Endophytic Bacillus pacificus from Cissus quadrangularis L.: A Source of Bioactive Flavonoids and Terpenoids

*Corresponding author: Preetha S S Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. Endophytic Bacillus pacificus from Cissus quadrangularis L.: A Source of Bioactive Flavonoids and Terpenoids Preetha S S 1, * and Kaladevi V 2 1 Department of Botany, Mar Ivanios College Trivandrum. 2 Department of Environmental sciences, St. John's College, Anchal. World Journal of Biology Pharmacy and Health Sciences, 2025, 23(03), 333-340 Publication history: Received on 10 August 2025; revised on 17 September 2025; accepted on 20 September 2025 Article DOI: https://doi.org/10.30574/wjbphs.2025.23.3.0847 Abstract Cissus quadrangularis L. is a renowned medicinal plant with established fracture-healing and antioxidant properties, often attributed to its phytochemicals. However, the role of its endophytic bacteria as a source of these bioactive compounds remains largely unexplored. This study aimed to isolate, characterize, and identify endophytic bacteria from C. quadrangularis stems and analyze their potential for producing therapeutic secondary metabolites.Endophytic bacteria were isolated from surface-sterilized stem segments of C. quadrangularis collected from Kerala, India. A predominant isolate (QS1) was characterized morphologically, biochemically, and via 16S rRNA gene sequencing. Secondary metabolites were extracted from the isolate using ethyl acetate and subjected to qualitative phytochemical screening and quantitative analysis of total flavonoid content (TFC) using the aluminum chloride colorimetric method. The isolate QS1 was identified as Bacillus pacificus through polyphasic characterization. Phytochemical screening of its crude extract confirmed the presence of flavonoids and terpenoids, while alkaloids, phenolics, tannins, saponins, and proteins were absent. Quantification revealed a high flavonoid yield of 18.62 mg Quercetin Equivalents (QE) per mL of crude extract. The endophytic bacterium Bacillus pacificus QS1, isolated from C. quadrangularis, is a prolific producer of flavonoids and terpenoids. This finding provides a novel microbiological perspective for the plant's documented osteotherapeutic and antioxidant activities, suggesting that its endophytic microbiome is a significant and sustainable source of bioactive compounds. This research underscores the potential of endophytic bacteria as alternative producers of valuable pharmaceuticals, reducing the reliance on direct plant extraction. Keywords: Cissus quadrangularis; Endophytic bacteria; Bacillus pacificus; 16S rRNA sequencing; Secondary metabolites; Flavonoids; Bone-healing; Antioxidant 1. Introduction Endophytes represent a significant reservoir of untapped bioactive compounds with considerable potential for pharmaceutical applications (Fazilath et al., 2019). These microorganisms reside within the internal tissues of healthy plants without causing apparent harm to their hosts. The exploration of endophytes, particularly those associated with medicinal plants, offers a promising strategy for discovering novel metabolites, as the therapeutic properties of such plants may often be attributed to their microbial inhabitants rather than the plant’s own constituents (Strobel et al., 2003; Alwin et al., 2014). This approach also provides a sustainable alternative for the large-scale production of bioactive compounds through microbial fermentation, reducing the need for extensive plant harvesting (Kusari et al., 2011). Cissus quadrangularis L., a perennial plant from the Vitaceae family, is a well-known ethnomedicinal shrub native to tropical regions, including India and Sri Lanka. Commonly referred to as devil’s backbone or hadjod, it is esteemed in World Journal of Biology Pharmacy and Health Sciences, 2025, 23(03), 333-340 334 Ayurveda for its ability to promote bone health, accelerate fracture healing, and treat conditions such as osteoporosis, arthritis, and digestive disorders (Stohs & Ray, 2013; Kameshwaram et al., 2018). Scientific investigations have validated its antioxidant, antimicrobial, anti-inflammatory, and anticancer properties, which are linked to its rich phytochemical profile comprising vitamins, steroids, flavonoids, and minerals (Chidambara et al., 2003; Suresh et al., 2019). While most research has focused on the bioactivities of the plant itself or its endophytic fungi, the endophytic bacteria of C. quadrangularis remain largely unexplored (Nishanthi et al., 2016). Endophytic bacteria, residing interor intracellularly, can significantly enhance plant growth, stress tolerance, and disease resistance. They are prolific producers of diverse secondary metabolites with antibiotic, antiviral, and anticancer activities (Lodewyckx et al., 2002; Singh et al., 2021). Given the medicinal importance of C. quadrangularis, its endophytic bacterial community may be a valuable source of novel bioactive agents. The necessity of this work is underscored by the escalating global challenge of antimicrobial resistance (AMR), which necessitates the urgent discovery of novel antibacterial compounds with unique mechanisms of action (World Health Organization, 2021). Endophytic bacteria, evolving in close association with their host plants, represent a frontier in the search for such new chemical entities. Their ecological niche suggests they produce a diverse array of antimicrobial secondary metabolites as a survival strategy to compete with other microbes and aid their host plant's defense, making them a highly relevant and promising source for new antibiotics (Singh and Dubey, 2022). Furthermore, this research is particularly relevant for the sustainable utilization of medicinal plants. Over-harvesting of popular medicinal species like C. quadrangularis for their bioactive compounds threatens their natural populations and ecological balance. By isolating and characterizing the endophytic bacteria responsible for producing these therapeutic metabolites, this study paves the way for the development of an alternative, microbial-based production platform. This approach aligns with the principles of green chemistry and bioprospecting, offering a renewable and environmentally friendly method to harness valuable compounds without further depleting natural plant resources (Kaul et al., 2023). This study aims to isolate, identify, and characterize endophytic bacteria from the stem tissues of C. quadrangularis collected from Alappuzha, a region with limited prior investigation. The objectives include the morphological, biochemical, and molecular identification of bacterial isolates, followed by preliminary phytochemical screening and spectroscopic analysis of their secondary metabolites to evaluate their antibacterial, antioxidant, and cytotoxic potential. 2. Materials and Methods 2.1. Plant Material Collection and Surface Sterilization Healthy Cissus quadrangularis L. plants were collected from Chengannur, Alappuzha district, Kerala. Disease-free specimens were carefully uprooted using sterile gloves and tools to minimize contamination. The samples were immediately placed in sterile, sealed bags and transported to the laboratory under controlled conditions. Surface sterilization was performed following established protocols with modifications (Suryanarayanan et al., 2018). Healthy stems were washed under running tap water to remove debris, air-dried, and transferred to a biosafety cabinet. Under aseptic conditions, stems were cut into 1 cm segments. The sterilization protocol involved sequential immersion in 70% ethanol (1 min), 2% sodium hypochlorite (5 min), and three rinses with sterile distilled water. The sterilized segments were blotted dry on sterile filter paper to be used for isolation. 2.2. Isolation of Endophytic Bacteria The surface-sterilized stem segments were aseptically placed on Nutrient Agar (NA) plates. The plates were incubated at 37°C for 24–48 hours. Bacterial colonies emerging from the tissue segments were sub-cultured onto fresh NA plates through successive streaking to obtain pure isolates. Pure cultures were maintained on NA slants at 4°C for further study (Verma et al., 2021). World Journal of Biology Pharmacy and Health Sciences, 2025, 23(03), 333-340 335 2.3. Characterization of Bacterial Isolates 2.3.1. Morphological and Microscopic Characterization The morphological characteristics (shape, size, colour, margin, and elevation) of the isolates were recorded. Gram staining and motility tests (hanging drop method) were performed according to standard microbiological procedures (Beveridge, 2001). 2.3.2. Biochemical Characterization The isolates were subjected to a series of biochemical tests for preliminary identification, including: • Indole Test: To detect the production of indole from tryptophan (MacFaddin, 2000). • Methyl Red (MR) and Voges-Proskauer (VP) Tests: To determine glucose fermentation pathways (Cappuccino & Welsh, 2020). • Catalase Test: To identify the presence of the catalase enzyme (MacFaddin, 2000). 2.4. Molecular Identification by 16S rRNA Gene Sequencing 2.4.1. Genomic DNA Extraction Genomic DNA was extracted from overnight bacterial cultures using the standard phenol-chloroform method (Sambrook & Russell, 2001). The extracted DNA was quantified and qualified using 0.8% agarose gel electrophoresis. 2.4.2. PCR Amplification and Sequencing The 16S rRNA gene was amplified using universal primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1544R (5′- AAGGAGGTGATCCAGCCGCA-3′) (Weisburg et al., 1991). The 30 µL PCR reaction mixture consisted of: 1X PCR buffer, 1.5 mM MgCl₂, 0.2 mM dNTPs, 10 pmol of each primer, 1 U of Taq DNA polymerase, and 50–100 ng of template DNA. Amplification was performed in a thermal cycler with the following conditions: initial denaturation at 94°C for 2 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 52°C for 60 s, and extension at 68°C for 90 s; followed by a final extension at 68°C for 7 min. The amplicons (~1500 bp) were visualized on a 1.5% agarose gel and sent for commercial sequencing. 2.4.3. Phylogenetic Analysis The obtained sequences were compared to reference sequences in the GenBank database using the BLASTn algorithm (Johnson et al., 2008). Phylogenetic trees were constructed using the MEGA software (version 11) with the NeighborJoining method to confirm taxonomic affiliation (Tamura et al., 2021). 2.5. Extraction of Secondary Metabolites Bacterial isolates were grown in 50 mL of Nutrient Broth in 250 mL Erlenmeyer flasks at 30°C with shaking at 150 rpm for 5–7 days. The culture broth was centrifuged at 8000 rpm for 15 min to obtain a cell-free supernatant. Secondary metabolites were extracted from the supernatant using an equal volume of ethyl acetate (3 times). The combined organic phases were dried over anhydrous sodium sulphate and concentrated to dryness under reduced pressure using a rotary evaporator. The crude extract was stored at 4°C for further analysis (Kaur et al., 2022). 2.6. Preliminary Phytochemical Screening The crude ethyl acetate extract was resuspended in methanol and screened for major phytochemical classes using standard qualitative assays (Harborne, 1973; Sofowora, 1993): • Phenolics: Ferric chloride test (formation of a dark green colour). • Alkaloids: Mayer’s test (formation of a creamy white precipitate). • Terpenoids: Salkowski test (formation of a reddish-brown ring at the interface). • Proteins: Biuret test (colour change from blue to violet). • Tannins: Lead acetate test (formation of a white precipitate). • Saponins: Foam test (formation of stable froth for 60-120 seconds). World Journal of Biology Pharmacy and Health Sciences, 2025, 23(03), 333-340 336 2.7. Quantification of Total Flavonoid Content (TFC) The TFC of the extract was estimated using the aluminium chloride colorimetric method (Zhishen et al., 1999). Briefly, 0.1 mL of the extract was mixed with 0.3 mL methanol, 0.02 mL of 10% AlCl₃, 0.02 mL of 1 M CH₃COOK, and 0.56 mL distilled water. After 30 min incubation at room temperature, the absorbance was measured at 415 nm. A standard curve was prepared using quercetin, and the results were expressed as mg of Quercetin Equivalents (QE) per gram of dry extract (mg QE/g). 3. Results 3.1. Plant Collection and Surface Sterilization Healthy and disease-free specimens of Cissus quadrangularis were successfully collected from Chengannur, Alappuzha district, Kerala. The plants were carefully uprooted using sterile tools to minimize external contamination. Surface sterilization of the stem segments using a sequential treatment with 70% ethanol and 2% sodium hypochlorite was effective, as confirmed by the absence of microbial growth on control plates inoculated with the final rinse water. This validated the efficacy of the protocol in eliminating epiphytic microorganisms, ensuring that subsequent isolates were of endophytic origin. 3.2. Isolation and Morphological Characterization of Endophytic Bacteria Following surface sterilization, the stem segments were plated on Nutrient Agar. After 24–48 hours of incubation at 37°C, distinct bacterial colonies emerged from the plant tissues. One predominant isolate, designated QS1, was selected for further characterization based on its robust growth. The colony was circular, cream-colored, and exhibited a smooth texture and entire margin. 3.3. Microscopic and Biochemical Characterization of Isolate QS1 3.3.1. Gram Staining and Motility Microscopic examination after Gram staining revealed that isolate QS1 is a rod-shaped (bacillus), Gram-negative bacterium. The hanging drop method confirmed that the isolate is motile, exhibiting true, directional movement distinct from Brownian motion. 3.3.2. Biochemical Tests A series of biochemical tests were conducted for the preliminary identification of isolate QS1. The results are summarized in Table 1. Table 1 Biochemical profile of endophytic bacterial isolate QS1 Biochemical Test Observation Result Interpretation Indole Test Red ring formed with Kovac's reagent Positive Produces indole from tryptophan Methyl Red (MR) Test Red color developed Positive Mixed-acid fermentation Voges-Proskauer (VP) Test No color change Negative Does not produce acetoin Catalase Test Rapid effervescence observed Positive Produces catalase enzyme 3.4. Molecular Identification and Phylogenetic Analysis Genomic DNA was successfully extracted from isolate QS1, and the 16S rRNA gene was amplified via PCR, yielding a single amplicon of approximately 1500 bp. Sanger sequencing of the amplicon produced a 1379 bp sequence. BLASTn analysis of this sequence against the NCBI database revealed a 100% sequence similarity with Bacillus pacificus strain MTCC 13015 (Accession Number: NR_182261.1), with a query coverage of 100% and an E-value of 0.0. The high score (2347) and maximum identity confirm the isolate as Bacillus pacificus. World Journal of Biology Pharmacy and Health Sciences, 2025, 23(03), 333-340 337 3.5. Extraction and Phytochemical Screening of Secondary Metabolites Secondary metabolites were extracted from the cell-free supernatant of an B. pacificus QS1 culture using ethyl acetate. The crude extract was subjected to qualitative phytochemical screening, the results of which are presented in Table 2. Table 2 Results of preliminary phytochemical screening of the ethyl acetate extract from B. pacificus QS1 Phytochemical Class Test Performed Observation Result Terpenoids Salkowski test Reddish-brown ring formed Present Flavonoids AlCl₃ test Yellow coloration Present Phenolic compounds Ferric Chloride test No dark green color Absent Alkaloids Mayer’s test No precipitate formed Absent Proteins Biuret test No violet color Absent Tannins Lead Acetate test No white precipitate Absent Saponins Foam test No persistent froth Absent 3.6. Quantification of Total Flavonoid Content (TFC) The Total Flavonoid Content of the crude extract was quantified using a quercetin standard curve. The calibration curve showed a strong linear relationship (R² > 0.99) between quercetin concentration and absorbance at 415 nm. The absorbance of the sample (0.450) corresponded to a concentration of 18.62 mg Quercetin Equivalents (QE) per mL of the crude extract, indicating a high flavonoid yield. Table 3 Total Flavonoid Content of the ethyl acetate extract from B. pacificus QS1 Sample Absorbance at 415 nm Concentration from Graph (mg QE/mL) Bacterial Extract 0.450 18.62 4. Discussion The present study successfully isolated and characterized an endophytic bacterium, Bacillus pacificus strain QS1, from the stem of the medicinal plant Cissus quadrangularis. The identification was confirmed through a polyphasic approach combining morphological, biochemical, and molecular (16S rRNA sequencing) analyses. The primary objective was to explore this endophyte for its potential to produce bioactive secondary metabolites, a rationale supported by the plant's renowned ethnomedicinal properties, particularly in bone fracture healing (Stohs & Ray, 2013). The hypothesis that the therapeutic efficacy of medicinal plants may be partly attributable to their endophytic microbiome (Strobel & Daisy, 2003) is central to this investigation. The isolation of a Bacillus species from C. quadrangularis aligns with recent findings by Deka et al. (2023), who also reported Bacillus subtilis and Bacillus velezensis as endophytes in the same host plant. Bacillus species are ubiquitous endophytes renowned for their metabolic versatility and ability to produce a vast array of antimicrobial, antioxidant, and anticancer compounds (Zhao et al., 2022). The biochemical profile of QS1—positive for indole, methyl red, and catalase tests—suggests a robust metabolic capacity for producing various organic acids and enzymes, which are often precursors or indicators of broader secondary metabolic pathways. A significant finding of this study was the production of flavonoid and terpenoid compounds by B. pacificus QS1, as confirmed by qualitative phytochemical screening and quantitative analysis. The high total flavonoid content (18.62 mg QE/mL) is particularly noteworthy. Flavonoids are potent antioxidants and are implicated in promoting osteoblast differentiation and bone formation (Trzeciakiewicz et al., 2020). This finding provides a plausible microbiological basis for the observed bone-healing properties of C. quadrangularis extracts. It suggests that endophytes like B. pacificus could be symbiotic contributors to the host plant's pharmacological effects by producing bone-active flavonoids. This complements the work of Awari et al. (2024), who isolated endophytic fungi (Colletotrichum and Phoma spp.) from C. quadrangularis that produced phytosterols and exhibited significant World Journal of Biology Pharmacy and Health Sciences, 2025, 23(03), 333-340 338 antioxidant activity. While their study focused on fungal endophytes and phytosterols, our work on bacterial endophytes and flavonoids expands the understanding of the microbial contribution to the plant's bioactivity. The absence of alkaloids in our extract contrasts with the study by Anwar et al. (2020), who identified an endophytic Pseudomonas sp. (CqB14) from C. quadrangularis that produced alkaloids among other metabolites with anti-osteosarcoma potential. This discrepancy highlights the vast metabolic diversity among endophytic communities, even within the same host plant. Different endophytes occupy distinct ecological niches and may contribute unique sets of bioactive compounds to the plant's overall metabolome. This underscores the importance of isolating and characterizing multiple endophytic species from a single host to fully exploit its pharmaceutical potential. The results of this study are further supported by broader research on endophytes from other medicinal plants. For instance, Hastuti et al. (2024) isolated eleven endophytic fungi from Michelia champaca that produced flavonoids, alkaloids, tannins, saponins, and terpenoids, demonstrating the protective role these metabolites play for the host plant. Similarly, a recent review by Verma et al. (2023) emphasized that endophytic Bacillus species are a goldmine for novel antimicrobials and antioxidants, often mimicking the host plant's bioactivity. Our isolation of a flavonoidproducing Bacillus from a plant known for its antioxidant and bone-healing properties is a direct validation of this concept. This study confirms that the stem of Cissus quadrangularis harbors endophytic bacteria with significant potential for producing bioactive compounds. The identification of Bacillus pacificus QS1 and its production of flavonoids and terpenoids provides a scientific basis for further exploration. The findings align with the growing paradigm that endophytes are valuable, sustainable sources of novel pharmaceuticals that can reduce the harvesting pressure on medicinal plants. 5. Conclusion In conclusion, this study successfully isolated and identified the endophytic bacterium Bacillus pacificus QS1 from the stem of the medicinal plant Cissus quadrangularis. The isolate was found to produce significant quantities of flavonoid and terpenoid compounds, which are associated with the plant's renowned bone-healing and antioxidant properties. This work provides crucial evidence that the therapeutic benefits of medicinal plants may be intrinsically linked to their symbiotic endophytic microbes. Consequently, endophytic bacteria represent a promising and sustainable source of novel bioactive compounds for future pharmaceutical development, particularly in bone disorder therapeutics, offering an alternative to direct plant extraction. Compliance with ethical standards Acknowledgement The authors are grateful to Principal, Mar Ivanios College and St. John’s College for providing necessary help during this work. Disclosure of conflict of interest No conflict of interest to be disclosed. References [1] Fazilath U, Aswathy JM, Pandey A. Endophytic microbes as a novel source of bioactive compounds: A review. Ind Biotechnol. 2019;15(1):1-15. 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