Phytochemical Profiling and In-Vitro Evaluation of Biological Activities of Solanum diphyllum L

INTRODUCTION

The Solanaceae family, comprising approximately 98 genera and 3000 species, is a particularly fascinating group of angiosperms. Its members show a diverse range of morphological as well as ecological characteristics and have a cosmopolitan distribution, largely due to their utilisation as food, decorative plants, and medicinal resources. Members of the Solanaceae family are beneficial to people’s health as well as nutrition. They consist of tobacco, jimson weed seeds, black nightshade, aubergine, tomato, potato and capsicum (commonly known as chilli peppers). Throughout growth and during post-harvest distribution, these plants emit positive compounds along with potentially hazardous compounds. These active compounds consists of glycosides and alkaloids [1]. While some of these substances can be beneficial, such as antioxidants, others may offer health hazards if ingested in excess. Understanding the balance between nutritional advantages and potential toxicity is critical for safe consumption and use in food systems. Antiherpes and cancer drugs are produced from Solanum nigrum and Solanum lyratum. Glycosides like spirosolane, solanidane, spirostane, and furostane are abundant in 45 Solanum plant species, according to extensive research on these plants. The extract displayed considerable cancer-cell-growth inhibition across different cell lines and showed promising anti-herpes [2]. In conventional Indian medicine, this shrub is extensively to treat skin infections, respiratory issues, and intestinal parasites. The genus Solanum L. is the largest in the Solanaceae family. It has almost 1500 species and lives in tropical and subtropical parts of the world. They are found mostly in the south and southeast of Brazil [3]. Originally in Mexico and extending south to Costa Rica in Central America, the medicinal herb species Solanum diphyllum L., also referred to as two-leaf nightshade, has evaded cultivation and naturally spread over many tropical and subtropical regions of the world. S. diphyllum was originally discovered in India in 1995 in two lactation sites in West Bengal’s Howrah District [4]. There are no published scientific data on its micromorphological characteristics. Against breast and colon cancer cell lines, the plant showed encouraging cytotoxicity, and it may be a powerful source of anticancer compounds. The ultrastructural characteristics of the pollen, seed sporoderm, and leaf were studied using scanning electron microscopy. The morphology and anatomy of Solanum diphyllum L. were similar to those of its genus’ near relatives. The presence of lenticel-like structures may be a response to the plant’s defence needs and/or a result of an adaptation to its surroundings; for example, the plant discards excess storage materials to reduce the effect of stored osmolytes, and the presence of epidermal wax and tannins may aid the plant’s acclimatisation to the surrounding light intensities. Solanum diphyllum L. shows remarkable cytotoxicity against colon (HCT 116) and breast cancer cells (MCF7) indicating a potential source of anticancer agents [5]. Only a few number of phytochemicals from solanaceous plants are studied for biological activity, even though many have been isolated and identified. In their study, they describe the identification and isolation of the steroidal alkaloid that was recently found in Egyptian flora (Solanum diphyllum) and its potent cytotoxic action from (rel. int.) 575 [m], 557 [m-h2o], 542 [m-h2o-ch3], and 396 [m-c6h11o6] [6]. Solanum diphyllum L. exhibits anti-inflammatory, anticancer, antiulcer, antioxidant, antimicrobial, anti-infection, anti-alpha amylase, and anti-alpha glucosidase activities, as well as immunomodulatory effects, and additionally encourages cytotoxic effects on breast and colon cancer cell lines, indicating its potential as an important reservoir of anticancer compounds [7]. The objective of this investigation is to identify secondary metabolite classes using phytochemical tests and GC-MS Analysis, and characterisation of Solanum diphyllum L. species, as well as to evaluate in vitro antibacterial efficacy and cytotoxicity on different tumor cells.

Experimental Study

Procurement and processing of plant samples

A plant, Solanum diphyllum L.It was collectedfrom the Uttara Kannada district, Karnataka, India. The plant surface was thoroughly cleaned using running tap water; the plant’s surface was thoroughly cleaned to get rid of any impurities and particulates. Ground the samples after shade drying. A Soxhlet extraction was performed on about 25 g of plant powder using Distilled Water and Methanol as solvents at varying temperatures. To obtain an adequate extract for additional research, the extraction procedure was repeated multiple times. The semisolid extract was kept in glass vials for further analysis.

Qualitative phytochemical analysis

The following standard protocols were used to assess the extract for the determination of bioactive compounds from [8] [9].

Protein test:

Millon test

When two mL of Millon reagent were combined with the initial extract, a white sediment was formed that turned red when heated slightly, signifying the presence of protein.

Test for Ninhydrin

 The crude extract becomes violet when heated with two mL of a 0.2% Ninhydrin solution, indicating the existence of amino acids and proteins.

Test for Carbohydrates:

Benedict’s reagent test

When extract was heated along with 2 ml of Benedict’s reagent, carbohydrates are showed up, by forming a reddish-brown precipitate.

Molisch’s reagent test

2 ml of extract was thoroughly agitated with Molisch’s reagent, then concentrated H₂SO₄ (2 mL) added. At the interphase, a violet ring was formed suggesting the existence of a carbohydrate.

Flavonoids test

Alkaline test

The extract was combined with 2% NaOH (2 mL) solution. A strong yellow tint was produced; the yellow colour becames colorless when few drops of neutralised acid was added, shows the existence of flavonoids.

Saponins test

Foam test

The steady foam was formed by combining the extract with 5 mL deionized water by vigorous shaking indicated the existence of saponins.

Test for steroids

Urine test

After combining crude, extract with two mL of chloroform and concentrated suphuric acid (H₂SO₄ along sides of the test tube). An crimson hue formed at the bottom layer of the chloroform suggesting the steroids are present.

Terpenoids test

Burchard Test

After the extract was dissolved in 2 ml of chloroform, and dried off.  To this, add concentrated H₂SO₄ and boil for roughly 2 minutes. Terpenoids were identified by their greyish colour.

Test for alkaloids

Dragendroff’s test

After the extract was dissolved in 2 ml of chloroform, and dried off.  To this, add concentrated H₂SO₄ and boil for roughly 2 minutes. alkaloids were identified by their greyish colour.

Wagner’s reagent test

When the extract was combined with Wagner’s reagent, the formation of reddish-brown sediment indicates the existence of alkaloids.

Gas chromatography and mass spectrometry (GC-MS)

The chemical composition of S. diphyllum L. leaf extract was characterized by GC–MS studywere performed using QP2010S, Shimadzu, Japan, mass spectrometry (MS)  and gas chromatography (GC) enhanced with an electron ionisation detector. An EC-5 with 30-meter-long column with a 0.25 mm diameter was employed, using a 0.25 µL injection volume and a 2 mm direct injector were included with the chromatograph. A 2 mm direct injector liner was used to inject 2 µL of the sample. The initial temperature was 60°C for two minutes, then increased to 450°C in a level of 20°C per minute [10]. All specimens were injected straight into the capillary column using a 2 mm injector. The identification of the chromatographic peaks was achieved by their relative retention times, and the mass spectrometric analysis was conducted by precisely identifying the compounds through matching the spectral data with the NIST library [11].

Fourier Transform Infrared Spectroscopy (FTIR)

A spectrophotometer, Thermo Fisher Scientifics USA (Waltham, MA, USA), was employed for the analysis of FTIR, to identify the bioactive constituents. The extracts of S. diphyllum were paired with potassium bromide (KBr) and formed into pellets. The absorption spectra will be recorded from the 400–4000 cm⁻¹ region. The spectral analysis confirms the presence of functional biomolecules in the samples, enabling thorough characterisation of the chemical composition of the nanoparticles [12]

Cytotoxic assay

Cell culture technique

The NCCS (National Centre for Cell Sciences) provided the HeLa and A549 cell lines for this study. The cells are proliferated in DMEM, provided with 2 mM L-glutamine, and 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), along with 1.5 g/L sodium carbonate (Na₂CO₃), has important amino acids of 0.1 mM. For cell proliferation, sodium pyruvate (1mM) and glucose (1.5 g/L) are used in order to avoid contamination from microbes. 10% foetal bovine serum (GIBCO, USA) was added; the antibiotics streptomycin and penicillin were added in 100 IU/100 µg per ml; a moist incubator with 5% carbon dioxide (CO₂) was used to enhance and maintain their physiological conditions for growth and survival; and the cells were kept at 37^°C [13].

Evaluation of cytotoxicity

The fifty percent maximum concentration of inhibition measured by the MTT test and the value expressed in IC₅₀ to measure the cytotoxicity. A 96-well microplate was loaded with cultured cells (1×10¹), and incubated at 37°C for 48 hours with 5% CO₂ moistened incubator. Plant extracts of S. diphyllum with 100 µL of distinct concentrations (0–160 µg/mL) after incubation were added to each well after the monolayer was rinsed with fresh media. The cells were kept under the same settings for additional stimulations. Each well is specified with 100 µL of MTT reagent after discarding of culture media. After that, the incubation was carried out for an extra 4 hours at the same temperature. That ensures precise measurement; to dissolve the formazon crystals 100 µL of (DMSO) was added after removal of supernatant [14].

Glucose Uptake Assay

α-Glucosidase Inhibitory activity

A technique outlined by [15]. It was used to assess the samples’ α-glucosidase inhibitory activity. 50 mM of phosphate buffer with a pH of 6.9 contains 50 µL of α-glucosidase 1 U/mL from yeast (SRL, Bangalore, India) are pre-incubated for 10 minutes at 37°C with varying level of samples (0–100 µg/mL). We added 50 µL of a 5 mM p-nitrophenyl-α-D-glucopyranoside solution to the phosphate buffer to start the reaction. For thirty minutes, the catalytic action was conducted at 37°C. Na₂CO₃ (1 M) was added to terminate the reaction, and transmittance was recorded at 405 nm. The formula (OD of blank – OD of test/OD of blank) * 100 was employed to calculate the inhibition percentage, and the findings were shown in IC₅₀.

Antimicrobial Activity

The samples’ antibacterial activity was tested using the agar well diffusion method, according to reports of [16], with small alterations. Escherichia coli (MTCC-7410) Gram-negative, and  Staphylococcus aureus (MTCC-7443), Gram-positive, are used. Two fungus species: Aspergillus flavus (MTCC-9606) and Aspergillus niger. Using sterile saline solution, the cell suspension was set to approximately 5 × 105 CFU/mL. Samples were suspended in 20 mg/mL DMS stock solution and added at volumes ranging from 100 µg to 400 µg for each well. The Muller Hinton agar is used for bacteria, and for fungal psecies, Czapek s-Dox agar is used. Bacteria were incubated at 37°C, while fungal species are incubated at 28°C for 72 hours, and the zone of inhibition (mm) was evaluated in diameter.

RESULTS AND DISCUSSIONS

Preliminary Qualitative Analysis of S. diphyllum

In a phytochemical study. How the solvent selection effects the extraction of compounds in leaf extracts of S. diphyllum, the methanolic extract reveals the existence of polysaccharides, terpenoids, flavonoids, proteins, alkaloids, steroids, along with saponins, indicating that it has therapeutic potential because of its cytotoxic, antibacterial, and antioxidant qualities. The lack of steroids in the Aqueous extract and methanol extract suggests that they rely on organic solvents for extraction (Table 1). Similarly, it was reported that S. diphyllum, the stem and root extract reveals that occurrence of phenols, glycosides, flavonoids, alkaloids, saponins and carbohydrates, while histochemical studies showed the existence of calcium, tannins, starch and mucilage cells ^[17].The investigation by [18] the species Solanum Dasyphyllum fruit extract reported to possesses alkaloids, tannins, steroids and cardiac glycosides.

Gas chromatography and mass spectrometry (GC-MS)

GCMS profiling of S. diphyllum identified the range of compounds with variable area of percentage and Retention time, as demonstrated in (Table 2). And (Fig 2 & 3).  The methanol extract shown the total 9 compounds, 2,2-Dimethoxybutane RT 3.192, Neophytadiene RT 18.409, Squalene RT 32.886, Stigmasterol RT 38.565, Yohimbine RT 38.898, Diosgenin RT 39.233, 5H-3,5a-Epoxynaphth[2,1-c]oxepin, dodecahydro-3,8,8,11a- RT 40.010, Lup-20(29)-en-3-ol, acetate, (3.beta.)- RT 40.745 and Methyl (19alpha, 20alpha)-11-methoxy-19-methyl-16, 17-dide RT 42.016. Moreover, the aqueous extract has compounds like  2-Pentanone, 4-hydroxy-4-methyl-RT- RT 4.384, Diphenyl sulfone RT 18.681, Triphenylphosphine oxide RT 28.714 (Table 3). The solanum villosum the ethanol leaf extract shown the existence of 12 bioactive compounds the principal chemical compounds are phytol, N-Hexadecanoic acid, 2H-1-Bezopyran-6-ol, 11,14.17-Eicosatrienoate, 4-(3,5-Di-Tert-Butyl-4-Hydoxy Phenyl) Butyle Acrylate, 4,3-Dihydro-2,5,7,8-Tetra methyl-2(4,8,12-Trimethyl Treidecyl)-Acetate and Ethyl ester these compounds are antiflammatory, anticoronary, antieczemic, hepatoprotective, antimicrobial, lypocholestrolemic activity were reported[19]. Solanum kakasianum, the leaf and root extract contains 16 and 32 compounds, such as heptadecane-9–hexyle and stigmasterol, compound have antibacterial activity [20].

Fourier Transform Infrared Spectroscopy (FTIR)

FTIR spectra. specifies the functional groups of chemically active compounds. The significant peaks were found between 4000 cm^-1 to 600cm^-1. The methanolic extract revealed that O-H strong, sharp, O-H very strong, very broad, C-O strong, C-H medium, sharp, S=Ostrong, sharp, C=C medium, and C-H medium to strong. Corresponding to the Alkane, alkene, alkyl halide, alcohol, secondary alcohol, aromatic substitution, phenol, carboxylic acid, and sulfoxide. The Aqueous revealed that O-H strong, broad, O-H very strong, very broad, C=C medium, CO-O-CO medium, C-Cl strong, sharp, and C-CL strong. Corresponding to Alcohol, alkane, alkyl halide, carboxylic acid, cyclic alkene, anhydride, and halo compound illustrated in (Fig. 4), (Table 4, and 5). It was previously reported that the leaf, seed, and pollen of S. diphyllum exhibited the functional peaks, and have the functional groups like aliphatic amine corresponding to peak 1041, 2361 is nitrile, 3118 to aromatic alkene, 3700 with N-H stretch amide group, and this species has nitro compounds and aromatic amino acids in the range of 1550 to 1600 [21]. The studies of S. lycopersicum displayed the presence of nitro-compound, carboxylic acid. Imine/oxime, aldehydes and sulfonyl chloride, ketones and alkyl aryl, sulfoxide, correspond to their peaks at 828.888, 821.527(550-850), 700.998 (610-700), and 590.111 (515-690) [22].

Evaluation of Cytotoxicity Effect from S. diphyllum

HeLa and A549 cancer cell lines in a dose-dependent manner.  The percentage of Aqueous extract for A549 is 48%, 59%, 65%, 73%, and 84%. At 100, 200, 300, 400, and 500 µg/ml levels, the IC₅₀ value is 454.58 µg/ml, relative to reference cisplatin, while HeLa cells have IC₅₀ value of 191.58 µg/ml at the same volumes, with percentages of inhibition of 38%, 46%, 65%, 55%, 64%, and 71%. The methanolic extract had no activity against the A549 cancer cell line at the tested doses (Fig. 5). The percentages of inhibition are 41%, 53%, 66%, 77%, and 86% at the volumes 100, 200, 300, 400, and 500 (µg/ml). Sequentially, the inhibition concentration (IC50 value) is 318.07 µg/ml for HeLa cell line, compared to the standard camptothecin.  The plant S. diphyllum with steroidal alkaloid 3-O-(beta-D-glucopyranosyl) etioline [(25S)-22, 26-epimino-3beta-(beta-D-glucopyranosyloxy) cholesta-5, 22(N)-dien-16alpha-ol], which shows strong Anti-proliferative effects against the HeLa cell line reported by [23]. From previous reported that S. melangona L fruit peel methanol extract, in-vitro studies shows the successful inhibition against tested five different cancer cell lines; (HCT116),   (HEP2), (MCF7), (HeLa) and (HEPG2) cell lines and isolated compounds are subjected for in-vivo studies which shows significant cell death against (HEP2), (MEP) with IC₅₀ value 2.14±0.35 at volume levels of 100 and 200 mg/ml  [24]. In contrast, S. jasminoides, the alkaloid fractions show dose-dependent strong cytotoxic activity against the tested animals [25].

Glucose Uptake Assay

Methanolic extracts of S. diphyllum showed concentration-dependent inhibition at concentrations of 20 μL, 40 μL, 60 μL, 80 μL, and 100 μL, with percentages of inhibition of 8%, 25%, 41%, 55%, and 73%. The inhibition concentration was (IC₅₀ value) was determined to be 71.492 µg/mL (Graph 1), which is displayed in Table 4. The IC₅₀ value for Aqueous is 112.25 µg/mL. With inhibition percentages of 3%, 16%, 29%, 43%, and 57%, respectively, at quantities of 25 μL, 50 μL, 75 μL, 100 μL, and 125 μL (Fig. 3). In comparison to standard acarbose, percentage inhibition ranged from 13% to 85% at extract volumes, with 29.51 µg/mL IC₅₀ value, These findings imply that plants may have antidiabetic qualities based on earlier research. The α-Amylase activity of S. incanum L. extract of root showed the percentage of inhibition at 75.95% at volume 700 µg/ml with IC₅₀ 495 µg/ml [26].

Antimicrobial activity of S. diphyllum

Staphylococcus aureus and Escherichia coli pathogens against S. diphyllum extract showed increased noticeable antibacterial activity. (Figure 5). The least zone of inhibition was shown by the Aqueous, measuring 8 mm and 12 mm at concentrations of 20 µg and 30 µg for both bacterial strains, while methanolic extract with inhibition of 9 mm, 11 mm, and 14 mm at concentrations of 10 µg, 20 µg, and 30 µg for S. aureus and 8 mm, 11 mm, and 14 mm at the same concentrations demonstrated the greatest inhibition. S. diphyllum leaf extract displayed good results for S. aureus when compared to E. coli.From the studies of[27], Solanum nigram, the ethanol extract successfully inhibited the against the pathogensand Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli. Previous studies on S. nigram reported against these pathogens: Staphylococcus aureus, Staphylococcus pseudintermedius, Streptococcus agalacticae, Streptococcus canis, Bacillus cereus and Bacillus subtilis. Gram-positive bacteria, and Pseudomonas aeruginosa, Escherichia coli, Serratia marcescens, Pectobacterium atrosepticum, Erwinia chrysantemi, Candida albicans, and Candida parapsilosis among these bacteria, the highest inhibition was observed in Pectobacterium with a 7-11.0 mm zone of inhibition than antibiotics [28]. The strains of Aspergillus flavus and Aspergillus niger in antifungal activity S. diphyllum (Fig.5). The zone of inhibition expressed in concentrations (µg) of 100, 200, and 300 µg for A. niger and A. flavus is 10 mm and 9 mm at a dose of 300 µg, respectively. 10 mm and 9 mm at a dose of 300 µg exhibitted the inhibition zone by the methanol extract for A. niger and A. flavus. No activity is seen in distilled water extract at dose of 100 and 200 µg; the aqueous extract shows inhibition for A. flavus at a concentration of 300 µg. The inhibition zone ranges from 7 mm to 8 mm. S. aculeastrum showed the zone of inhibition at 5µg/ml. Acetone and methanol extracts both inhibited the fungal pathogen Aspergillus flavus and Penicillium notatum with 56 to 100µg of inhibition percentage [29]. It was reported that the species Solanum tomentosum methanol extract shows dose-dependent inhibition for Aspergillus niger and Fusarium oxysporum with 47.22 to 50.56% percentage of inhibition [30].

CONCLUSION:

From the investigations of the above study, Solanum diphyllum consists of diverse bioactive compounds and phytochemicals with a variety of physicochemical and pharmacological effects. Their existence supports Solanum diphyllum L. Traditional therapeutic value in treating a range of illnesses. These phytoactive compounds are related to numerous pharmacological and biological effects. The many substances found in GC-MS could be a useful source of new medications. The cytotoxic assay of S. diphyllum against A549 and HeLa cancer cell lines exhibits the effective  results. With IC₅₀ of 71.492 µg/ml and 112.25 µg/ml according to their standards, and the existence of the steroidal alkaloid 3-O-(beta-D-glucopyranosyl) etioline [(25S)-22,26-epimino-3beta-(beta-D-glucopyranosyloxy) cholesta-5,22(N)-dien-16alpha-ol] from earlier research, the different compounds are isolated and identified they are potential drug candidate the GC-MS study shows anticancer potential A549 and HeLa cancer cell lines  S. diphyllum has potential antidiabetic efficacy. Further additional studies are required to isolate, characterise, and investigate the pharmacological effects of reported compounds. Further investigation is needed on clinical trials, evaluations, and the creation of customised delivery systems should be the top priorities for future research.

ACKNOWLEDGMENT

The authors express their sincere gratitude and facilities to the Department of Studies in Botany, Davangere University, Shivagangotri, Davangere, Karnataka, India. Dr. Sudharshan B L Cheliyan Biotec Solutions, Mysore.  Additionally, Karnataka University Dharwad provides a Sophisticated Analytical Instrumentation Facility (SIAF) provides lab facilities for experiments.

6. Conflict of interest form

The authors declare that they have no conflict of interest regarding the publication of this manuscript.

REFERENCES

1. Friedman, M. (2006). Potato glycoalkaloids and metabolites: roles in the plant and in the diet. Journal of agricultural and food chemistry, 54(23), 8655-8681.
2. Nohara, T., Ikeda, T., Fujiwara, Y., Matsushita, S., Noguchi, E., Yoshimitsu, H., & Ono, M. (2007). Physiological functions of solanaceous and tomato steroidal glycosides. Journal of natural medicines, 61(1), 1-13.
3. de Jesus Matias, L., Rocha, J. A., Menezes, E. V., de Melo Júnior, A. F., & de Oliveira, D. A. (2019). Phytochemistry in medicinal species of Solanum L.(Solanaceae). Pharmacognosy Research, 11(1).
4. Singh, R. K., Jalal, J. S., & Jadhav, C. R. (2014). Solanum diphyllum (Solanaceae) in India. TAPROBANICA: The Journal of Asian Biodiversity, 6(2).
5. Hamada, F. A., Hamed, A. I., Sheded, M. G., & Shaheen, A. M. (2010). Macro, micro-morphological and bioactivity aspects of naturalised exotic Solanum diphyllum L. Al-Azhar Bulletin of Science.(ISCAZ 2010), 175-206.
6. El-Sayed, M. A., Mohamed, A. E. H. H., Hassan, M. K., Hegazy, M. E. F., Hossain, S. J., Sheded, M. G., & Oht, S. (2009). Cytotoxicity of 3-O-(β-D-glucopyranosyl) etioline, a steroidal alkaloid from Solanum diphyllum L. Zeitschrift für Naturforschung C, 64(9-10), 644-649.
7. Patel, A., & Avhad, A. Phytochemical and Nutritional Evaluation of Solanum diphyllum L.: A Potential Medicinal Plant. Botanical Insights: From Traditional Knowledge to Modern Science, 50.
8. Yadav, R. N. S., & Agarwala, M. (2011). Phytochemical analysis of some medicinal plants. Journal of phytology, 3(12).
9. Praveen T, Shreyas B, Prashanth K, Darshan R C, Siddappa B K . Phytochemical Screening, GC-MS Studies of Amorphophallus paeoniifolius (Dennst) Nicolson, and its Antimicrobial Activity. Bull. Env. Pharmacol. Life Sci., Vol 14[2] January 2025: 15-23.
10. Praveen, T., & Siddappa, B. (2025). Phytochemical Profiling and Biological Activities of Strobilanthes urens (B. Heyne ex Roth) JRI Wood. Leaf Extract (Acanthaceae). Sciences of Phytochemistry, 4(2), 107-115.
11. RC, D., & Kakkalameli, S. B. (2025). Phytochemical screening, proximate analysis and phyto-synthesis of zinc oxide metallic nanoparticles from the leaf of Abutilon persicum (Burm. f.) Merr. and their biological applications. Authorea Preprints.
12. Praveen, T., & Kakkalameli, S. B. (2025). Studies on phytochemical, GC-MS analysis in vitro cytotoxic assay and their biological Activities of Cynarospermum asperrimum (Nees) Vollesen.(Acanthaceae).
13. Tolosa, L., Donato, M. T., & Gómez-Lechón, M. J. (2014). General cytotoxicity assessment by means of the MTT assay. In Protocols in in vitro hepatocyte research (pp. 333-348). New York, NY: Springer New York.
14. Pandey, G., Prajapati, K. K., Shukla, L., Pandey, V. N., & Pandey, R. (2024). Anticancer activity and new drug discovery in Solanum species: a review. INTERNATIONAL JOURNAL OF PLANT AND ENVIRONMENT, 10(03), 48-58.
15. Kandimalla, R., Kalita, S., Choudhury, B., & Kotoky, J. (2015). A review on anti-diabetic potential of genus Solanum (Solanaceae). J Drug Deliv Ther, 5(1), 24-27.
16. Ranjitha, N., Krishnamurthy, G., Manjunatha, M. N., Naik, H. B., Pari, M., NK, V., … & Pradeepa, K. (2023). Electrochemical determination of glucose and H2O2 using Co (II), Ni (II), Cu (II) complexes of novel 2-(1, 3-benzothiazol-2-ylamino)-N-(5-chloro-2-hydroxyphenyl) acetamide: Synthesis, structural characterisation, antimicrobial, anticancer activity and docking studies. Journal of Molecular Structure, 1274, 134483.
17. Verma, R., & Dutta, S. (2020). Phytochemical and histochemical studies on Solanum diphyllum L.-An exotic potent weed. International Journal of Fundamental and Applied Sciences (IJFAS), 9(1), 1-3.
18. Matthew, O., James, A., Akogwu, I., Fabunmi, T., Idoko, O., Ebun, B., … & Dorathy, O. (2021). Evaluation of in vitro antioxidant, phytochemical and GC-MS analysis of aqueous extract of Solanum Dasyphyllum fruits. J Med Bio Sci Res, 7(3), 10-14.
19. Oza, C. (2025). International Journal of Advanced Pharmaceutical Sciences and Research (IJAPSR. International Journal of Advanced Pharmaceutical Sciences and Research (IJAPSR).
20. Chirumamilla, P., Dharavath, S. B., & Taduri, S. (2022). GC–MS profiling and antibacterial activity of Solanum khasianum leaf and root extracts. Bulletin of the National Research Centre, 46(1), 127.
21. Anilkumar, V. S., Aswathy, P. J., Sunila, A. V., & Murugan, K. (2014). Characterization of Solanum diphyllum L. medicinally potential species from the southern Western Ghats in Kerala, using SEM and FTIR spectroscopy.
22. Ramya, S., Chandran, M., King, I. J., Jayakumararaj, R., Loganathan, T., Pandiarajan, G., … & Aruna, D. (2022). Phytochemical screening, GCMS and FTIR profile of bioactive compounds in Solanum lycopersicum wild fruits collected from palani hill ranges of the Western Ghats. J. Drug Deliv. Ther, 12(6), 56-6.
23. Ishwar Prakash Sharma. (2019). Solanum diphyllum L.: a new record for the flora of Uttarakhand and Central-Western Himalaya, india
24. Salama, M., & Ezzat, S. M. (2013). In vitro and in vivo anticancer activity of the fruit peels of Solanum melongena L. against hepatocellular carcinoma.
25. Porwal, O., Nanjan, M. J., Chandrasekar, M. J. N., Srinivasan, R., & Gupta, S. (2014). Anticancer potential of solanum jasminoides. International Journal of Pharmaceutical Sciences and Research, 5(9), 3768.

• Andargie, Y., Sisay, W., Molla, M., & Tessema, G. (2022). Evaluation of antidiabetic and antihyperlipidemic activity of 80% methanolic extract of the root of Solanum incanum linnaeus (solanaceae) in mice. Evidence‐Based Complementary and Alternative Medicine, 2022(1), 4454881.
• Shahiladevi, S., and Jegadeesan, M. 2007. Antimicrobial Activity of Black Fruit Variant of Solanum nigram L. C. International Journal of Current Microbial Applied Science, 6(5): 2706- 2713. doi:https://doi.org/10.20546/ijcmas.2017.605.303
• Gębarowska, E., Łyczko, J., Rdzanek, M., Wiatrak, B., Pląskowska, E., Gołębiowska, H., … & Gębarowski, T. (2022). Evaluation of antimicrobial and chemopreventive properties and phytochemical analysis of Solanum nigrum L. aerial parts and root extracts. Applied Sciences, 12(14), 6845.
• Koduru, S., Grierson, D. S., & Afolayan, A. J. (2006). Antimicrobial Activity of Solanum aculeastrum. Pharmaceutical biology, 44(4), 283-286.
• Aliero, A. A., & Afolayan, A. J. (2006). Antimicrobial activity of Solanum tomentosum. African Journal of Biotechnology, 5(4), 369-372.

5. Figures and tables

Figures

1. Fig. 1:  Showing S. diphyllum A. Habitat, B. Twig, C. Fruit, D. Flower, E. Crude extract.
2. Fig 2: Chromatogram of S. diphyllum methanol leaf extract.
3. Fig 3: Chromatogram of S. diphyllum distilled water leaf extract.
4. Fig 4: FTIR chromatogram illustrating peak positions of the bioactive functional groups present in S. diphyllum A. Aqueous, B. Methanol
5. Fig 5: Cytotoxic assay showing different concentrations of S. diphyllum leaf extracts against A549 cell line.
6. Fig 6: Cytotoxic assay showing different concentrations of S. diphyllum leaf extracts against the HeLa cell line.
7. Fig 7: Showing Cytotoxic assay of IC50 value of aqueous and methanolic leaf extracts of S. diphyllum leaf extracts against HeLa and A549cell lines.
8. Fig 8: Microscopic images showing the cytotoxic effects (% cell viability) of methanolic and Aqueous leaf extracts of S. diphyllum in Fig A: Aqueous extract against A549 cancer cell line. Fig B: Methanol extract against A549 cancer cell line, Fig C: dist. water extract against HeLa cancer cell line, Fig D: Methanol extract against the HeLa cancer cell line
9. Fig 9: showing Glucose uptake assay of S. diphyllum leaf extract by α-Glucosidase inhibition assay.
10. Fig 10. Antimicrobial activity of S. diphyllum methanol and distilled water leaf extracts.