HPLC-Based Elucidation of Tannins from the Tissue and Callus Culture Extracts of Selected Medicinal Plants

1. INTRODUCTION

Callus is a mass of mostly unorganized and undifferentiated cells. The plant tissue culture method provides a continuous and reliable source of natural products. The standardization of plant products is critical considering the growing demand for natural goods as medicines [1]. The production of secondary metabolites in cell culture relies on the amount/types of PGRs, carbon source, and other climatic conditions including light, temperature and gas composition [2].

Chromatographic fingerprinting presents a practical and effective solution to the global need for a stronger quality assessment method for traditional medicine [3]. This optimized technique enables the representation of chemical constituents distributed in plant materials, creating a “chemical database” that can be referenced in future research [4]. Tannins, also known as tannic acid, play an important biological role and have diverse applications. Their anticarcinogenic and antimutagenic effects are believed to stem from their antioxidant properties, which help protect cells from oxidative damage, including lipid peroxidation. Tannins also exhibit significant antibacterial activity. They suppress the growth of different microorganisms such as fungi, yeasts, bacteria, and viruses. Notably, propyl gallate and tannic acid but not gallic acid have been shown to inhibit the growth of aquatic bacteria, and microbes responsible for producing undesirable flavors [5].

The hydrolysis of ester linkages between gallic acid and polyols hydrolyzed during the maturing of any edible fruit is presumably linked to their antimicrobial capabilities. Tannins in these fruit, therefore, act as a natural barrier against microbial diseases [6]. Tannins demonstrate additional physiological effects, including lowering serum cholesterol, blood pressure, increasing blood coagulation and altering immune response [7]. The selected medicinal plants contain tannins, which have traditionally been used to treat a range of ailments. To demonstrate their effectiveness, it is essential to conduct both their qualitative analysis and quantification of tannin compounds present in various plant parts, using systematic scientific methods and comparisons with standard tannin compounds.

Achyranthes aspera L. belongs to the family Amaranthaceae.  A. asperas is used to treat diarrhea, piles, heart disease, dyspepsia, vomiting, ascites, abdominal enlargement, and enlargement of the cervical gland [8-10]. Ipomoea hederacea of Convolvulaceae are known as ivy leaf morning glory or kaladana and habbunil. It is used to cure abdominal diseases, bronchitis, eye inflammation, gout, scabies, headache, constipation, fever, flatulence, leucoderma, eye disorders, splenopathy and hepatopathy [11]. Ocimum basilicum of Lamiacae/Labiatae is commonly known as basil or niazbo. It is used to treat stomachache and diarrhea due to its diuretic, demulcent and antipyretic properties [12]. While callus cultures provide a reliable source of secondary metabolites, there is limited standardized methodology employed for optimizing culture conditions with the aim to maximize tannin production. Moreover, despite the known medicinal potential of tannins in selected plants, comprehensive chromatographic profiling and quantitative analysis using HPLC remains underexplored for quality assurance and therapeutic validation. Hence, this study aims to use High Performance Liquid Chromatography (HPLC) to identify tannin compounds in selected medicinal plants by testing different mobile phase gradients and run times.

2. MATERIALS AND METHODS

2.1. Collection and Identification of Selected Plants

Healthy seeds of Achyranthes aspera L. (Voucher No. GC. Herb. Bot. 3492), Ipomoea hederacea (L.) Jacquin. (Voucher No. GC. Herb. Bot. 3493), and Ocimum basilicum L. (Voucher No. GC. Herb. Bot. 3491) were purchased from the market and identified by a taxonomist from GC University Lahore.  The entire study was carried out at the Plant Biotechnology Laboratory, GC University of Lahore.

To prevent microbial contamination during in vitro culture, seeds were subjected to sterilization using 3% sodium hypochlorite solution for 1 minute and then rinsed thrice with sterile distilled water. A total of 10 seeds from each species were aseptically placed in sterile Petri plates containing moist cotton pads to facilitate germination in the growth room.

Following successful germination, young seedling tissues (leaf and stem) were excised under sterile conditions and used for callus induction. Murashige and Skoog (MS) medium was supplemented with various concentrations of auxins (e.g.NAA, 2-4D) and cytokinins (BAP, KIN) to establish callus biomass production. Cultures were maintained at 25 ± 2°C under a 16-hour light/8-hour dark photoperiod provided by cool white; fluorescent lights to mimic optimal physiological conditions for callus development.

Developed callus tissues, along with the original leaf and stem tissues, were harvested and oven-dried at 40°C for one week to remove moisture while preserving bioactive compounds. Dried samples were ground into fine powder using a mechanical grinder. One gram of each powdered sample (leaf, stem, and corresponding callus) was weighed for tannin extraction, following the HPLC protocol outlined by reference [13].

2.4.1. Preparation of Mobile Phase. A binary mobile phase consisting of methanol and water in a 1:1 ratio (50:50 mL) was prepared. The solvent mixture was thoroughly filtered using a 0.2 µm syringe filter to remove the particulate matter. Then, it was subjected to sonication for 20 minutes to ensure degassing and homogeneity, which improved peak resolution during chromatographic separation [14]

2.4.2. Preparation of Standard. Tannic acid was used as a standard and dissolved into 10 ml of mobile phase to form stock solution. A series of dilution (20 µg/ml, 30 µg/ml, 40 µg/ml and 50 µg/ml) was prepared and diluted with 10 ml of mobile phase. A calibration curve was plotted against the area.

2.4.3. Preparation of Sample. One gram of each plant tissue and macerated callus of leaf and stem were soaked in 10 mL of mobile phase and kept for 12 hrs with stirring. Then extracts were filtered using 0.2 µm syringe filters and subjected to 20 minutes of sonication to enhance solubility and remove trapped air. The resulting clear extracts were injected into the HPLC system for profiling and quantification.

Table 1. Chromatographic Instrumentation of Quantitative Determination of Tannins

3. RESULTS

The leaf and stem explants of A. aspera, I. hederacea and O. basilicum were grown on MS media containing different combinations of plant growth regulators (PGRs). The effects of different concentrations of PGRs were recorded for the callogenic response of different explants of I. hederacea, that is, leaf, stem and root. The maximum callogenic response was exhibited by MS medium supplemented with 2.0 mg/l 2,4-D in combination with 4.0 mg/l NAA for the leaf of A. aspera as compared to other explants as the leaf callus had green colour and granular morphology (Figure 1A). The minimum callogenesis response was observed in MS medium supplemented with 0.5 mg/l 2,4-D, in combination with 0.5 mg/l BAP, for the stem of I. hederacea as compared to other explants. Stem callus was brown and had granular morphology. Stem explants of MSDB and MSDK of I. hederacea and O. basilicum respectively responded for callus induction with a low callus induction percentage (Table 2).

Figure 1. Effects of Different Concentrations of PGRs on Callus Induction (A) Leaf Explant of A. aspera on MSDN Medium (B) Stem Explant of A. aspera on MSDN Medium (C) Leaf Explant of I. hederacea on MSDB Medium (D) Stem Explant of I. hederacea on MSDB Medium (E) Leaf Explant of O. basilicum on MSDK Medium (F) Stem Explant of O. basilicum on MSDK Medium.

The combination of media is given in table 2 below.

Table 2. Effect of Different PGRs on Callus Induction of Different Parts of Selected Plants

3.1. HPLC-Based Comparison of Tannin Extraction from Leaf, Stem, and Callus Tissue of A. aspera, I. hederacea, and O. basilicum

Chromatogram of selected plants showed the retention time of  2.9 minutes at 270 wavelengths for tannin using methanol and water mobile phase. The tannins compound was analyzed for 5 minutes run time. The maximum area of sample was shown by O. basilicum callus culture extract, that is, 9365.56 among all explants and callus culture samples. The highest tannin content was exhibited by O. basilicum leaf callus culture extract, that is, 95.04 (Table 3).

Table 3. Quantitative Estimation of Tannin Content in Leaf and Stem of Selected Medicinal Plants

aspera leaf seedling extract showed the highest area (1,245,086), indicating abundant bioactives. Ocimum basilicum leaf extract had the highest concentration (22.90 mg/mL), suggesting strong extractability. A. aspera stem tissue showed maximum recovery (135.45%), Further, its callus culture had the highest average signal (101.25), pointing to enhanced in vitro metabolite production. Conversely, Ipomoea hederacea stem seedling had the lowest area (41,976), indicating low phytochemical yield. The highest standard deviation (32.19) and RSD (23.97%) in I. hederacea extracts indicated low precision, requiring optimization (Table 4).

Table 4. HPLC Profiling of Selected Medicinal Plants

Figure 2. HPLC Profile of Standard Drug (Tannic Acid)

HPLC chromatograms at 270 nm display the tannin profiles extracted from the leaf, stem, and callus tissues of Achyranthes aspera, Ipomoea hederacea, and Ocimum basilicum, revealing distinct differences in the tannin content across tissue types and species. In all three plants, leaf extracts consistently exhibited the highest and sharpest peaks, particularly around the retention time of ~2.6 minutes, indicating a greater concentration of tannins (Figure 3). Stem extracts showed moderate peak intensities, reflecting lower tannin levels as compared to leaf tissues (Figure 4). In contrast, callus cultures displayed slightly reduced peak intensities, suggesting minimal tannin accumulation under in vitro conditions. Among the species, O. basilicum leaf extract showed the highest peak intensity, indicating it as the richest tannin source, followed by A. aspera and I. hederacea. These findings highlight that natural, differentiated tissues, especially leaves are superior to stem and callus tissues for tannin extraction and confirm species- and tissue-specific variation in tannin biosynthesis.

Figure 3. HPLC Profile of Seedling Leaf Tissue (Left Side) and Callus Culture (Right Side) of A. aspera, I. hederacea and O. basilicum. Leaf Extracts Exhibit Higher and Sharper Peaks, Especially Around ~2.6 Min, Indicating Greater Tannin Concentrations

Figure 4. HPLC Profile of Seedling Stem Tissue (Left Side) and Callus Culture (Right Side) of A. aspera, I. hederacea and O. basilicum. Stem Extracts Show Moderate Peak Intensities

Figure 5. Comparative Analysis of the Heat Maps of HPLC Parameters of Selected Plants. The Colors Blue and Red Show the Value from the Lowest to the Highest, respectively.

The heat map illustrates the relative recovery or concentration of tannin compounds across various medicinal plant extracts, as analyzed by HPLC. Each row represents a specific extract from Ocimum basilicum, Achyranthes aspera, and Ipomoea hederacea, differentiated by plant part (leaf or stem) and tissue type (seedling tissue or callus culture). The color gradient from blue (low) to red (high) indicate the abundance of target compounds. Notably, the extracts from seedling tissues, particularly O. basilicum leaf and I. hederacea stem callus cultures, show higher compound recovery, while callus cultures, especially from A. aspera, generally exhibit lower recovery levels. This suggests that differentiated plant tissues may be more effective sources of bioactive tannins than callus cultures under the tested conditions (Figure 5).

4. DISCUSSION

The influence of different combinations of PGRs on the callogenic response of explants (leaf and stem) of the selected plants was assessed. Among the tested combinations, the maximum callus biomass production was noted in the leaf explants of Achyranthes aspera on MS medium fortified with 2.0 mg/L 2,4-D and 4.0 mg/L NAA. The resulting callus was green with a granular texture (Figure 1A), highlighting a robust morphogenic response as compared to other explants. Previous studies also highlighted the effectiveness of leaf explants in callus induction for A. aspera. Reference [15] reported successful callus formation using leaf explants cultured on MS medium supplemented with various concentrations of 2,4-D and NAA. The most pronounced callogenic response was achieved with a combination of 2 mg/L 2,4-D, IBA, BAP, IAA, and 4 mg/L NAA. Similarly, [16] also demonstrated efficient callus induction using leaf explants of A. aspera on MS medium enriched with varying concentrations of 2,4-D and NAA, further reinforcing the potential of this explant-PGR combination for the in vitro morphogenesis. In contrast, the lowest callogenic response was noted in stem explants of I. hederacea cultured on MS medium supplemented with 0.5 mg/L 2,4-D and 0.5 mg/L BAP. This callus exhibited a brown coloration with a granular morphology, indicating limited cellular proliferation. Callus initiation was typically observed within 13 to 17 days of incubation. Notably, stem explants from MSDB and MSDK variants of I. hederacea and O. basilicum demonstrated only modest callus induction, reflected by a relatively low callus formation percentage.

Chromatography, central to phytochemistry, is essential to obtain pure compounds for therapeutic development by enabling the separation, identification, and structural analysis of bioactive substances. It also plays a vital role in quality control and standardization of phytotherapeutics, primarily through HPTLC/HPLC fingerprinting and quantification of specific chemical markers. These techniques not only help in species identification and classification but also support the isolation and characterization of marker compounds, estimate genetic diversity, and offer real-time analyte detection through spectrum scanning and post-chromatographic derivatization[17-20].

HPLC settings were optimized to produce excellent chemical information and chromatograms with the best separation of neighboring peaks, which could be used to quantify the bioactive chemicals under investigation. The influence of different column temperatures, such as 25°C, 30°C, and 35°C on the separation process was also investigated. At 35°C, the majority of the peaks in HPLC chromatograms were well resolved. As a result, this temperature was chosen as the fingerprint analysis column temperature [21].

 To examine the number of peaks and their spacing, detection wavelengths of 280, 254, and 360 nm were used. Finally, the wavelength of 280 nm was chosen, since this wavelength yielded more peaks than 254 or 360 nm. According to the current study, HPLC fingerprinting involves the characterization of bio active compounds. Mobile phase and column play an important role in isolating the compounds. Standards and extracts from seedling leaf, stem tissue, and callus cultures of selected plants showed a consistent baseline and a distinct peak characteristic of tannins, with 270 nm identified as the optimal wavelength for their detection. The tannin content of seedling leaf, stem tissue, callus culture extracts of selected plants exhibited a peak and retention time between 0 to 15 minutes. The HPLC profiling of seedling leaf, stem tissue and callus culture extracts of the leaf and stem of selected plants established a significant peak of tannin fraction at 2 to 2.9 retention time (Figure 3 and 4), (Table 1, 2). A previous study demonstrated that P. tuberosa showed best peak of tannic acid at 270 nm wavelength [22]. The presence of additional minor peaks further supports the occurrence of diverse polyphenolics, such as catechol and benzoic acid, as commonly reported in similar phytochemical analyses. Such findings were observed in the HPLC analysis of whole plant extracts of E. campestre, which revealed the presence of various polyphenolic compounds such as catechol and benzoic acid, further supporting the identification of tannins and related phenolics at the optimal detection wavelength of 270 nm [14]. The HPTLC analysis of ethanolic extracts from C. nilgirensis, C. gigantea, and C. crinita also revealed remarkable results, highlighting the presence of a diverse range of phytochemicals, including phenolics, flavonoids, and tannins. These results coincide with the current observations [23]. Another study [24] also confirmed the presence of polyphenols in the methanolic leaf extract of Synsepalum dulcificum. This finding also coincides with the current results.

aspera leaf tissue extract exhibited the highest chromatographic area (1,245,086), highlighting a rich abundance of bioactive compounds and confirming its potential as a valuable phytochemical source. In contrast, O. basilicum leaf extract recorded the highest concentration recovered (22.9031 mg/mL), suggesting it is chemically potent and highly extractable. Remarkably, A. aspera stem seedling tissue demonstrated the highest recovery percentage (135.45%), while its callus culture produced the strongest average signal (101.25), both pointing toward enhanced metabolite accumulation, likely due to favorable in vitro culture conditions stimulating biosynthesis. On the other hand, I. hederacea stem seedling tissue showed the lowest area (41,976), indicating a low phytochemical yield from that specific tissue. The highest standard deviation (32.19) and relative standard deviation (RSD) (23.97%) were observed for I. hederacea leaf callus and stem seedling extracts, respectively. These value reflected low analytical precision and signaled the need for optimization to improve reproducibility (Table 4).

The heat map shows that tannin compound recovery varied across different plant extracts, with higher levels found in seedling tissues especially in Ocimum basilicum leaves and Ipomoea hederacea stem callus cultures. In contrast, callus cultures, particularly from A. aspera, showed lower recovery. This indicates that differentiated tissues may be more suitable for extracting bioactive tannins, as compared to undifferentiated calluses under the conditions tested. These findings highlight the importance of tissue type in optimizing compound yield for medicinal plant research (Figure 5).

The HPLC fingerprints of these standard phenolic compounds acquired using the procedures outlined above might be used as standards for future research. Comparison with such standard chromatograms would provide both qualitative and quantitative analysis of the real phenolic compounds present in any unknown plant sample. This would allow the identification and confirmation of the presence of any of these 9 typical phenolic compounds in the study sample [25]. [26] used HPLC to extract condensed tannins from the bark of four tree species in Mexico; namely Arbutus xalapensis Kunth, Prunus serotina Ehrh., Quercus crassifolia Humb. & Bonpl, and Quercus laurina Humb. & Bonpl. In that study, the total tannin content was also isolated from the Citrus limon extract using HPLC, which aligns with the methodology employed in the current study.

The current HPLC data revealed distinct trends among species and tissue types. A. aspera emerged as a metabolically rich plant, especially in its seedling and stem tissues, while O. basilicum displayed exceptional reproducibility and high chemical recovery, particularly in its callus forms. I. hederacea, although showing potential, demonstrates higher variability, especially in its stem extracts. This profiling not only underscores the phytochemical potential of these plants but also identifies which culture conditions and plant parts yield the best results, paving the way for future standardized extraction and pharmacological applications.

Author Contribution

Madieha Ambreen: conceptualization, investigation, methodology, writing – original draft. Safdar Ali Mirza: supervision, visualization, writing – review & editing. Zahida Bano: software formal analysis, data curation.

Conflict of Interest

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

Data Availability Statement

Data sharing is not applicable to this article as no new data were generated during the study.

Funding Details

No funding has been received for this research.

Generative AI Disclosure Statement

The authors did not used any type of generative artificial intelligence software for this research.

Acknowledgment

The authors express their gratitude to Dr. Zaheer-ud-din Khan from the Department of Botany, GC University Lahore, Pakistan, for his assistance in the identification of plant specimens