s reagent test showed a positive result only for the cyclohexane extract. Thus, these results imply that alkaloids are not uniformly distributed across the extracts, with only specific compounds present. The presence of carbohydrates in the six solvent extracts of Tragia plukentii leaves was evaluated using the Molish’s and Benedict’s tests. Molish’s test, which detects carbohydrates through violet ring formation, was positive for the ethyl acetate, water, and cyclohexane extracts, indicating the presence of carbohydrates. Conversely, methanol, glacial acetic acid, and benzene extracts tested negative, suggesting a lack of detectable carbohydrates in these extracts. Benedict’s test, which identifies reducing sugars, showed positive results for the methanol, ethyl acetate, water, and cyclohexane extracts, indicating the presence of reducing sugars.

However, the glacial acetic acid extract tested negative for reducing sugars, whereas the benzene extract tested positive, confirming the presence of reducing sugars. The presence of reducing sugars in the solvent extracts of Tragia plukentii leaves was assessed using the Fehling and Benedict tests. Fehling’s test, which detects reducing sugars through the formation of a red precipitate of cuprous oxide, yielded positive results for methanol, glacial acetic acid, benzene, and cyclohexane extracts, indicating the presence of reducing sugars in these samples. The ethyl acetate and water extracts tested negative, suggesting that reducing sugars were either absent or present at levels below the detection threshold. Conversely, Benedict’s test, which identifies reducing sugars by a color change from blue to various shades after heating, also showed positive results for methanol, glacial acetic acid, benzene, and water extracts, thereby confirming the presence of reducing sugars. However, the cyclohexane extract tested negative in this assay, suggesting a lack of detectable reducing sugars. This disparity between the tests highlights variations in sensitivity and indicates that reducing sugars may be unevenly distributed or present in varying amounts across different solvent extracts. The presence of flavonoids in the six solvent extracts of Tragia plukentii leaves was assessed by several tests. The alkaline reagent test showed positive results for all the extracts (methanol, ethyl acetate, glacial acetic acid, benzene, water, and cyclohexane), indicating that flavonoids were present across all the samples. The ferric chloride test was positive only for the methanol and cyclohexane extracts, suggesting that specific types of flavonoids in these extracts reacted with ferric chloride, whereas the other extracts did not react. The lead acetate test was positive for methanol, ethyl acetate, glacial acetic acid, benzene, and water but negative for cyclohexane, indicating that flavonoids are present in these extracts but possibly absent or undetectable in cyclohexane. The ammonia test was positive for all extracts, except for cyclohexane, which did not react, suggesting that flavonoids in the latter may differ in type or concentration. Finally, the Conc. sulfuric acid test yielded a positive result only for cyclohexane, indicating that the flavonoids in this extract reacted with concentrated sulfuric acid, whereas the other extracts did not. This variability in the results reflects the differences in flavonoid types and their reactivities with different reagents. The presence of glycosides in the six solvent extracts of Tragia plukentii leaves was evaluated using Borntrager’s, Legal’s, and Keller Kiliani tests. Borntrager’s test, which detects glycosides through pink or red color formation, was positive only for the benzene extract, suggesting that glycosides were present in this extract. A color test, in which glycosides are identified by a color change to pink or red, was positive for the methanol and glacial acetic acid extracts, indicating that glycosides are present in these solvents. The Keller-Kiliani test, which revealed the formation of reddish-brown glycosides, was positive for glacial acetic acid and benzene extracts. The other extracts (methanol, ethyl acetate, water, and cyclohexane) consistently tested negative across the tests, suggesting that glycosides may be present in varying amounts or types that are not detectable in these extracts. The presence of tannins and phenolic compounds in the solvent extracts of Tragia plukentii leaves was assessed by ferric chloride and iodine solution tests. The ferric chloride test, which detects phenolic compounds by forming a colored complex, yielded positive results for the methanol, ethyl acetate, water, and cyclohexane extracts, indicating the presence of phenolic compounds in these solvents. Glacial acetic acid and benzene extracts tested negative, suggesting lower or undetectable levels of phenolic compounds in these samples. Similarly, the iodine solution test, which identifies tannins through the formation of a blue or blue–black color, was positive for the methanol, ethyl acetate, water, and cyclohexane extracts, again indicating the presence of tannins in these extracts. Glacial acetic acid and benzene extracts tested negative, reflecting a lack of detectable tannins. Overall, the methanol, ethyl acetate, water, and cyclohexane extracts consistently contained both tannins and phenolic compounds, whereas the glacial acetic acid and benzene extracts exhibited negative results, suggesting that these compounds were less prevalent or absent in these solvents. The presence of phytosterols in the six solvent extracts of Tragia plukentii leaves was evaluated using Salkowski’s method and acetic anhydride tests. Salkowski’s test, which identifies phytosterols by a red or pink reaction with concentrated sulfuric acid, was positive only for the glacial acetic acid extract, indicating the presence of phytosterols in this sample. All other extracts (methanol, ethyl acetate, benzene, water, and cyclohexane) tested negative, suggesting the absence or undetectable levels of phytosterols in these solvents. Conversely, the acetic anhydride test, which detects phytosterols through a color change to blue or green with acetic anhydride and sulfuric acid, showed positive results for the methanol and glacial acetic acid extracts, confirming the presence of phytosterols in these solvents. The remaining extracts (ethyl acetate, benzene, water, and cyclohexane) tested negative, indicating that phytosterols were undetectable in these samples. Thus, phytosterols were identified in the methanol and glacial acetic acid extracts, with specific confirmation in the glacial acetic acid extract from Salkowski’s test, whereas other extracts showed minimal or no detectable phytosterols. The presence of proteins in the six solvent extracts of Tragia plukentii leaves was assessed by ninhydrin, Millon’s, and xanthoproteic tests. The ninhydrin test, which detects proteins by the formation of a purple or blue color, was positive for the methanol and cyclohexane extracts, indicating the presence of proteins in these solvents. Similarly, Millon’s test, which identifies proteins by a red color reaction with Millon’s reagent, was positive for the methanol and cyclohexane extracts, confirming the presence of proteins in these samples. The xanthoproteic test, which reveals proteins through yellow color formation upon reaction with nitric acid, also showed positive results for the methanol and cyclohexane extracts. All other extracts (ethyl acetate, glacial acetic acid, benzene, and water) tested negative in all three tests, suggesting that proteins were either absent or present at undetectable levels in these solvents. Thus, the methanol and cyclohexane extracts consistently demonstrated the presence of proteins, whereas the other solvents did not. The presence of amino acids and terpenoids in the six solvent extracts of Tragia plukentii leaves was assessed using ninhydrin and Salkowski tests. The ninhydrin test, which detects amino acids by producing a purple or blue color, was positive only for the methanol and glacial acetic acid extracts, indicating that amino acids were present in these solvents. The other extracts (ethyl acetate, benzene, water, and cyclohexane) tested negative, suggesting either the absence or undetectable levels of amino acids in these samples. The Salkowski test, which is used to identify terpenoids by a red or pink color reaction with sulfuric acid, was positive for glacial acetic acid and benzene extracts, confirming the presence of terpenoids in these solvents. The remaining extracts (methanol, ethyl acetate, water, and cyclohexane) did not contain detectable levels of terpenoids. Amino acids were identified in the methanol and glacial acetic acid extracts, whereas terpenoids were found in the glacial acetic acid and benzene extracts, with other extracts showing minimal or no presence of these compounds. The presence or absence of these compounds in the extracts influences their biological characteristics and therapeutic capabilities. For example, alkaloids and flavonoids are often associated with antioxidant and antimicrobial functions, whereas steroids and glycosides potentially exhibit antiinflammatory or cytotoxic effects. Tannins and phenols may have contributed to the astringent and antioxidant properties of the extracts, whereas terpenoids have been acknowledged for their wide range of pharmacological benefits, including anticancer and antimicrobial properties.

A thorough phytochemical examination of Tragia plukentii leaf extracts revealed notable differences in the composition of the bioactive compounds when different solvents were used. Alkaloids were occasionally found, appearing only in the glacial acetic acid fraction and cyclohexane extracts. Carbohydrates and reducing sugars were detected at varying concentrations, particularly in methanol, ethyl acetate, water, and cyclohexane, whereas glacial acetic acid and benzene showed inconsistent results. Flavonoids, with the exception of cyclohexane, were identified in all the extracts, with different tests revealing a variety of types. Glycosides were present mainly in the methanol, glacial acetic acid, and benzene extracts. Tannins and phenolic compounds are found in methanol, ethyl acetate, water, and cyclohexane but are absent in glacial acetic acid and benzene. Phytosterols were primarily found in the glacial acetic acid and methanol extracts, whereas proteins were identified in both the methanol and the cyclohexane extracts. Amino acids and terpenoids were detected in methanol, glacial acetic acid, and benzene extracts. This variability highlights the complex chemical composition of Tragia plukentii and suggests that different extracts may provide distinct therapeutic benefits on the basis of their phytochemical properties, including antioxidant, antimicrobial, antifungal, antiinflammatory, and other pharmacological effects.

4.5 Metabolite profiling

Discussion: Tragia plukentii A.R. sm Plant Among the identified compounds, 2-(2,6- hydroxyphenyl)-3,5,7-trihydroxy-4H-chromene-4-one presented a retention time (RT) of 12.34 minutes and a molecular weight (MW) of 302.0427, indicating moderate interaction with the stationary phase. (1R,21S,23R)-6,7,8,11,12,13,22,23-octahydroxy- 3,16-dioxo-2,17,20-trioxatetracyclo displays a significantly lower retention time of 8.32 minutes and a high molecular weight of 634.0806, suggesting that it is a larger, more polar molecule with extensive interactions. Norharman and NP-001787 share similar patterns, with retention times of 9.41 and 18.32 minutes and molecular weights of 168.0687 and 320.0532, respectively. L(-)-Pipecolinic acid, which has a very low retention time of 2.52 minutes and a molecular weight of 129.0790, is a small, highly polar molecule. Maltol (RT: 5.13, MW: 126.0317) and NP-016582 (RT: 22.26, MW: 305.2719) exhibited considerable differences in retention time despite their comparable molecular weights, reflecting differences in polarity and interactions. Corilagin and quercetin, with retention times of approximately 8.85–8.88 and molecular weights of 634.0806 and 302.0427, respectively, are highly polar compounds with substantial molecular weights. Indole-3-acetic acid (RT: 2.67, MW: 175.0633) and 3-(2-hydroxyethyl) indole (RT: 3.08, MW: 161.0841) are small polar molecules. Salsolinol (RT: 7.37, MW: 179.0946) and caprolactam (RT: 32.6, MW: 113.0841) showed significant differences in retention times, reflecting varied column interactions. High-molecular-weight compounds such as (1S,3R,4R,6S,8S,9R,13R,14R,16R)-3,4,6,9,14-pentahydroxy-5,5,9,14-tetramethyltetracyclo[11.2.1.0 a,0 b]hexadecan-16-yl acetate (RT: 16.23, MW: 412.2461) and thromboxane B1 (RT: 26.35, MW: 372.2512) have significant retention times, highlighting extensive interactions and complex structures. Low-molecular-weight compounds, such as methylhippuric acid (RT: 9.9, MW: 193.0739) and octyl hydrogen phthalate (RT: 20.03, MW: 278.1518), contributed to the broad spectrum observed, similar to xanthohumol (RT: 16.9, MW: 354.1467) and tetranor-12(R)-HETE (RT: 16.25, MW: 266.1882). The glacial acetic acid extract of Tragia plukentii revealed substantial medicinal potential through its 121 bioactive compounds, including carbohydrates, reducing sugars, flavonoids, proteins, and phenolic derivatives. OHR-LCMS analysis revealed diverse profiles of these compounds with varying retention times and molecular weights, highlighting their chemical complexities and interactions. Notable compounds, such as 2-(2,6-hydroxyphenyl)-3,5,7-trihydroxy-4H-chromene-4-one and (1R,21S,23R)-6,7,8,11,12,13,22,23-octahydroxy-3,16-dioxo-2,17,20-trioxatetracyclo, exhibited moderate to strong interactions with the chromatographic column, whereas smaller, highly polar molecules, such as L(-)-picoecolinic acid and indole-3-acetic acid, presented lower retention times. This diverse range of molecular weights and retention times highlights the broad bioactive potential of the extract, supporting its traditional medicinal use and suggesting promising avenues for the isolation and development of new therapeutic compounds with significant health benefits.

4.6 Antifungal activity

Discussion: The antifungal activity of extracts from Tragia plukentii against Trichophyton rubrum (strain ATCC28188) was assessed, and the results are presented in Table 7. Figures 3 and 4 show the bar and line graphs, respectively, of the antifungal activity of the T. plukentii leaf extracts. The following six extracts were tested: methanol, glacial acetic acid, water, ethyl acetate, cyclohexane, and benzene. Among these extracts, the extract obtained using glacial acetic acid presented the strongest antifungal activity, surpassing the standard drug terbinafine, which has a larger inhibition zone of 45 mm than does terbinafine (29 mm). The aqueous (11 mm), cyclohexane (11 mm), and methanol (10 mm) extracts showed moderate antifungal activity, with inhibition zones smaller than those of terbinafine. The ethyl acetate (8 mm) and benzene (7 mm) extracts exhibited the weakest activity, with smaller inhibition zones than did terbinafine. This study revealed that the glacial acetic acid extract of Tragia plukentii has superior antifungal efficacy against tinea, causing Trichophyton rubrum, compared with the standard drug terbinafine.

The present study demonstrated that Tragia plukentii plant extracts possess notable antifungal properties against Trichophyton rubrum (strain ATCC28188). Among the various extracts tested, glacial acetic acid exhibited the most potent antifungal activity, outperforming the standard drug terbinafine. This extract exhibited a significantly greater inhibition zone, indicating its superior efficacy. These findings suggest that the glacial acetic acid extract of Tragia plukentii could be a more effective alternative to terbinafine for treating tinea infections caused by Trichophyton rubrum.

This study highlights the unique characteristics, physicochemical properties, extraction yields, and phytochemical composition of Tragia plukentii. Unique dark green leaves with a pungent and bitter taste are important identifiers of Tragia plukentii and are important for plant identification and classification. Low moisture and ash contents ensure the quality, safety, and efficacy of Tragia plukentii leaf powder. These properties are fundamental for the stability and prevention of microbial contamination and for meeting modern pharmacognosy standards. This study revealed that different solvents produced extracts with different physical properties and yields. This variability highlights the importance of solvent selection for optimizing the extraction of the desired bioactive compounds. Tragia plukentii is rich in diverse bioactive compounds, such as carbohydrates, flavonoids, proteins, tannins, phenolic compounds, alkaloids, glycosides, and terpenoids. These compounds have potential applications in pharmaceuticals, nutraceuticals, and cosmetics, thereby emphasizing their versatility and value. The glacial acetic acid extract, containing 121 bioactive compounds, showed excellent antifungal activity against Trichophyton rubrum, outperforming the standard drug terbinafine. Tragia plukentii is an appropriate choice for the development of new antifungal drugs, as demonstrated by its antifungal activity in glacial acetic acid extracts. These findings demonstrate its potential as a powerful antifungal drug, necessitating additional research on the extraction and use of these bioactive chemicals for therapeutic purposes. Furthermore, this study establishes a precedent for the significance of solvent selection in bioactive chemical extraction, which could influence future research and standardization processes in the development of herbal medicines. Overall, this investigation provides insights into the pharmacological potential and practical applications of Tragia plukentii in modern healthcare practices.

In this study, we demonstrated that the glacial acetic acid extract of Tragia plukentii exhibited antifungal activity superior to that of the commercially used drug terbinafine. These promising results suggest that this extract is a potential candidate for the development of novel antifungal agents. Future studies should focus on isolating and characterizing the active compounds in glacial acetic acid extracts to better understand their mechanism of action. Additional in vivo studies and clinical trials are needed to evaluate the safety, efficacy, and potential benefits of this extract over existing antifungal treatments. It is also important to optimize the extraction process and formulate the extract as a stable and effective pharmaceutical product. Ultimately, the goal of this research was to develop new antifungal agents that are more effective and have fewer side effects than currently available drugs.

OHR-LCMS = Orbitrap High Resolution Liquid Chromatography and Mass Spectroscopy; SAIF = Sophisticated Analytical Instrument Facility; RT = Retention Time; MF = Molecular Formula; MW = Molecular Weight; IIT = Indian Institute of Technology; g/mol = Grams per Mole; g = Gram; Q-TOF = Quadrupole Time of Flight; mm = Micrometer; ATCC = American Type Culture Collection; cm = Centimeter; ml = Milliliter; ^。C = degree Celsius; LOD = Loss on Drying; V = Voltage; KV = Kilo voltage; % = Percent; FWHM = Full-Width at Half Maximum; ESI = Electrospray Ionization; HMDB = Human Metabolome Database; .sdf = Structured Data File; .mol = molecule (Molfiles are text files that contain structural information for a single-molecule compound. ); fu/cm = Foaming Units per Centimeter; w/w = Wight by Weight.

Ethics approval and consent to participate

”Not applicable”

Consent for publication

All the authors agreed with the content and gave explicit consent to submit, and they obtained consent from the responsible authorities at the institute/organization where the work had been carried out before the work was submitted.

Availability of data and material

“The data sets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.”

All the authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript. The authors have no financial or proprietary interests in any material discussed in this article.

Competing interests

The authors declare that they have no competing interests.”

Funding

“The authors declare that no funds, grants, or other support was received during the preparation of this manuscript.”

Author contributions

“All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Mr. Sayyad Sumer Sharif and Dr. Santosh Ramrao Butle. The first draft of the manuscript was written by Dr. Santosh Ramrao Butle and Manish Purushottam Deshmukh, and all the authors commented on previous versions of the manuscript. All the authors have read and approved the final manuscript.”

Acknowledgments

The authors thank the Director, School of Pharmacy, Swami Ramanand Teerth Marathwada University, Nanded, for providing the essential facilities for this research. The authors acknowledge the DST, FIST School of Pharmacy, Swami Ramanand Teerth Marathwada University, Nanded, and the Director, Innovation Incubation and Linkages (IIL), Coordinator, Central Instrumentation Center for Multidisciplinary Research and Innovation (CICMRI), and RUSA Center at Swami Ramanand Teerth Marathwada University, Nanded, for providing sophisticated analytical instrument facilities to conduct this research. The authors also thank the Datta Meghe Institute of Higher Education and Research (DMIHER, DU), Sawangi (Meghe), Wardha for the necessary support in the publication of this research work. The authors thank the Director of IIT Mumbai for providing OHR‒LCMS analysis of samples at the Sophisticated Analytical Instrumentation Facility (SAIF).”

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No competing interests reported.

Additionalfile1.docx
Additional file 1: This article has an accompanying supplementary file named “Additional file 1”, published alongside the electronic version of this article in BMC Complementary Medicine and Therapies, which includes images depicting the antifungal activity of T. plukentii leaf extracts against T. rubrum, a detailed list along with chemical structures of all bioactive compounds identified by OHR-LCMS analysis in positive and negative ionization mode, and a plant authentication certificate of T. plukentii. Appendix A: Images depicting the antifungal activity of T. plukentii extracts against Trichophyton rubrumfungi Images depicting the antifungal activity of T. plukentii extracts against Trichophyton rubrum are provided in Appendix A. Appendix B: Bioactive compounds identified in the glacial acetic acid extract of Tragia plukentiiby OHR-LCMS analysis (positive ionization mode) This section includes a detailed list of bioactive compounds identified in the glacial acetic acid extract of Tragia plukentii by OHR-LCMS analysis, along with structures in positive ionization mode. Appendix C: Bioactive compounds identified in the glacial acetic acid extract of Tragia plukentiiby OHR-LCMS analysis (negative ionization mode) This section includes a detailed list of bioactive compounds identified in the glacial acetic acid extract of Tragia plukentii by OHR-LCMS analysis, along with structures in negative ionization mode. Appendix D: Plant authentication certificate of Tragia plukentii The plant authentication certificate of Tragia plukentii is provided in this section.
Additionalfile2Graphicalabstract.pptx
Additional file 2: Graphical abstract
Additionalfile3ImageofT.plukentiiplant.jpg
Additional file 3: Image of Tragia plukentiiA.R. sm Plant
Additionalfile4OHRLCMSspectrum.jpg
Additional file 4: Chromatogram of Tragia plukentiiA.R. sm analyzed by Orbitrap High Resolution LC-MS
Additionalfile5Graphofantifungalactivity.jpg
Additional file 5: Bar graph showing the antifungal activity results of T. plukentii leaf extracts against Trichophyton rubrum
Additionalfile6Linegraphofantifungalactivity.jpg
Additional file 6: Line graph showing antifungal activity results of T. plukentii leaf extracts against Trichophyton rubrum

Phytochemical Analysis, OHR-LCMS Assisted Metabolite Profiling, and Antifungal Activity of Natural Products from the Medicinal Plant Tragia Plukentii A.R. sm as Antitinea Agents

Status:

Version 1

Abstract

Background

Methods

Results

Conclusions

The trial registration number (TRN):

Figures

1 Background

2 Methods

2.1 Chemicals and reagents

2.2 Collection of plant material

2.2.1 Drying and powdering of Tragia plukentii leaves

2.2.2 Preparation of extracts

2.3 Equipment and software

2.4 Fungal strains

2.5 Morphological study of plant leaves

2.6 Physicochemical study of powdered drug

2.7 Physical characterization of the plant extracts

2.8 Preliminary phytochemical screening of plant extracts

2.9 Orbitrap high resolution-liquid chromatography mass spectrometry (OHR-LCMS) analysis

2.9.1 Data processing and identification

2.9.2 Building the bioactive metabolite database

2.10 Determination of the antifungal activity of T. plukentii plant leaf extracts against Trichophyton rubrum

3 Results

3.1 Morphological study of plant leaves

3.2 Physicochemical study of powdered drug

3.3 Physical characteristics of the plant extracts

3.4 Preliminary phytochemical screening

3.5 Metabolite profiling

3.6 Antifungal activity

4 Discussion

4.1 Morphological study of plant leaves

4.2 Physicochemical study of powdered drug

4.3 Physical characteristics of the plant extracts

4.4 Preliminary phytochemical screening

4.5 Metabolite profiling

4.6 Antifungal activity

5 Conclusions

6 Future Scope

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1