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2024 Volume 3
Article Contents
Abstract
Introduction
Material and methods
Plant material, bacterial strain, and vector
Seed germination and preparation of explants
Preparation of explants, culture medium, and culture conditions
Optimization of parameters affecting transformation
Transient and stable GUS gene assay
Statistical data analysis
Effect of inoculation time with bacterial suspension
Effect of acetosyringone concentration
Effect of the co-cultivation period
Effect of BAP concentration
Effect of co-cultivation temperature
Effect of co-cultivation medium pH
Effect of cotyledon explant condition
Effect of MES buffer
GUS gene assay
Results and discussion
Effect of inoculation time with bacterial suspension
Effect of acetosyringone concentration
Effect of co-cultivation period
Effect of BAP concentration
Effect of co-cultivation temperature
Effect of co-cultivation medium pH
Effect of sonication of cotyledon explants
Effect of MES buffer
Development of healthy T. alexandrinum plant
Conclusions
Understanding host-pathogen interactions
Enhancing vector design
Exploring novel Agrobacterium strains
Refining tissue culture protocols
Incorporating CRISPR-Cas technology
Utilizing omics technologies
Studying epigenetic regulation
Addressing biosafety concerns
Author contributions
Data availability
Acknowledgments
Conflict of interest
Rights and permissions
References
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In silico exploration of Elaeocarpus ganitrus extract phytochemicals on STAT3, to assess their anticancer potential

  • 1.

    Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi-110025, India

  • 2.

    Department of Chemistry, Government Degree College, Baramulla-193101, India

More Information
  • Elaeocarpus ganitrus Rox of the Elaeocarpaceae family is a broad-leaved medicinal plant and exhaustively used in orthodox systems of treating diseases. However, its anticancer impact and propensity to STAT3 has not yet been analyzed. The plant's extracts were in vitro assayed on the HeLa cell line and subsequently, GC-MS chromatogram of the methanolic, and chloroform extracts of the plant revealed that 106 compounds were present in the extracts. Subsequent filtration using Lipinski rules resulted in 81 phytochemicals being selected for the docking process with pre-selected receptor STAT3 (6NJS). Twenty-six out of 81 phyto-ligands showed high binding energy. Many drugs have weak pharmacokinetic properties and cellular toxicity and consequently, cannot pass through clinical trials. Hence, it is essential to determine the pharmacokinetic parameters of the phytoligands showing preferred binding with receptor 6NJS to consider the apparent bioavailability. The data for pharmacokinetics behavior, bioavailability extent, drug-likeness properties, medicinal chemistry friendliness, and toxicity of 26 phytochemicals with referenced inhibitors was explored. These 26 compounds were further checked for their ADMET properties by using the swissADME and PROTOX-II web server with the known inhibitors plumbagin and sanguinarine to determine the lead phytocompounds. The predictions of ADMET properties obtained six suitable phytocompounds (EG-9, EG-12, EG-13, EG-15, EG-16 and EG-26) of E. ganitrus, and found to be a perfect fit in the bioavailability radar. 2D and 3D interaction of phytoligands with the STAT3 show that the binding is through lys97, suggesting NH2-terminal domain binding of STAT3 with ligands which is the main mono-ubiquitin conjugation spot. Most of the phytoligands interactions exist in the Linker domain and Transactivation domain of the STAT3.

  • Introduction

    Trifolium alexandrinum (Berseem) earns its moniker as the Fodder Crop Champion due to its exceptional nutrient profile, wide adaptability, and the ability to be harvested multiple times[1,2]. Typically sown in October, it yields between 100 and 150 tons of fresh biomass per hectare over five to six harvests extending through November and May. Its remarkable nitrogen-fixing capability significantly enhances soil fertility by fixing 297−400 kg of atmospheric nitrogen per hectare[1]. Trifolium incorporation into crop rotations plays a pivotal role in enriching the soil with nitrogen to prepare it for subsequent summer crops like cotton and rice, necessitating planting at least once every two years[3]. It is recognized as a premier Rabi (winter season) forage crop in the entire North West Zone, Hill Zone, and portions of the Central and Eastern Zones of India, it spans over 20 lakh hectares[4]. Efficient in vitro plant regeneration and transformation systems are imperative for successfully introducing desired foreign genes into plants. Low genetic variations pose a significant obstacle to the advancement of forage legumes, necessitating genetic transformation to introduce new genes or cultivate new varieties. Biotechnological approaches offer potent alternatives for enhancing crop genetics, including elevating nutritional content and bolstering resistance to biotic and abiotic stresses. While progress has been made in creating genetic variations in T. alexandrinum, genetic transformation remains limited in this legume fodder crop, especially for Indian varieties due to genotype-dependent low regeneration frequency, poor susceptibility to Agrobacterium, and response to selective agents[1,3,5]. Only a few reports are available on this species in vitro culture and genetic transformation[6,7] (Table 1). The proper selection of suitable explants, optimization of Agrobacterium infection and regeneration conditions can overcome the limitations in the genetic transformation of this species. Therefore, in this study, the various parameters affecting genetic transformation were optimized using transient GUS activity in cotyledon explants (Fig. 1) to establish a reliable, and efficient Agrobacterium-mediated genetic transformation of T. alexandrinum to improve its high biomass production, resilience to adverse climatic and biotic stresses, substantial nitrogen fixation and more nutritious and palatable fodder.

    Table 1.  Trifolium species with explant used, gene transferred, antibiotic for selection, the vector used, analysis method, and transformation efficiency.
    S. no. Species Explant Gene transferred Vector and Agrobacterium strain Transformation efficiency Selection Analysis Ref.
    1 Trifolium
    Alexandrinum    		 Cotyledon uid A gene p7i-UG (EHA105) − − GUS assay [1]
    2 Trifolium
    repens    		 − WXP1 and GUS pCAMBIA3301
    (AGL1)    		 − Phosphinothricin GUS staining, Northern blot, PCR and RT-PCR [8]
    3 Trifolium occidentale Cotyledon Bar selection gene and uid A gene pHZBar-intGUS (GV3101) 7.5% Ammonium glufosinate and Timentin Southern blot and GUS assay [9]
    4 Trifolium subterranean Hypocotyl Bar, uid A, nptII, ∝−al pMCP3
    (AGL1)    		 − Phosphinothrycin Southern blotting, Nothern blotting, GUS assay, and α-AI immunoblot assay [10]
    5 Trifolium subterranean
    		Cotyledon uid A gene and hyg gene pH35
    (AGL0)    		 5.2% Hygromycin and Cefotaxime PCR [11]
    6 Trifolium
    Pratense    		 Callus generated from cotyledon and hypocotyl IFS gene pRI101-AN-IFS
    (LBA4404)    		 − Kanamycin and cephalosporin RT-PCR [12]
     | Show Table
    DownLoad: CSV
    Figure 1.  Optimization of different parameters for Agrobacterium-mediated transformation of Trifolium alexandrinum.
    DownLoad: Full-Size Img PowerPoint

    Material and methods

    Plant material, bacterial strain, and vector

    Mature seeds of T. alexandrinum variety Mescavi were obtained from the Chaudhary Charan Singh Haryana Agriculture University, Hisar, Haryana, India. Agrobacterium tumefaciens strain EHA105 harboring a binary vector pCAMBIA2301 that carries a scorable marker, uid A gene encoding for β glucuronidase (GUS), and a selectable marker, nptII gene encoding for neomycin phosphotransferase was used for transformation. Both genes are controlled by CaMV35S promoter (Fig. 2).

    Figure 2.  T-DNA region of the plant binary vector pCAMBIA2301 used for transformation. LB: Left border, GUS: uid A, nptII: neomycin phosphotransferase-II selectable marker genes and their respective promoters plus restriction enzyme sites, NOS terminator: nopaline synthase terminator, RB: Right border.
    DownLoad: Full-Size Img PowerPoint

    Seed germination and preparation of explants

    Healthy seeds were surface sterilized by stirring them in an aqueous 0.2% mercuric chloride solution for 3 min in a laminar air-flow chamber. Afterward, the seeds were washed five to six times with autoclaved distilled water to remove all traces of mercuric chloride. The seeds were then germinated on MSB5 medium[13] containing salts of Murashige and Skoog medium and vitamins of B5 medium[14], and 3% sucrose for 3 d at 24 ± 2 °C under a 16-h photoperiod of cool-white fluorescent light with an intensity of 80 μEm−2·s−1.

    Preparation of explants, culture medium, and culture conditions

    The cotyledon explants with petioles (~6 mm in length) were cut closely to the embryonic axis using a sterilized scalpel and forceps inside a laminar airflow cabinet. The cotyledons with intact petioles were cultured on MSB5 semi-solid media with varied amounts of BAP (2−4 mg/L) by slightly embedding the proximal cut end in the medium. The pH of the medium was maintained at 5.8 before autoclaving for 20 min at 121 °C and 15 psi. The cultures were kept at 24 ± 2 °C and exposed to 16 h of cool-white fluorescent light.

    Optimization of parameters affecting transformation

    Several variables affecting transformation efficiency were optimized using transient GUS activity to establish an efficient transformation protocol for T. alexandrinum. Three-day-old cotyledon explants excised from in vitro-raised seedlings were incubated in A. tumefaciens (pCAMBIA2301) solution and/or sonicated in a bath-type sonicator (at 40 kHz) for 30 s and gently rotated at 80 rpm for 10–40 min (Fig. 3). The explants were air-dried on sterile filter paper and co-cultivated on filter paper hydrated with liquid MSB5 co-culture medium containing MES buffer, 50−100 μM acetosyringone, 0−4 mg/L BAP at 5.2−5.8 pH under 22−28 °C and dark conditions for 1−4 d. Sixty explants were used for each experimental parameter and each experiment was carried out in triplicate (Fig. 4).

    Figure 3.  A. tumefaciens mediated transformation of T. alexandrinum. (a) T. alexandrinum seeds (bar = 1 mm). (b) T. alexandrinum 3-d old seedling (bar = 10 mm). (c) Cotyledons with petiole used as explant (bar = 2 mm). (d) Explants in co-cultivation medium (bar = 10 mm). (e) Sonication of explants (bar = 6 cm). (f) Transformed explant showing transient GUS activity while untransformed (control) explant showed no GUS activity after dipping in GUS-solution, (bar = 6 mm). (g) Agrobacterium treated explants on selection media (bar = 10 mm). (h) Explants regenerating on selection medium (bar = 10 mm) and arrow showed completely bleached non-transformed shoots on selection medium. (i) Explant with multiple shoots (bar = 0.5 mm). (j) Transformed regenerated shoots showing GUS activity and non-transformed regenerated shoots without GUS activity (bar = 1 cm).
    DownLoad: Full-Size Img PowerPoint
    Figure 4.  Stable GUS expression in the cotyledonary explant. (a) Shoots regenerated from untransformed explant (bar = 2 mm). (b) Shoots regenerated from transformed explants showing GUS activity (bar = 2 mm).
    DownLoad: Full-Size Img PowerPoint

    Transient and stable GUS gene assay

    The cocultured explants were thoroughly washed with sterilized distilled water and a final wash with cefotaxime antibiotic to kill all bacteria attached to the surface of the explants, and dried on a sterile filter paper. The explants were either incubated in a freshly prepared X-Gluc solution at 37 °C for approximately 24 h[15] or transferred onto MSB5 medium containing 2 mg/L BAP for shoot regeneration under culture conditions as mentioned in the seed germination section. The explants were treated with 70%−90% ethanol to remove their chlorophyll and examined under a stereo-zoom microscope. The frequency of GUS activity was determined by dividing the number of cotyledon explants showing blue color with the total number of cotyledon explants incubated in X-Gluc.

    Statistical data analysis

    Statistical analysis was performed for each parameter and experiment using one-way ANOVA in Graph Pad Prism 9.4. Data was recorded on 60 explants per analysis and was replicated three times. The 'Honest Significant Difference' test by Tukey was utilized to determine major variations among different groups at various levels of significance, * p < 0.05, ** p < 0.01, *** p < 0.001.

    Effect of inoculation time with bacterial suspension

    The cotyledons with petiole explants were cut from young seedlings and incubated with the co-culture medium. The number of explants that expressed GUS following Agrobacterium infection depends on the co-incubation time. In this period, Agrobacterium had the opportunity to invade meristematic cells. This study examined various (20, 30, and 40 min) co-cultivation periods.

    Effect of acetosyringone concentration

    Naturally, wounded plant cells release a phenolic compound, acetosyringone, which stimulates Agrobacterium for attachment with wounded plant cells and induces the Vir gene. Vir gene expression controls the T-DNA transfer in the plant cell[16]. It has been demonstrated that GUS is more frequently used for the genetic transformation of many plant species. The effect of different acetosyringone concentrations (50, 75, and 100 μM) on transient GUS expression was investigated by co-cultivating cotyledonary explant for 3 d in the dark.

    Effect of the co-cultivation period

    The co-cultivation incubation period influenced the genetic transformation. In legume (Cicer arietinum), the co-cultivation period was carried out for 1–4 d and optimized that 2 d of co-cultivation was better for transformation[17]. A short period is not beneficial because bacteria need sufficient time to stick to and invade plant cells. In contrast, a long period causes necrosis and, as a result, reduces T-DNA transfer. In this study, explants were incubated in co-cultivation media (pH 5.5) in the dark for 2−4 d to optimize the co-cultivation period for transient genetic expression in Trifolium alexandrinum.

    Effect of BAP concentration

    In earlier studies[18,19], it was underlined how crucial it was to include phytohormone in the co-culture medium to increase transformation incidence. A thorough investigation found that phytohormone in the medium may aid the transformants to survive in intense stress brought on by wounding and exposure to Agrobacterium. The most common compound utilized in different legume species was 6-benzyl aminopurine (BAP)[20]. For maximum transformation efficiency in Trifolium alexandrinum, different concentrations of BAP (2, 3, and 4 mg/L) were used with MsB5 media.

    Effect of co-cultivation temperature

    The Agrobacterium-mediated transformation was affected by co-cultivation temperature in Trifolium pratense[12]. Lower temperature enhanced pilus construction and increased the number of pili on the cell surface[21]. The enhanced transformation may have been partly caused by the T-DNA transfer machinery's Vir B-Vir D4 components and improved performance at low temperatures[22]. In the present study, the co-cultivation temperature was optimized between 22−24 °C to increase the transformation efficacy.

    Effect of co-cultivation medium pH

    The pH level of the co-cultivation medium plays a pivotal role in determining the virulence and transformation efficiency of Agrobacterium. The expression of Vir genes is influenced by the pH of the surrounding environment, with optimal transformation efficiency observed within the pH range of 5.0 to 6.0 in the co-cultivation medium[23]. To investigate this, three specific pH values (5.2, 5.5, and 5.8) were assessed, each with approximately 60 explants assigned to the respective treatment groups.

    Effect of cotyledon explant condition

    For genetic transformation in Trifolium, cotyledonary explants having meristematic cells are the primary goal, and mechanical damage to the meristematic region increases the potential of foreign gene uptake. Making tiny wounds and sonication enables Agrobacterium to enter meristematic cells quickly. The transformation effectiveness gradually increased with sonication cycles lasting up to 30 s.

    Effect of MES buffer

    MES buffer initiates the Agrobacterium-mediated genetic transformation[24]. In the present study, the impact of MES buffer, either being present or not in the transient GUS experiment was investigated.

    GUS gene assay

    The explants were co-cultured in liquid MSB5 co-culture medium for 1−3 d at 22−28 °C under dark conditions, were washed with sterilized distilled water and antibiotic to clean extra bacteria attached on the surface of explants, and later patted dry on a sterile filter paper, and incubated in X-Gluc dye at 37 °C for approximately 24 h[15]. The tissue should be de-coloured in 70%−90% ethanol the day after the assay solution is removed. There were 60 explants employed for each experimental parameter, and each experiment was carried out in triplicate.

    FrequencyofGUSexpression=ThenumberofcotyledonexplantsshowingabluecolorTotalnumberofcotyledonexplantsincubatedinX-Gluc

    Where the gene has been actively expressed can be seen as remarkable blue spots by the GUS-induced stain. Thus, robust promoter activity results in significant staining, whereas weak promoter activity results in minimal staining. Optimized Agrobacterium transformation protocol led to a high frequency of transformation (up to 96%) and yielded high blue spots on explants after following selected best parameters that effect transformation. Microscopic observation of the blue spots revealed transient uid A gene incorporation into the explant; besides, no blue spots are visible in control explants (without Agrobacterium treatment) (Fig. 4). Thus, following these optimized parameters gave the highest transformation efficiency in Trifolium alexandrinum.

    Results and discussion

    Agrobacterium-mediated genetic transformation has been the most common method for Trifolium species transformation[9−11,25]. A. tumefaciens and A. rhizogenes have been used for genetic transformation[5,26]. However, an efficient Agrobacterium-mediated genetic transformation for an Indian cultivar of T. alexandrinum has very few reports. The barriers to efficient gene transfer in this legume can be overcome with methodological adjustments, particularly in co-cultivation conditions and explant selection. The significant improvement in the efficiency of Agrobacterium-mediated transformation of T. alexandrinum presents a promising avenue for genetic improvement of this species.

    Effect of inoculation time with bacterial suspension

    Three-day-old cotyledon explants with petiole incubated in Agrobacterium suspension for 10, 20, 30, and 40 min showed significant differences in transient GUS expression. Explants incubated for 20 min in Agrobacterium suspension exhibited a maximum frequency of GUS expression, i.e. 91.11%, and those with higher incubation time, i. e., 30- and 40-min reduced percent explants with GUS expression. Therefore, the incubation period of 20 min was optimal in cotyledonary explants of T. alexandrinum (Fig. 5a), and a similar effect was observed in Trifolium repens[27], Vigna mungo L. Hepper[28]. Further increase in incubation time, decreased the transformation frequency with excessive bacteria growth that were difficult to eliminate. In some other plant species and legumes, explants were incubated for 20−40 min[16,27,29].

    Figure 5.  Optimization of Agrobacterium-mediated genetic transformation of Egyptian clover (Trifolium alexandrinum). (a) Effect of Agrobacterium inoculation time in minutes. (b) Effect of different acetosyringone concentrations (μM). (c) Effect of co-cultivation duration. (d) Effect of BAP concentrations in cocultivation medium. (e) Effect of temperature during co-cultivation. (f) Effect of co-cultivation medium pH. (g) Effect of explant condition (intact and sonicated). (h) Effect of presence of MES buffer in cocultivation medium. Significant differences were obtained at * p < 0.05, ** p < 0.01, *** p < 0.001 levels.
    DownLoad: Full-Size Img PowerPoint

    Effect of acetosyringone concentration

    The addition of a phenolic compound, acetosyringone (50, 75, and 100 μM) in the cocultivation medium significantly increased the transformation efficacy with an increase in its concentration. The maximum GUS expression frequency (90%) was observed at 100 μM of acetosyringone (Fig. 5b). The presence of acetosyringone in the bacterial suspension and co-cultivation medium is essential for the T. alexandrinum notable transformation. Researchers also successfully used acetosyringone at a concentration of 100 μM to increase transformation efficiency like in Cicer arietinum, Citrullus lematus Pisum sativum, and Vigna unguiculata L. Walp[16,29−31] whereas in T. subterranean and Sesamum indicum 20 μM acetosyringone was used for genetic transformation[10,32].

    Effect of co-cultivation period

    The Agrobacterium-inoculated explants were co-cultured for 1−4 d in the dark. The co-cultivation duration had a significant effect on GUS expression frequency. The 3-d co-cultivation period was the most effective, with a high transformation frequency (89.4%) for T. alexandrinum (Fig. 5c) as the same co-cultivation period was used in Vigna unguiculata L. Walp, Vigna radiata[31,33]. Co-cultivation for short periods of 1 and 2 d was not sufficient to improve GUS gene expression and extending the cocultivation beyond 4 d caused necrosis due to the excessive growth of Agrobacterium resulting in a decline of transformation frequency and explants regeneration. Most Trifolium species like T. subterranean[10], T. pratense[12], and other legumes[34,35] required a 2−7-d co-cultivation period for the maximum transformation frequency.

    Effect of BAP concentration

    As reported earlier, BAP was the most effective cytokinin for shoot regeneration in Trifolium species[1,3]. In this study, explants incubated with different BAP concentrations, 2, 3 and 4 mg/L showed significant differences in transient GUS expression. BAP might have helped the explants to survive under bacterial stress conditions. Maximum transformation efficacy (81.66%) was observed in those explants incubated on a co-cultivation medium with 2 mg/L BAP (Fig. 5d). TDZ, kinetin, and zeatin were also used in different concentrations in some other plant species[10,12,36].

    Effect of co-cultivation temperature

    Co-cultivation at 25 °C gave the maximum transitory GUS expression in Trifolium subterraneum[10] and at 26 °C produced the best transformation in both Trifolium species, T. repens, T. pratense, and Medicago[37]. Among the three temperatures (22, 24, and 28 °C) evaluated in the current study, T. alexandrinum cotyledonary explants transformation had the maximum effectiveness, i.e., 80.55% GUS expression at 24 °C, (Fig. 5e) indicating that a suitable low temperature during co-cultivation can enhance Agrobacterium-mediated transformation.

    Effect of co-cultivation medium pH

    In the present study, the effect of the co-cultivation medium at different pH (5.2, 5.5, and 5.8) was tested on transient GUS activity. The maximum GUS transformation frequency of 80.55% was at pH 5.5 (Fig. 5f) while the minimum frequency of 51.6% was at pH 5.2. Co-cultivation medium pH is the essential factor responsible for the activation of vir gene expression. So, the pH should be adjusted appropriately when preparing a co-cultivation medium. pH 5.3 and 5.4, were also used in genetic transformation studies in Trifolium alexandrinum and Cowpea[7,31].

    Effect of sonication of cotyledon explants

    Sonication of the cotyledonary explants at 40 kHz for 30 s significantly improved the transformation efficiencies to 91.1% which was 1.5-fold of the non-sonicated (intact) explants (Fig. 5g). However, sonication treatment longer than 30 s reduced the transformation efficiency. Sonication treatments considerably boosted the efficacy of genetic transformation in several plant species[38]. More phenolic compounds may be released from the micro-wounds made by sonication, enabling Agrobacterium to enter the tissue deeper and increase the effectiveness of plant transformation[39,40].

    Effect of MES buffer

    The pH of the Agrobacterium inoculation and co-cultivation medium affected the transformation efficacy of T. alexandrinum in the present study. The use of MES buffer in the co-cultivation medium maintained or retained the pH and without MES buffer use, the pH was changed to the lower side[41]. MES buffer improves the attachment of Agrobacterium to the plant leading to better T-DNA transfer and integration[16]. The MES buffer presence in the cocultivation medium improved the transformation efficiency by 82.7% (Fig. 5h) than without an MES buffer, it was only 52.2%. In the genetic transformation of several legumes, an MES buffer is also added to the cocultivation medium.

    Development of healthy T. alexandrinum plant

    Cocultured 3-day-old cotyledonary explants with petiole (Fig. 6a, b) on transfer onto shoot regeneration medium (MS medium having 2 mg/L BAP) containing 80 mg/L kanamycin and 250 mg/L cefotaxime regenerated healthy shoots (Fig. 6c, d). The regenerated shoots were elongated on MS medium containing 0.15 mg/L BAP, 80 mg/L kanamycin, and 250 mg/L cefotaxime (Fig. 6e). The elongated shoots were transferred to MS medium containing 1.5 mg/L IBA, 200 mg/L cefotaxime, and 30 mg/L kanamycin for rooting (Fig. 6f, g). Rooted shoots were grown in pots containing sterilized soil for acclimatization and flowering (Fig. 6h).

    Figure 6.  Development of Trifolium alexandrinum plants. (a) Three-day-old seedling, (bar = 10 mm), (b) excised cotyledonary explant with petiole (bar = 2 mm), (c), (d) young multiple shoots emerging from the petiolar region of cotyledon explants (bar = 0.3 cm, bar = 0.7 cm), (e) multiple shoots arising from cotyledon explant (bar = 0.8 cm), (f) elongated shoots on elongation medium (bar = 2 cm), (g) rooted shoot on rooting medium (bar = 2 cm), (h) mature plant with white flowers (bar = 9 cm).
    DownLoad: Full-Size Img PowerPoint

    Conclusions

    Optimizing factors that enhance Agrobacterium virulence for T-DNA transport and improves the ability to survive and regenerate transformed cells is crucial for legume crops like Berseem. In the current study, standardized parameters have been established for an efficient Agrobacterium-mediated transformation strategy, focusing on T. alexandrinum cotyledonary explants. Through experimentation with various variables, including bacterial inoculation time, co-cultivation duration, acetosyringone concentration, sonication-induced injuries, co-cultivation medium pH, presence of MES buffer, BAP concentrations, and co-cultivation temperature, we observed increased transformation frequencies. After thorough optimization, it was determined that cotyledonary explants with petioles, subjected to injury and inoculated in a co-cultivation medium with a pH of 5.5 for 20 min, supplemented with acetosyringone (100 μM), BAP (2 mg/L), MES buffer, and incubated for 3 d at 24 ± 2 °C, exhibited enhanced transformation efficiency. The inclusion of MES buffer maintained a constant pH in the co-cultivation medium. This optimized procedure holds promise for efficient genetic modification of Trifolium alexandrinum and other leguminous plants.

    Despite advancements, challenges persist in achieving optimal transformation efficiency in T. alexandrinum. This paper explores potential future directions and strategies to overcome these challenges, ultimately advancing the genetic improvement of this valuable forage crop.

    Understanding host-pathogen interactions

    Investigating the molecular mechanisms underlying the interaction between T. alexandrinum and Agrobacterium tumefaciens is crucial. A comprehensive understanding of factors influencing transformation efficiency, such as host cell receptivity, bacterial virulence, and genetic compatibility, will inform targeted optimization strategies.

    Enhancing vector design

    Development of improved binary vectors tailored specifically for T. alexandrinum transformation can enhance efficiency. This involves optimizing promoter and terminator sequences, incorporation of tissue-specific regulatory elements, and exploring alternative selectable marker genes to minimize pleiotropic effects and increase transformation frequency.

    Exploring novel Agrobacterium strains

    Screening and characterization of diverse Agrobacterium strains for their suitability in T. alexandrinum transformation could lead to the identification of strains with enhanced virulence and transformation efficiency. Additionally, engineering Agrobacterium strains to improve their capacity for DNA delivery and integration into the plant genome could be a promising avenue.

    Refining tissue culture protocols

    Fine-tuning tissue culture conditions, including explant type, hormone concentrations, and co-cultivation parameters, can significantly impact transformation efficiency. Optimization of these parameters based on the specific requirements of T. alexandrinum will contribute to consistent and reproducible transformation outcomes.

    Incorporating CRISPR-Cas technology

    Integration of CRISPR-Cas genome editing technology with Agrobacterium-mediated transformation can facilitate precise genetic modifications in T. alexandrinum. Harnessing CRISPR for targeted gene knockouts, gene editing, and regulatory element manipulation offers unprecedented opportunities for trait improvement with high specificity and efficiency.

    Utilizing omics technologies

    Leveraging transcriptomics, proteomics, and metabolomics approaches can provide insights into the molecular responses of T. alexandrinum to transformation and identify key regulatory nodes. Integrating omics data with transformation experiments enables a holistic understanding of the genetic and biochemical pathways involved, guiding rational design strategies for enhanced transformation efficiency.

    Studying epigenetic regulation

    Investigating the role of epigenetic modifications in modulating gene expression during T. alexandrinum transformation could uncover epigenetic targets for manipulation to improve transformation efficiency and stability of transgene expression.

    Addressing biosafety concerns

    As genetic modification technologies advance, addressing biosafety and regulatory considerations are paramount. Proactive engagement with regulatory bodies and stakeholders, along with thorough risk assessment of transformed T. alexandrinum lines ensures responsible deployment and acceptance of genetically modified crops.

    In conclusion, advancing the efficiency of Agrobacterium-mediated transformation in T. alexandrinum requires a multidisciplinary approach integrating molecular biology, microbiology, bioinformatics, and plant physiology. By addressing key challenges and implementing innovative strategies, researchers can unlock the full potential of genetic transformation for improving this vital forage crop.

    Author contributions

    The authors confirm contribution to the paper as follows: performing experiment and writing manuscript: Prajapati M, Chaudhary D; assisting in experiment and manuscript: Ahlawat YK, Jaiwal PK, Jaiwal R; project supervision: Chaudhary D, Jaiwal PK; study conception: Ahlawat YK, Chaudhary D. All authors reviewed the results and approved the final version of the manuscript.

    Data availability

    All data generated or analyzed during this study are included in this published article.

    Acknowledgments

    Mukta Prajapati and Darshna Chaudhary are grateful to the ClR-SRF and Department of Biotechnology, New Delhi for the financial support (09/382(0235)/2019-EMR-1 and BT/INF/22/SP43043/2021) respectively.

  • The authors declare that they have no conflict of interest.

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  • Cite this article

    Mehnaj, Bhat AR, Athar F. 2024. In silico exploration of Elaeocarpus ganitrus extract phytochemicals on STAT3, to assess their anticancer potential. Medicinal Plant Biology 3: e009 doi: 10.48130/mpb-0024-0010
    Mehnaj, Bhat AR, Athar F. 2024. In silico exploration of Elaeocarpus ganitrus extract phytochemicals on STAT3, to assess their anticancer potential. Medicinal Plant Biology 3: e009 doi: 10.48130/mpb-0024-0010
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In silico exploration of Elaeocarpus ganitrus extract phytochemicals on STAT3, to assess their anticancer potential

  • 1.

    Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi-110025, India

  • 2.

    Department of Chemistry, Government Degree College, Baramulla-193101, India

  • Received: 08 December 2023
  • Revised: 25 March 2024
  • Accepted: 18 April 2024
  • Published online: 16 May 2024
Medicinal Plant Biology  3,  Article number: e009  (2024)  |  Cite this article

Abstract: Elaeocarpus ganitrus Rox of the Elaeocarpaceae family is a broad-leaved medicinal plant and exhaustively used in orthodox systems of treating diseases. However, its anticancer impact and propensity to STAT3 has not yet been analyzed. The plant's extracts were in vitro assayed on the HeLa cell line and subsequently, GC-MS chromatogram of the methanolic, and chloroform extracts of the plant revealed that 106 compounds were present in the extracts. Subsequent filtration using Lipinski rules resulted in 81 phytochemicals being selected for the docking process with pre-selected receptor STAT3 (6NJS). Twenty-six out of 81 phyto-ligands showed high binding energy. Many drugs have weak pharmacokinetic properties and cellular toxicity and consequently, cannot pass through clinical trials. Hence, it is essential to determine the pharmacokinetic parameters of the phytoligands showing preferred binding with receptor 6NJS to consider the apparent bioavailability. The data for pharmacokinetics behavior, bioavailability extent, drug-likeness properties, medicinal chemistry friendliness, and toxicity of 26 phytochemicals with referenced inhibitors was explored. These 26 compounds were further checked for their ADMET properties by using the swissADME and PROTOX-II web server with the known inhibitors plumbagin and sanguinarine to determine the lead phytocompounds. The predictions of ADMET properties obtained six suitable phytocompounds (EG-9, EG-12, EG-13, EG-15, EG-16 and EG-26) of E. ganitrus, and found to be a perfect fit in the bioavailability radar. 2D and 3D interaction of phytoligands with the STAT3 show that the binding is through lys97, suggesting NH2-terminal domain binding of STAT3 with ligands which is the main mono-ubiquitin conjugation spot. Most of the phytoligands interactions exist in the Linker domain and Transactivation domain of the STAT3.

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    Introduction

    • Cancer is a cluster of diseases and STAT3 protein has significant roles in all types of cancer. Signal transducer and activator of transcription (STAT), belong to the family of cytoplasmic transcription factors, activate and transduce extracellular growth factor, and also affect cytokine signals and affect gene transcriptional events. STAT3 mutant intrinsically alone is enough to instigate oncogenic transformation, and tumorigenesis[1−3]. A survey of the current literature reveals that STATs have transactivated domains and play a significant role in cancer migration and invasion. Hampering of c-Src kinase activity or downregulation of STAT3 signaling stimulates apoptosis[4]. The study of chemical interactions between STAT3 receptor and phytochemicals assist in drug designing and hence in cancer therapy[5]. There are a variety of phytochemicals that have a high propensity to modulate directly or indirectly the STAT3 signaling pathway. Triterpenoids like betulinic acid, polyphenols curcuminoids, plumbagin a naphthoquinone, diosgenin a steroid, hydroxycinnamic acid, and thymoquinone are the phytochemicals that suppress STAT3 expression[6]. Many plant-derived phytochemicals manifest high anticancer activity and lead researchers to adopt integrated multifaceted research techniques[7−11]. Though, Elaeocarpus ganitrus Roxb. (also known as Rudraksha) constitutively placed in Ayurvedic system, also has anticancer potential[12]. Recently its silver nanoparticle has been assessed for anticancer and antiproliferative activities[13]. Its impact on STAT is yet to be explored. In the last few decades, phytochemical composition of Elaeocarpus genus has been extensively investigated. Phytochemicals of various extracts of different parts of the plant showed the presence of alkaloids, flavonoids, carbohydrates, glycosides, proteins, quinine, coumarins, tannins, minerals, vitamins, saponins, phenolic compounds, and fixed oils in a high concentration, thus adding to its medicinal value[14]. The pharmacological screening of metabolites like polyphenols, alkaloids, terpenoids and flavonoids have been explored to demonstrate cancer pathways to ascertain possible mechanism[15−18]. As stated in the literature, the beads and the bark of the plants have been extensively studied while the leaves of the E. ganitrus have not been studied for their anticancer efficiency. Besides, leaves of the plants were shown to have good antioxidant potential[19,20]. The emphasis of the study is to identify phytochemicals retrieved from Elaeocarpus ganitrus leaf research data and GC-MS profiling. To accentuate, the binding role of Elaeocarpus ganitrus phytochemicals with STAT3 receptor, their ADME properties and pharmacokinetic studies were investigated.

    Materials and methods

      Cell lines, chemicals and reagents

    • The chemicals and solvents used in the extraction and phytochemical analysis were of analytical grade, sourced from Sigma-Aldrich. MTT (3-[4,5-dimethylthiazol-2-1yl]-2,5 diphenyl tetrazolium bromide) was also procured from Sigma-Aldrich. HeLa cells were obtained from the National Centre for Cell Sciences (NCCS), Pune, India. Fetal bovine serum and Dulbecco's Modified Eagle's Media were acquired from Gibco-life technologies.

      Procurement of plant material

    • Fresh leaves of E. ganitrus were purchased from Patanjali Herbal Garden Nursery in Panchayanpur, Uttarakhand, India. Authentication of the Elaeocarpus ganitrus was conducted by the Department of Botany, Jamia Hamdard, New Delhi, India, and the voucher specimen was deposited at the University.

      Method to prepare extracts

    • Leaves of E. ganitrus were carefully washed, air-dried for ten days, and ground to a fine powder. A sample of 1,000 grams of powder was exhaustively extracted three times with 100% methanol (10 times weight/volume) at room temperature for 72 h using a soxhlet apparatus. The resulting crude methanol extract was fractionated successively with solvents in increasing polarity order: heptane, chloroform, ethyl acetate, methanol, and water. The residue was air-dried and utilized for the subsequent solvents. The fractions obtained from each solvent were filtered, dried under vacuum using a rotary evaporator, and stored at 40 °C until use[21].

      Qualitative phytochemical analysis

    • The presence or absence of phytochemicals such as terpenoids, steroids, saponins, flavonoids, glycosides, tannins, and phenols in the chloroform and methanol extracts of E. ganitrus leaves was determined following the standard methodology[22].

      Cellular studies on HeLa cancer cell line

    • The HeLa cell line was stored in Dulbecco's Modified Eagle's Medium which is rich in 10% Fetal Bovine Serum, 1% antibiotic solution, 25 mM sodium bicarbonate, and 10 mM HEPES in a 5% CO2 humidified atmosphere at 37 °C in an air jacketed incubator. The stock culture was perpetuated in the exponentially growing phase by passaging as, monolayer culture with 0.02% EDTA. Dislodged cells suspended in complete medium were routinely reseeded.

      Cytotoxicity assay/MTT assay

    • The cytotoxic effects of the various fractions of E. ganitrus leaf on the HeLa cancer cell line were evaluated using the MTT assay. Cells were seeded overnight, and exposed to different concentrations of the prepared fractions (ranging from 50 to 200 μg/ml), and incubated for 48 h. After treatment, cells were incubated with MTT solution and the formazan crystals were solubilized and the absorbance was read at 570 nm[23].

      In silico investigation of phytochemicals obtained from leaf extracts

    • Binding energies of phytochemicals retrieved from plant leave extract with STAT3 were calculated by using software InstaDock for molecular docking. Discovery Studio Visualizer, and PyMOL, were used to visualize the chemical interactions of ligands and proteins. SWISS-ADME tool and ProTox-II were used for pharmacokinetic profiling studies. The X-ray crystal structure of STAT3 (PDB ID: 6NJS) was downloaded from Protein Data Bank (PDB). All co-crystallized hetero atoms and attached water molecules and co-crystallized ligands, were eliminated from the original coordinates. The Polar hydrogen atoms were inculcated, the residue structures having lower occupancy were removed, and the incomplete side chains were then substituted by using ADT. Three-dimensional structures of phytocompounds were sketched using Chem3D.

      Drug-likeness properties prognosis

    • Determination of the analogous behavior to the drug of phytoligands with the help of cheminformatics was done using online tool SwissADME developed by the Molecular Modelling Group, Swiss Institute of Bioinformatics[24]. The computation of pharmacokinetics and physicochemical molecular properties help medicinal chemists in their routine drug discovery processes. Significant basic molecular information can be excavated from the chemical structure. The methods were preferred over other methods because of the speed, but also for the ease of interpretating results by fingerprinting method to enable researchers move through translation to medicinal chemistry and in molecular designing[25].

      Molecular docking-based virtual screening

    • The rationale behind molecular docking is to steer medicinal chemists for translational research. The affinity of a molecule to the receptor changes with small structural changes in the molecule[26]. For molecular docking, STAT3 core complex PDB id (PMID: 31715132) was remodeled to ascertain binding energies with the best conformational poses of Elaeocarpus ganitrus leaves phytoligands. The InstaDock software is used to dock phytoligands with blind search space having a grid size of 110, 70, and 108 Å for X, Y, Z coordinates, correspondingly. The center of the grid was confined to X: 63.09, Y: 14.98, and Z: −76.91 axis, which covers all the heavy atoms embedded in the protein. The conformational site selected was so that the movement of the ligands was free to probe their best binding coordinates. Default docking specifications were employed to calculate various parameters. All the docking conformational poses were generated using PyMOL, a molecular visualization system and Discovery Studio Predictor.

      Pharmacokinetic and toxicity prediction

    • Physicochemical parameters, water solubility, lipophilicity, pharmacokinetics, and drug-likeness were elicited from SwissADME. To retrieve the toxicological profile of the phytoligands ProTox-II servers were employed[27]. Early estimation of the Absorption, Distribution, Metabolism, Excretion and Toxicity abbreviated as ADMET imperative to ascribe the pharmacodynamics success of the lead phytoligands. (SMILES) strings to encode chemical structures were imported from PubChem, open chemistry database and implemented in SWISS-ADME tool[24] to auspicate lipophilicity to show hydrophobic and hydrophilic nature, water solubility, necessary for absorption across membranes, and drug-likeness rules to assess metabolic profiles. Toxicology prediction of phytoligands is a crucial and fundamental aspect in the drug discovery process. ProTox-II is used to estimate computational toxicity, to accelerate the course to drug discovery, compute animal toxicity, and also help to attenuate animal experiments. In the PROTOX-II web server, toxicity classes are designated into four segments. Category I comprised of chemical entities with LD50 (LD = lethal Dose) values (LD50 ≤ 5) mg/kg, Category II comprised of compounds with LD50 values (5 < LD50 ≤ 50) mg/kg, Category III comprised of chemical entities having LD50 values (50 < LD50 ≤ 300) mg/kg, Category IV comprised of compounds which have LD50 values (300 < LD50 ≤ 2,000) mg/kg, Category V comprised of compounds with LD50 values (2,000 < LD50 ≤ 5,000) mg/kg and Category VI comprised of compounds showing LD50 values (LD50 > 5,000) mg/kg[28]. Category I and II manifested high toxicity, Category III and IV are comparatively less toxic and Category V and VI are considered to be non-toxic.

    Results and discussion

    • The solvent extraction technique is usually employed to prepare extracts from plant materials attributable to its convenience to operate. The importance lies in that a large amount of plant material can be extracted with minimal solvent[26]. Fresh leaves of Elaeocarpus ganitrus were purchased from Patanjali Herbal Garden Site Nursery located in Panchayanpur, Uttarakhand 249405, India. The confirmation of the authenticity of the Elaeocarpus ganitrus was done by the Department of Botany, Jamia Hamdard, New Delhi, India, and the leaf specimens deposited in the University. The crude methanol extract was unintermittedly fractionated in the solvents heptane, chloroform, ethyl acetate, methanol, and water according to their increasing polarity[16]. The anticancer activity of extracts was analyzed on the basis of their IC50 values. Cancerous HeLa cell line when treated with E. ganitrus leaf extracts exhibited a substantial inhibition of cells. The half maximal inhibitory concentration of chloroform and methanol extracts of E. ganitrus was (IC50 = 304.39 μg/ml) and (IC50 = 308.59 μg/ml) respectively followed by water (IC50 = 340.14 μg/ml), ethyl acetate (IC50 = 350.72 μg/ml) and heptane (IC50 = 381.76 μg/ml) extracts (Fig. 1a−e & Fig. 2). The qualitative investigation using standard methodology[22] of chloroform and methanol fractions of E. ganitrus leaves disinterred the presence of major phytochemicals namely steroids, saponins, terpenoids, tannins, phenols, glycosides and flavonoids Table 1. GC-MS analysis of the chloroform and methanolic fractions was done based on their lowest half maximal inhibitory concentration to get a complete profiling of the plant compounds. The peaks in the total ion current (TIC) chromatogram of GC-MS profile of the phytoligands commensurate with the spectrum of known chemical databases stockpiled in the GC-MS library. The gas chromatogram depicts the relative concentrations of different phytoligands getting eluted according to the retention time. The heights of the peak represent the comparative concentrations of the compounds present in the plant appear as peaks at different m/z ratios. The components present with their retention time, molecular formula, molecular weight and concentration (peak area %) are provided in Tables 2 & 3 showing the presence of 56 and 50 bioactive phytochemicals in the chloroform and methanol extracts respectively. Of 106 phytoligands obtained from chloroform and methanol extracts of E. ganitrus leaves, 81 phytoligands were identified has having the best drug-like properties following Lipinski's rule of five. Lipinski's rule states that molecular properties, physical or chemical of a compound are significant for a drug's pharmacokinetics behavior inside a biological system. The drug molecules that go along with the RO5 have fewer attrition rates when undergoing clinical trials. The cheminformatics study to identify potential chemical entities having propensity for predefined biological targets is called virtual screening[28]. To endeavor in vitro experiments time diminution, molecular docking-based virtual screening of 81 selected compounds with two reference inhibitors having substantial binding energies with 6NJS were preferred for further analysis. The STAT3 has dual nature as an oncogene or as a tumor suppressor during cancer progression. It has a SH2 domain, linker domain, DNA binding domain, and all-alpha domain. The total energy of binding, Vander Waals forces, hydrogen bonding, electrostatic attraction, desolvation, and also a number of rotatable bonds present in the phytoligand, contribute to observe the free energy of binding of phytoligands with the receptor. Twenty-six (EG-1 to EG-26) compounds were selected as having appreciable binding affinities towards the 6NJS receptor (Table 4).

      Figure 1. 

      Effects of (a) heptane, (b) chloroform, (c) methanol, (d) ethyl acetate and (e) water fractions of E. ganitrus leaves on the human cancer cell lines HeLa using MTT assay.

      Figure 2. 

      IC50 values of different extracts of E. ganitrus leaves against human cancer cell lines HeLa.

      Table 1.  Qualitative analysis of phytochemicals in E. ganitrus leaf extracts.

      Tested compounds Chloroform extractMethanol extract
      Steroids++
      Terpenoids++
      Saponins++
      Glycosides++
      Tannins++
      Flavonoids++
      Phenols++
      + → Present; − → Absent.

      Table 2.  GC–MS analysis of chloroform fraction of E. ganitrus leaves.

      Peak no.R. TimeAreaArea %Name
      17.32833924513.60Phenol, 2-methoxy-4-(2-propenyl)-
      27.4944325420.46Cyclododecane
      37.92550188745.32Bicyclo[7.2.0]undec-4-ene,4,11,11-trimethyl-8-methylene-
      48.3922645730.281,4,8-Cycloundecatriene, 2,6,6,9-tetramethyl-,(e,e,e)-
      59.13715685461.66Phenol, 3,5-bis(1,1-dimethylethyl)-
      610.01627271302.891-Heptadecene
      712.22129599333.141-Octadecene
      812.6701789630.19Neophytadiene
      912.7821478930.162-Pentadecanone, 6,10,14-trimethyl-
      1013.4961275110.147,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione
      1113.59421053742.23Hexadecanoic acid, methyl ester
      1213.8011860960.20Isophytol
      1314.00325829872.74Dibutyl phthalate
      1414.2331264020.141-Nonadecene
      1515.1432629120.281-Octadecanol
      1615.1964093250.439,12-Octadecadienoic acid (z,z)-, methyl ester
      1715.25614188331.509,12,15-Octadecatrienoic acid, methyl ester, (z,z,z)-
      1815.39682671938.76P-menth-1-ene-3,3-d2
      1915.77642684864.53Cholest-24-ene, (5.alpha.,20.xi.)-
      2016.08118351251.95Behenic alcohol
      2117.0155746820.61Glycidyl palmitate
      2217.5023567620.384,8,12,16-Tetramethylheptadecan-4-olide
      2317.79215482661.64N-tetracosanol-1
      2418.5078929660.95Glycidyl oleate
      2518.6333870750.41Pentacosane
      2618.88511608121.23Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester
      2718.94925201262.671,2-Benzenedicarboxylic acid
      2819.38317228801.83Hexacosyl pentafluoropropionate
      2919.9972957890.31Carbonic acid, propyl 3,5-difluophenyl ester
      3020.15216559811.76Tetracontane
      3120.2877183500.769-Otadecenoic acid (z)-, 2,3-dihydroxypropyl ester
      3220.43716334241.73Octadecanoic acid, 2,3-dihydroxypropyl ester
      3320.87585309029.04Carbonic acid, eicosyl prop-1-en-2-yl ester
      3421.22511697911.24 .alpha.-tocospiro b
      3521.37114757101.56 .alpha.-tocospiro b
      3621.56627907932.96Tetracosane
      3721.6195665540.601-Heptacosanol
      3821.9782082850.22Tetracontane
      3922.24431468313.34Tetracontane
      4022.3391988570.21Triacontyl acetate
      4122.7586025580.64 .gamma.-tocopherol
      4222.98737696614.00Tetracontane
      4323.0833075630.33Octacosanol
      4423.3688043680.852,5,7,8-Tetramethyl-2-(4,8,12-trimethyltridecyl)-3,4-dihydro-2h-chromen-6-yl hexofuranoside
      4523.83228257813.00Hexatriacontane
      4624.4422247490.24Ergost-5-en-3-ol
      4724.6971463260.162,6,10,15,19,23-Hexamethyl-tetracosa-2,10,14,18,22-pentaene-6,7-diol
      4824.81625792062.73Tetracontane
      4925.34444036834.67 .gamma.-sitosterol
      5025.8972474510.26Phenol, 2,4-bis(1,1-dimethylethyl)-, phosphite (3:1)
      5125.97110509901.11Tetracontane
      5227.36910622631.13Tetracontane
      5329.04944683384.74Benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy-,octadecyl ester
      5431.0618597260.91Tetrapentacontane
      5533.5056890170.73Tetrapentacontane
      5636.5155702640.60Tetrapentacontane

      Table 3.  GC–MS analysis of methanol fraction of E. ganitrus leaves.

      Peak no.R. timeAreaArea %Name
      14.56217160622.754h-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-
      25.5451328910.211,5-Dimethyl-1-vinyl-4-hexenyl 2-aminobenzoate
      36.130664200.11E-6-octadecen-1-ol acetate
      46.658982694115.754-Hydroxy-3-methylacetophenone
      57.421592600.091-Undecanol
      67.7462900200.46Methyl2,3,6,7-tetra-o-acetyl-4-o-methyl-.beta.-glycero-d-glucoheptopyranoside
      78.93519327693.10Guanosine
      89.1785358220.861,3:2,5-Dimethylene-l-rhamnitol
      99.9494638700.74Octadecanoic acid
      1010.1442790410.451,2-Benzenedicarboxylic acid, diethyl este
      1110.37016839662.70 .alpha.-methyl-l-sorboside
      1210.60613007272.08 .alpha.-d-galactopyranoside, methyl
      1310.9202805730.45Butanoic acid, 3-methyl-, hexahydro-4- methylspiro[cyclopenta[c]pyran-7(1h),2'-oxirane]-1,6-diyl ester
      1411.090803800.13Tricyclo[7.2.0.0(2,6)]undecan-5-ol, 2,6,10,10-tetramethyl- (isomer 3)
      1511.2241616060.26 .alpha.-d-galactopyranoside, methyl
      1611.4921894850.30Octadecanoic acid, methyl ester
      1712.4372606340.422(4h)-benzofuranone, 5,6,7,7a-tetrahydro-6- hydroxy-4,4,7a-trimethyl-, (6s-cis)-
      1812.6431237680.20Neophytadiene
      1913.56528660454.59Hexadecanoic acid, methyl ester
      2013.780340440.051-hexadecen-3-ol, 3,5,11,15-tetramethyl-
      2113.910475350.08Silane, ethenylethyldimethyl-
      2214.555735350.12Pentadecanoic acid, methyl ester
      2315.18817688992.839,12-Octadecadienoic acid (z,z)-, methyl ester
      2415.24959714079.57(9e,12e)-9,12-octadecadienoyl chloride #
      2515.385793321212.711,1'-Bicyclohexyl, 2-methyl-, cis-
      2615.4819100731.46Methyl stearate
      2715.758823962913.21Cholest-24-ene, (5.alpha.,20.xi.)-
      2816.0753964310.64Methyl octadeca-9,12-dienoate
      2916.444772460.12Methyl 4-(dimethylamino)bicyclo[2.2.2]oct- 5-ene-2-carboxylate
      3016.612548090.09Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester
      3116.9992156190.3517-octadecynoic acid
      3217.2492190610.35Eicosanoic acid, methyl ester
      3318.1272324800.37Oleoyl chloride
      3418.5093011250.48Undec-10-ynoic acid, undec-2-en-1-yl ester
      3518.7051353320.22Hexadecanoic acid, 1-(hydroxymethyl)-1,2-ethanediyl ester
      3618.89946078647.38Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester
      3719.6551063120.17Hexadecanoic acid, methyl ester
      3820.3005806710.93Oleoyl chloride
      3920.4629943041.59Octadecanoic acid, 2,3-dihydroxypropyl ester
      4020.91235840275.749-octadecenamide
      4121.2252214500.35 .alpha.-tocospiro b
      4221.3783841650.62 .alpha.-tocospiro b
      4321.6261759730.28Eicosyl heptafluorobutyrate
      4421.803899870.14Hexacosanoic acid, methyl ester
      4522.7691609600.26 .gamma.-tocopherol
      4622.989867570.14Tetracontane
      4723.174924420.15Stigmast-5-en-3-ol, (3.beta.)-
      4823.38010301431.65Vitamin e
      4925.37212372631.98 .gamma.-sitosterol
      5027.0841838260.29Di-o-acetyltetrahydrostapelogenin

      Table 4.  Docking results of 81 phytoligands.

      S. no.Name of the ligandBinding free energy
      (kcal/mol)      		pKiLigand efficieny (kcal/mo/non-H atom)Torsional energy
      1Phenol, 2-methoxy-4-(2-propenyl)-–5.64.110.46671.2452
      2Cyclododecane–5.94.330.49170
      3Bicyclo[7.2.0]undec-4-ene,4,11,11-trimethyl-8-methylene-–6.64.840.440
      41,4,8-Cycloundecatriene, 2,6,6,9-tetramethyl-,(e,e,e)-–6.54.770.43330
      5Phenol, 3,5-bis(1,1-dimethylethyl)-–6.54.770.43330.9339
      61-Heptadecene–4.63.370.27064.3582
      71-Octadecene–42.930.24.0469
      8Neophytadiene–5.94.330.2954.0469
      92-Pentadecanone, 6,10,14-trimethyl-–5.43.960.28423.7356
      107,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione–6.54.770.3250.6226
      11Hexadecanoic acid, methyl ester–4.93.590.25794.6695
      12Isophytol–4.93.590.23334.3582
      13Dibutyl phthalate–5.23.810.263.113
      141-Nonadecene–4.93.590.25794.9808
      151-octadecanol–53.670.26325.2921
      169,12-Octadecadienoic acid (z,z)-, methyl ester–53.670.23814.6695
      179,12,15-Octadecatrienoic acid, methyl ester, (z,z,z)-–5.43.960.25714.3582
      18P-Menth-1-ene-3,3-d2–4.93.590.490.3113
      19Behenic alcohol–4.73.450.20436.5373
      20Glycidyl palmitate–5.43.960.28423.7356
      214,8,12,16-Tetramethylheptadecan-4-olide–6.34.620.27393.7356
      22N-tetracosanol-1–4.43.230.1767.1599
      23Glycidyl oleate–4.63.370.19175.6034
      24Pentacosane–4.73.450.1886.8486
      25Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester–4.63.370.26.226
      261,2-Benzenedicarboxylic acid–5.74.180.4751.2452
      27Carbonic acid, propyl 3,5-difluophenyl ester–6.14.470.40671.5565
      289-Octadecenoic acid (z)-, 2,3-dihydroxypropyl ester–53.670.26.5373
      29Octadecanoic acid, 2,3-dihydroxypropyl ester–4.93.590.1966.8486
      30Carbonic acid, eicosyl prop-1-en-2-yl ester–5.13.740.18896.8486
      31Tetracosane–4.93.590.20426.5373
      321-Heptacosanol–5.13.740.18218.0938
      33Triacontyl acetate–3.92.860.11479.339
      34Gamma.-tocopherol–6.84.990.22674.0469
      35Octacosanol–4.23.080.14488.4051
      362,5,7,8-Tetramethyl-2-(4,8,12-trimethyltridecyl)-3,4-dihydro-2h-chromen-6-yl hexofuranoside–7.25.280.17146.226
      37Ergost-5-en-3-ol–7.35.350.25171.8678
      382,6,10,15,19,23-Hexamethyl-tetracosa-2,10,14,18,22-pentaene-6,7-diol–64.40.18755.6034
      39Gamma.-sitosterol–96.60.32.1791
      404h-pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl-–53.670.50.6226
      411,5-Dimethyl-1-vinyl-4-hexenyl 2-aminobenzoate–6.44.690.322.4904
      42E-6-octadecen-1-ol acetate–4.73.450.21365.2921
      434-Hydroxy-3-methylacetophenone–5.74.180.51820.6226
      441-undecanol–4.53.30.3753.113
      45Methyl2,3,6,7-tetra-o-acetyl-4-o-methyl-.beta.-glycero-d-glucoheptopyranoside–5.54.030.19643.7356
      46Guanosine–6.84.990.26151.5565
      471,3:2,5-Dimethylene-l-rhamnitol–5.43.960.41540.3113
      48Octadecanoic acid–5.33.890.2655.2921
      491,2-benzenedicarboxylic acid, diethyl este–5.43.960.33751.8678
      50 .alpha.-methyl-l-sorboside–4.73.450.36151.8678
      51 .alpha.-d-galactopyranoside, methyl–5.23.810.41.8678
      52Butanoic acid, 3-methyl-, hexahydro-4- methylspiro[cyclopenta[c]pyran-7(1h),2'-oxirane]-1,6-diyl ester–6.84.990.22673.4243
      53Tricyclo[7.2.0.0(2,6)]undecan-5-ol, 2,6,10,10-tetramethyl- (isomer 3)–6.54.770.40620.3113
      54 .alpha.-d-galactopyranoside, methyl–5.33.890.40771.8678
      55Octadecanoic acid, methyl ester–4.13.010.19525.2921
      562(4h)-benzofuranone, 5,6,7,7a-tetrahydro-6- hydroxy-4,4,7a-trimethyl-, (6s-cis)-–6.54.770.46430.3113
      57Neophytadiene–53.670.254.0469
      58Hexadecanoic acid, methyl ester–4.93.590.25794.6695
      591-Hexadecen-3-ol, 3,5,11,15-tetramethyl-–5.74.180.27144.3582
      60Pentadecanoic acid, methyl ester–4.43.230.24444.3582
      619,12-Octadecadienoic acid (z,z)-, methyl ester–5.43.960.25714.6695
      62(9e,12e)-9,12-octadecadienoyl chloride #–4.73.450.2354.3582
      631,1'-bicyclohexyl, 2-methyl-, cis-–5.64.110.43080.3113
      64Methyl stearate–53.670.23815.2921
      65Cholest-24-ene, (5.alpha.,20.xi.)-–9.26.750.34071.2452
      66Methyl octadeca-9,12-dienoate–4.53.30.21434.6695
      67Methyl 4-(dimethylamino)bicyclo[2.2.2]oct- 5-ene-2-carboxylate–5.74.180.380.9339
      68Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester–4.83.520.20876.226
      6917-octadecynoic acid–5.13.740.2555.2921
      70Eicosanoic acid, methyl ester–4.83.520.20875.9147
      71Undec-10-ynoic acid, undec-2-en-1-yl ester–5.13.740.21255.9147
      72Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester–4.73.450.20436.226
      73Hexadecanoic acid, methyl ester–4.53.30.23684.6695
      74Oleoyl chloride–4.63.370.234.6695
      75Octadecanoic acid, 2,3-dihydroxypropyl ester–4.63.370.1846.8486
      769-octadecenamide–4.73.450.2354.6695
      77 .alpha.-tocospiro b–6.44.690.19394.3582
      78Eicosyl heptafluorobutyrate–5.64.110.16977.1599
      79Hexacosanoic acid, methyl ester–4.73.450.16217.7825
      80Stigmast-5-en-3-ol, (3.beta.)-–7.65.570.25332.1791
      81Vitamin e–7.15.210.2294.0469
      82Plumbagin–5.94.330.42140.3113
      83Sanguinarine–8.96.530.3560

      The absorption of drugs by the body is related to their pharmacokinetic properties and also cellular toxicity. The potency of the drug depends mostly on the pharmacokinetic parameters because ADME processes command the rate and extent of absorption when an administered dose of a drug approaches to its action site. Hence, in silico pharmacokinetic profile of filtered compounds was surveyed to gather the putative bioavailability data for receptor 6NJS. The cumulative findings for pharmacokinetics profiling, bioavailability data, drug-likeness properties and drug friendliness and toxicity effects of selected 26 phytoligands with known inhibitors (Plumbagin and Sanguinarine) are given in Tables 5−10. The prediction revealed that the six molecules (EG-9, EG-12, EG-13, EG-16, and EG-26) can be lead compounds for new drug candidates for anti-cancer phytomedicine. The Half maximal Inhibitory concentration of EG-13 was (IC50 = 254.29 µg/ml) further support our results (Fig. 3).

      Table 5.  Pharmacokinetics prediction of phytoligands established in E. ganitrus.

      S. no.Phytochemical
      		Gastro- intestinal absorptionBlood-brain permeantP-glycoprotein substrateCYP450 1A2 inhibitorCYP450 2C19 inhibitorCYP450 2C9 inhibitorCYP450 2D6 inhibitorCYP450 3A4 inhibitorSkin permeation as log Kp (cm/s)
      EG-1Cholest-24-ene, (5.alpha.,20.xi.)-LowNoNoNoNoYesNoNo–1.02
      EG-2gamma.-sitosterolLowNoNoNoNoNoNoNo–2.65
      EG-3Stigmast-5-en-3-ol, (3.beta.)-LowNoNoNoNoNoNoNo–2.20
      EG-4Ergost-5-en-3-olLowNoNoNoNoNoNoNo–2.50
      EG-52,5,7,8-Tetramethyl-2-(4,8,12-trimethyltridecyl)-3,4-dihydro-2h-chromen-6-yl hexofuranosideLowNoNoNoNoNoNoYes–3.60
      EG-6Vitamin eLowNoYesNoNoNoNoNo–1.33
      EG-7GuanosineLowNoNoNoNoNoNoNo–9.37
      EG-8gamma.-tocopherolLowNoYesNoNoNoNoNo–1.51
      EG-9Butanoic acid, 3-methyl-, hexahydro-4- methylspiro[cyclopenta[c]pyran-7(1h),2'-oxirane]-1,6-diyl esterHighYesNoNoNoNoYesYes–6.18
      EG-10Bicyclo[7.2.0]undec-4-ene,4,11,11-trimethyl-8-methylene-LowNoNoNoYesYesNoNo–4.44
      EG-111,4,8-Cycloundecatriene, 2,6,6,9-tetramethyl-,(e,e,e)-LowNoNoNoNoYesNoNo–4.32
      EG-12Phenol, 3,5-bis(1,1-dimethylethyl)-HighYesNoNoNoNoYesNo–4.07
      EG-137,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dioneHighYesNoNoYesYesNoNo–5.28
      EG-142(4h)-benzofuranone, 5,6,7,7a-tetrahydro-6- hydroxy-4,4,7a-trimethyl-, (6s-cis)-HighYesNoNoNoNoNoNo–6.79
      EG-15Tricyclo[7.2.0.0(2,6)]undecan-5-ol, 2,6,10,10-tetramethyl- (isomer 3)HighYesNoNoYesYesNoNo–4.75
      EG-161,5-Dimethyl-1-vinyl-4-hexenyl 2-aminobenzoateHighYesNoNoYesYesNoNo–4.54
      EG-17alpha.-tocospiro bHighNoNoNoNoNoNoNo–3.90
      EG-184,8,12,16-Tetramethylheptadecan-4-olideLowNoNoYesNoYesNoNo–2.70
      EG-19Carbonic acid, propyl 3,5-difluophenyl esterHighYesNoYesYesNoNoNo–5.37
      EG-202,6,10,15,19,23-Hexamethyl-tetracosa-2,10,14,18,22-pentaene-6,7-diolLowNoNoYesNoYesNoNo–2.37
      EG-21CyclododecaneLowNoNoNoNoNoNoNo–4.42
      EG-22NeophytadieneLowNoYesNoNoYesNoNo–1.17
      EG-231,2-Benzenedicarboxylic acidHighNoNoNoNoNoNoNo–6.80
      EG-244-Hydroxy-3-methylacetophenoneHighYesNoYesNoNoNoNo–6.54
      EG-251-Hexadecen-3-ol, 3,5,11,15-tetramethyl-LowNoYesNoNoYesNoNo–2.41
      EG-26Methyl 4-(dimethylamino)bicyclo[2.2.2]oct- 5-ene-2-carboxylateHighYesNoNoNoNoNoNo–6.65
      PlumbaginHighYesNoYesNoNoNoNo–5.82
      SanguinarineHighYesYesYesYesNoNoNo–5.17

      Table 6.  Bioavailability prediction of phytoligands established in E. ganitrus.

      Phyto-ligands Bioavailability scoreWater solubility as logSiLOGPXLOGP3WLOGPMLOGPSILICOS-IT
      EG-10.55Poorly soluble as –6.255.1210.628.428.327.14
      EG-20.55Poorly soluble as –6.194.758.867.965.807.04
      EG-30.55Poorly soluble as –6.194.799.348.026.737.04
      EG-40.55Moderately soluble as –5.794.928.807.636.546.63
      EG-50.55Poorly soluble as –7.376.148.896.313.498.12
      EG-60.55Poorly soluble as –9.165.9210.708.846.149.75
      EG-70.55Very Soluble as 0.51–0.23–1.89–3.00–2.76–2.22
      EG-80.55Poorly soluble as –8.795.7610.338.535.949.20
      EG-90.55Soluble as –2.863.873.342.932.073.34
      EG-100.55Soluble as –3.773.294.384.734.634.19
      EG-110.55Soluble as –3.523.274.555.044.533.91
      EG-120.55Soluble as –4.252.864.913.993.873.81
      EG-130.55Soluble as –3.812.913.813.592.873.82
      EG-140.55Very Soluble as –1.821.881.001.411.491.86
      EG-150.55Soluble as –3.183.014.093.613.813.40
      EG-160.55Moderately soluble as –4.283.374.834.123.633.75
      EG-170.55Poorly soluble as –7.194.947.246.583.677.85
      EG-180.55Poorly soluble as –6.314.157.866.524.966.99
      EG-190.55Soluble as –3.592.843.173.732.912.81
      EG-200.55Poorly soluble as –6.306.119.388.776.019.10
      EG-210.55Soluble as –3.213.014.104.685.004.00
      EG-220.55Poorly soluble as –6.115.059.627.176.217.30
      EG-230.85Soluble as –1.140.600.731.081.200.61
      EG-240.55Very Soluble as –2.531.540.951.901.442.14
      EG-250.55Moderately soluble as –5.514.978.026.365.256.57
      EG-260.55Very Soluble as –1.352.701.311.451.771.11
      Plumbagin0.55Soluble as –2.851.792.291.720.592.22
      Sanguinarine0.55Poorly soluble as –6.09–0.044.453.432.723.85

      Table 7.  Drug-likeness prediction of phytoligands established in E. ganitrus.

      Phyto-ligandsLipinski
      rule      		Ghose
      filter      		Veber
      filter      		Egan
      filter      		Muegge
      filter
      EG-1YesNoYesNoNo
      EG-2YesNoYesNoNo
      EG-3YesNoYesNoNo
      EG-4YesNoYesNoNo
      EG-5YesNoNoNoNo
      EG-6YesNoNoNoNo
      EG-7YesNoNoNoNo
      EG-8YesNoNoNoNo
      EG-9YesYesYesYesYes
      EG-10YesYesYesYesYes
      EG-11YesYesYesYesYes
      EG-12YesYesYesYesNo
      EG-13YesYesYesYesYes
      EG-14YesYesYesYesNo
      EG-15YesYesYesYesNo
      EG-16YesYesYesYesYes
      EG-17YesNoNoNoNo
      EG-18YesNoNoNoNo
      EG-19YesYesYesYesYes
      EG-20YesNoNoNoNo
      EG-21YesYesYesYesNo
      EG-22YesNoNoNoNo
      EG-23YesNoYesYesNo
      EG-24YesNoYesYesNo
      EG-25YesNoNoNoNo
      EG-26YesYesYesYesYes
      PlumbaginYesYesYesYesNo
      SanguinarineYesYesYesYesYes

      Table 8.  Medicinal chemistry prediction of phytoligands established in E. ganitrus.

      SI. No.PAINS structural alertBrenk structural alertLead-
      likeness      		Synthetic accessibility score
      EG-10125.61
      EG-20126.42
      EG-30126.30
      EG-40126.17
      EG-50037.10
      EG-60035.17
      EG-70003.86
      EG-80035.00
      EG-90225.59
      EG-100124.51
      EG-110123.66
      EG-120021.37
      EG-130014.35
      EG-140013.63
      EG-150023.77
      EG-160212.91
      EG-170036.76
      EG-180024.12
      EG-190112.23
      EG-200135.52
      EG-210022.21
      EG-220124.08
      EG-230011.00
      EG-240011.00
      EG-250123.89
      EG-260114.38
      Plumbagin2012.41
      Sanguinarine0212.59

      Table 9.  Toxicity prediction of phytoligands established in E. ganitrus.

      Phyto-ligandsLD50 (mg/kg)Toxicity classHepatotoxicityCarcinogenicityImmunotoxicityMutagenicityCytotoxicity
      EG-150005InactiveInactiveActiveInactiveInactive
      EG-28904InactiveInactiveActiveInactiveInactive
      EG-38904InactiveInactiveActiveInactiveInactive
      EG-48904InactiveInactiveActiveInactiveInactive
      EG-530005InactiveInactiveActiveInactiveInactive
      EG-650005InactiveInactiveInactiveInactiveInactive
      EG-7132InactiveInactiveInactiveInactiveInactive
      EG-850005InactiveInactiveInactiveInactiveInactive
      EG-980006InactiveActiveInactiveActiveInactive
      EG-1053005InactiveInactiveActiveInactiveInactive
      EG-1136505InactiveInactiveInactiveInactiveInactive
      EG-128004InactiveInactiveInactiveInactiveInactive
      EG-139004InactiveInactiveInactiveInactiveInactive
      EG-14342InactiveActiveInactiveInactiveInactive
      EG-1520505InactiveInactiveInactiveInactiveInactive
      EG-1642505InactiveInactiveInactiveInactiveInactive
      EG-173003InactiveInactiveInactiveInactiveActive
      EG-1844005InactiveInactiveInactiveInactiveInactive
      EG-1915004InactiveInactiveInactiveInactiveInactive
      EG-2043005InactiveInactiveInactiveInactiveInactive
      EG-217503InactiveActiveInactiveInactiveInactive
      EG-2250506InactiveInactiveInactiveInactiveInactive
      EG-2325305InactiveInactiveInactiveInactiveInactive
      EG-2428305InactiveInactiveInactiveInactiveInactive
      EG-253404InactiveInactiveInactiveInactiveInactive
      EG-2620004InactiveInactiveInactiveInactiveInactive
      Plumbagin162InactiveActiveInactiveActiveInactive
      Sanguinarine7784InactiveActiveActiveActiveInactive

      Table 10.  Bioavailability prediction of phytoligands established in E. ganitrus.

      Phyto-ligandLipophilicity
      (XLOGP3)      		Size
      (MW g/mol)      		Polarity
      (TPSA)      		Insolubility
      [Log S (ESOL)]      		Insaturation
      (Fraction Csp3)      		Flexibility
      (Num. rotatable bonds)
      EG-93.34368.4674.36–3.700.908
      EG-124.91206.3220.23–4.380.572
      EG-133.81276.3743.37–3.820.652
      EG-154.09222.3720.23–3.801.000
      EG-164.83273.3752.32–4.340.357
      EG-261.31209.2829.54–1.760.753
      Plumbagin2.29188.1854.37–2.770.090
      Sanguinarine4.45332.3340.80–5.240.150

      Figure 3. 

      IC50 values of EG-13 phytochemical of E. ganitrus leaves against human cancer cell lines HeLa.

      In Table 5, for pharmacokinetics prognostication, the gastrointestinal (GI) absorption rate was fetched for all preferred six phytoligands and both reference drugs. The blood-brain permeability was seen as positive for all the six phytoligands and both reference drugs. The prediction of bioavailability (Table 6) demonstrated that similar bioavailability scores were observed for all the filtered six phytoligands (0.55) like reference drugs. The water solubility data showed all the six compounds and plumbagin are soluble while Sanguinarine is poorly soluble. For drug-likeness prediction (Table 7), all the six compounds and both known inhibitors were obtained suitable for the Lipinski rule as zero violation. For Ghose, Veber, and Egan filter 0 violation was obtained for all the six phytoligands and both inhibitors. In the case of medicinal chemistry friendliness prediction (Table 8), the PAINS structural alert obtained 0 violations for all the six phytoligands and sanguinarine while two alerts for plumbagin. Table 9 shows EG-9 belongs to the non-toxic class VI, EG-15, and EG-16 also belong to the non-toxic class V, EG-12, EG-13, EG-26 and Sanguinarine belongs to the less toxic class IV while plumbagin belongs to the high-toxic class II. The bioavailability radar (Fig. 3) for phytoligands depicting bioavailability prognostic showed that all six phytoligands were found within the data range of lipophility nature (−0.7 < XLOGP3 < +5.0), molecule size (150 g/mol < MW < 500g/mol), polarity (20 Ų < TPSA < 130Ų), insolubility [−6 < LogS (ESOL) < 0], insaturation (0.25 < Fraction Csp3 < 1) and flexible bonds (0 < Num. rotatable bonds < 9) and colored part of radar while known inhibitors plumbagin and sanguinarine does not fit the bioavailability radar (Table 10). As mentioned in Table 5, all the phytoligands and reference compounds have higher gastrointestinal (GI) absorption rates, therefore they can instantly be absorbed by the human intestine. All phytoligands have the ability to pass the blood brain barrier (BBB permeant) and values for the aqueous solubility (log S) of the phytochemicals fall in the recommended range that is −1 to −5[29], thus, have improved absorption and distribution properties. The bioavailability scores were identical for all six molecules, standing at 0.55, similar to the reference drugs. In drug-likeness prediction, none of the six compounds and both known inhibitors violated the Lipinski rule, Ghose, Veber, and Egan filters. Regarding medicinal chemistry friendliness, the PAINS structural alert identified zero violations for all six phytoligands and Sanguinarine, whereas Plumbagin had two alerts. Table 9 revealed that EG-9 belonged to the non-toxic class VI, while EG-15 and EG-16 were in harmless class V. Other compounds EG-12, EG-13, EG-26 and sanguinarine was from less harmful class IV which could be modified to a non-toxic class during the lead optimization stage of drug discovery[30] while selected standard plumbagin showed high toxic class II. Drug-induced hepatotoxicity often lead to abrupt liver failure and drug rejections[31]. Drug-induced liver injury might be long-term or occur only once. Obviously, the selected compounds and standards are non-hepatotoxic. The bioavailability radar (Fig. 4) depicted that all six phytoligands were within the data range for oral bioavailability prediction. Conversely, standards plumbagin and sanguinarine did not fit within the bioavailability radar. The pink area shown in the radar corresponds to the most promising zone for all the bioavailability properties. In Table 10, all the phytochemicals satisfied 150 g/mol and 500 g/mol criteria for (SIZE) of good drug candidates. The polarity (POLAR) was observed with the Total Polarity Surface Area (TPSA) and all the phytochemicals show good TPSA values. Besides, the flexibility (FLEX) property evaluated by the number of rotatable bonds falls within the recommended range. Lipophilicity (LIPO) and insolubility (INSOLU) were evaluated and come in the range The Unsaturation (INSATU) was calculated using Fraction Csp3 falls within a recommended range of 0.25 < Fraction Csp3 < 1) for all phytoligands. However, Plumbagin and Sanguinarine exhibit lower values (0.09 and 0.15, respectively).

      Figure 4. 

      Bioavailability radar (pink area exhibits optimal range of particular property) for leading phytocompounds molecules. LIPO = lipophilicity as XLOGP3, SIZE = size as molecular weight, POLAR = polarity as TPSA (topological polar surface area), INSOLU = insolubility in water by log S scale, INSATU = insaturation as per fraction of carbons in the sp3 hybridization, and FLEX = flexibility as per rotatable bonds.

      2D and 3D interactions of the five phytoligands (EG-9, EG-12, EG-13, EG-15, EG-16 and EG-26) with 6njs are shown in Table 11. EG-9 divulged two assenting hydrogen bond interactions at the active site having amino acids of Glu96 and Lys97. In additon to that a non-classical C-H bond Vander Waals interaction was also noticed at the active site involving Arg93 residue and alkyl and pi-alkyl interactions were observed at Leu525 and Trp501 respectively. In EG-12 a conventional hydrogen bond interaction was observed at Asn538, a pi-pi T-shaped, two alkyl and a pi-alkyl interactions were observed at Tyr539, Ile522, Trp501 and Leu525 respectively. EG-13 showed one favorable hydrogen bond interaction and two hydrophobic alkyl interactions at the active site with the residue of Glu96, Leu95 and Lys97 respectively. EG-15 showed two alkyl and two pi-alkyl interactions at the active site of the residues of Leu95, Ile522, Trp501 and Tyr539 respectively. In EG-16 two conventional hydrogen bonds were observed at Leu731 and Thr716. EG-26 formed three favorable hydrogen bonds with Asp369, Asp370 and Asp371 at the active site of the receptor. Plumbagin showed a conventional hydrogen bond interaction, a pi-pi T-shaped and a carbon-hydrogen bond interaction at Tyr539, Trp501 and Ser540 respectively. Sanguinarine showed a carbon hydrogen bond, a pi-sigma, a alkyl, and a pi-alkyl interaction at the site of Glu696, Leu731, Pro769 and Pro695 respectively (Table 11). Previously it has been shown that residue at 97 could have amprospective ubiquitin acceptor position in STAT3 NH2 terminal domain, suggesting lysine amino acid may have a significant role and location in a sumolation/ubiquitination consensus sequence[32]. The majority of phytoligand interactions exist in the Linker domain and Transactivation domain of the STAT3.

      Table 11.  2D and 3D binding interactions between the receptor 6NJS and molecules.

      Phyto-ligands2D- Binding interaction3D- Binding interaction
      EG-9 (-6.8)
      EG-12 (-6.5)
      EG-13 (-6.5)
      EG-15 (-6.5)
      EG-16 (-6.4)
      EG-26
      (-5.7)
      Plumbagin
      Sanguinarine
    Conclusions

    • All the six compounds (EG-9, EG-12, EG-13, EG-15, EG-16 and EG-26) significantly bind with STAT3. The phytochemicals epitomized good in silico results as reflected by their promising binding affinity, considerable inhibitory constant with optimum protein-ligand stabilization energy. Consecutively, binding signifies that phytoligands interact with STAT3 by the NH2 terminal and boosts its transcriptional activity and interferes with the cellular proliferation process and apoptosis[32]. Bioavailability radar and toxicological profiles of the preferred phytoligands revealed that these compounds compel to have ample drug likeliness properties. Moreover, EG-9, EG-13, EG-15, EG-16 and EG-26 have not been explored for their anticancer potential and can be derivatized or have the probability of being used as lead compounds.

    Author contributions

    • The authors confirm contribution to the paper as follows: study design and draft manuscript preparation (equal): Mehnaj, Bhat AR, Athar F; supervision: Athar F; experimentation and writing of manuscript: Mehnaj; characterization and editing: Bhat AR. All authors reviewed the results and approved the final version of the manuscript.

    Ethical statement

    • This study involved the use of established human cell lines. The cell lines used in this research were obtained from the National Centre for Cell Sciences (NCCS), Pune, India and were used in accordance with institutional and national ethical standards. The cell lines have been previously published or validated, and no new human tissues were used in this study.

    Data availability

    • The supplementary data will be made available by the authors to all upon reasonable request.

    Acknowledgements

    • Miss Mehnaj is grateful to UGC for obtaining the non-NET fellowship allowing completion of this work.

      Conflict of interest

      • The authors declare that they have no conflict of interest.

      Rights and permissions
      • Copyright: © 2024 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (4)  Table (11) References (32)
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    Mehnaj, Bhat AR, Athar F. 2024. In silico exploration of Elaeocarpus ganitrus extract phytochemicals on STAT3, to assess their anticancer potential. Medicinal Plant Biology 3: e009 doi: 10.48130/mpb-0024-0010
    Mehnaj, Bhat AR, Athar F. 2024. In silico exploration of Elaeocarpus ganitrus extract phytochemicals on STAT3, to assess their anticancer potential. Medicinal Plant Biology 3: e009 doi: 10.48130/mpb-0024-0010
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  • Introduction
  • Materials and methods

  • Cell lines, chemicals and reagents

  • Procurement of plant material

  • Method to prepare extracts

  • Qualitative phytochemical analysis

  • Cellular studies on HeLa cancer cell line

  • Cytotoxicity assay/MTT assay

  • In silico investigation of phytochemicals obtained from leaf extracts

  • Drug-likeness properties prognosis

  • Molecular docking-based virtual screening

  • Pharmacokinetic and toxicity prediction

  • Results and discussion
  • Conclusions
  • Author contributions
  • Ethical statement
  • Data availability
  • Acknowledgements
  • Conflict of interest
  • Rights and permissions
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