Exploring the influence of organ differentiation on biosynthesis and accumulation of camptothecin in Ophiorrhiza rugosa var. decumbens

Abstract

Genus Ophiorrhiza has recently emerged as one of the promising sources of Camptothecin (CPT), an antitumour monoterpene indole alkaloid. It possesses CPT in its every part and has a relatively short life span. To determine whether differentiation plays any role in the synthesis and/or accumulation of CPT, the concentration of CPT was analyzed across various tissues of Ophiorrhiza rugosa var. decumbens obtained through both direct as well as indirect modes of regeneration. The results revealed that the plants obtained from both types of regeneration showed similar levels of CPT. It was also observed that with differentiation, the accumulation of CPT increases, as the callus, being an undifferentiated mass of cells, had only traces of CPT. In contrast, the completely differentiated in-vitro plant obtained from it showed a significantly higher percentage of CPT in shoots (0.22% dry weight) and roots (0.247% dw). The CPT when analyzed after hardening, varied among different organs of the plant. It was also observed that the inflorescence accumulated the highest concentration of CPT (0.348% dw) once the flowering began, accompanied by a decrease in remaining organs. This decrease may result from CPT being mobilized to the inflorescence as a chemical defense mechanism. These findings allowed us to determine the ideal plant harvesting age for CPT extraction. The findings could be used to decide the right stage of plant harvest, which is just before the onset of blooming.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-024-03999-4.

Introduction

Camptothecin (CPT), (4-ethyl-4-hydroxy-1H-pyrano[3ʹ,4ʹ:6,7]indolizino[1,2b]-quinoline-3,14 (4H,12H) dione) is a monoterpene indole alkaloid. It has been demonstrated that the CPT induces apoptosis and inhibits topoisomerase I, a key enzyme in DNA replication. Wang et al. (2016) discovered that CPT and its derivative drugs can bind to tubulin and prevent the production of microtubules in dividing cells, further enhancing the effect of DNA topoisomerase inhibition.

The low water solubility of CPT, fast blood clearance, and its ability to target non-tumor cells encouraged the synthesis of its derivatives, Irinotecan and Topotecan, which are used for treating lung, breast, uterine, cervical, colorectal, and ovarian malignancies (Venditto and Simanek 2010). These derivatives are made from plant-derived CPT that has been chemically altered (through methylation and/or hydroxylation) to increase the solubility, lessen its toxicity, and boost the activity. The safety and effectiveness of CPT have been greatly enhanced by more recent drug delivery techniques using copolymer and liposomal vehicles (Rajan et al. 2016).

Camptothecin is primarily isolated from two deciduous trees namely Camptotheca acuminata and Nothapodytes foetida. These plants have low productivity, slow growth rates, and a need for large cultivation areas. Worldwide, plants produce only 600 kg of CPT annually, which is insufficient to meet the estimated 3000 kg of yearly demand for CPT on the global market (Raveendran 2015). Among various screened plants, N. foetida produces the highest amount of camptothecin compared to other plants (Fulzele and Satdive 2005a). However, both these tree species grow slowly, building up biomass over time. Increasing demand and overharvest of N. foetida led to ecological issues, including damage to natural habitat (Cui et al. 2015).

Despite the capability of chemical synthesis, the spatial configuration necessary for the pharmacological activity of CPT limits their economical synthetic synthesis. As a result, plant sources found in nature are required for the production of pharmaceuticals based on CPT. This necessitates the search for additional, reliable plant-based sources or an artificial synthesis technique. Plants derived from tissue culture approach and grown under controlled conditions can serve as better sources of CPT. In this resource search for CPT, Ophiorrhiza is a potential group (Krishnakumar et al. 2020). The dicotyledonous genus Ophiorrhiza includes many small cultivable perennial herbaceous plants with short life spans and can be grown in a greenhouse or plant growth chambers with artificial lighting. In the Indian subcontinent, there are 47 species of this genus, primarily found in the Western Ghats and the northern states of India (Deb and Mondal 1997), whereas three varieties and 16 species are found in Kerala, India (Sibi et al. 2012).

The amount of secondary metabolite produced by a plant varies among different organs and is influenced by phases of development as well. Specifically, during the stages of fruit development or blossoming, alkaloids, which are defense compounds, increase in concentration (Lee et al. 2022). Similarly, Singh et al. (2020) showed that CPT biosynthesis in N. foetida starts only after root development. On the contrary, it is also suggested that CPT biosynthesis occurs constitutively since O. mungos’ absolute CPT content exhibited a positive connection with growth (Wetterauer et al. 2018). Furthermore, because the Ophiorrhiza plant shows the presence of CPT in all organs, whole plants can be used for CPT extraction.

The present research endeavors to examine the variability in camptothecin (CPT) concentrations during discrete stages of both direct and indirect regeneration, aiming to discern potential correlations between age, organ differentiation, and the synthesis of this vital secondary metabolite. Through quantitative analysis, this work sought to unravel the interplay between organ development and camptothecin biosynthesis, offering critical insights into the suitable time to harvest the plant to extract this compound and, in turn, optimal plant utilization.

Materials and methods

Chemicals and reagents

Camptothecin, methanol, and acetonitrile of analytical grade were procured from MERCK (India).

Plant materials

Fresh leaves of Ophiorrhiza rugosa var. decumbens (Deb and Mondal 1997) were collected from Medicinal Plants Nursery at Bhabha Atomic Research Centre, Mumbai, Maharashtra. The herbarium specimen of the collected sample has been submitted to the Bhabha Atomic Research Centre Herbarium (HBARC00006639) (Thiers 2024).

Young leaves from a three-month-old plant maintained at the Greenhouse facility, BARC, Mumbai (14 h/10 h light/dark photoperiod under natural light, relative humidity of 65–75% and at temperature 24.0 ± 1.5 °C) were used as the explant for the initiation of multiple shoots and callus via direct and indirect regeneration processes, respectively. The camptothecin content (% CPT) was estimated separately in both shoot and root of the obtained complete plantlet. Further, in-vitro plants were hardened and transferred to pots. Different parts of these plants were harvested for the detailed CPT analysis, including young leaves (first leaves below inflorescence), mature leaves (from the third node present below the inflorescence), roots, nodal and inter-nodal regions from the main stem of the four-month-old hardened plants prior to the onset of inflorescence. The same organs, along with inflorescence post onset of flowering in five-month-old plants, were also analyzed for CPT content (Fig. 1).

Fig. 1.

The young leaves and inflorescence of a five-month-old plant used for CPT analysis. Young leaves referred here were the first leaves below the inflorescence, whereas mature leaves were the ones obtained from the third node present below the inflorescence

Regeneration of plantlets through direct and indirect methods

Leaf discs (1 cm²) were cultured on ½ LS (Linsmaier and Skoog 1965) medium with 8.88 µM BA (Benzyl adenine) and 0.57 µM IAA (Indole-3-acetic acid) for multiple shoots regeneration (Kamble et al. 2013). For callus initiation, several media combinations of auxins and cytokinins were tried, and the best results were observed on MS (Murashige and Skoog 1962) with 21.49 µM NAA (Naphthalene acetic acid) and 2.32 µM Kn (Kinetin) (Table S1). For further multiple shoot regeneration from a callus, different combinations of MS media with Kn and IBA were tried (Table S2) with varying concentrations of agar. From all the media combinations, the best results were observed in 9.28 µM Kn and 2.46 µM IBA (Indole-3-butyric acid) with agar 0.9%. These multiple shoots obtained were separated and then transferred to ½ LS for further growth and were regularly subcultured every 18 to 20 days on the same medium. All the samples were cultured in sterile glass test tubes containing 20 mL of growth media. The in-vitro cultures were maintained at 16 h/8 h light/dark photoperiod under white fluorescent lights (50 μmol m⁻² s⁻¹, Phillips lamps), with a relative humidity of 65–75% and a temperature 24.0 ± 0.5 °C.

Quantification of camptothecin by HPLC

Different plant parts of O. rugosa such as leaves, stems, roots, inflorescence, callus etc. were collected and dried at 55 °C in an air dryer for 72 h. All samples were extracted with 100% methanol (100 mg dried powder per mL) for overnight, followed by sonication (Ultrasonic cleaner, Cole-Parmer) for 60 min. The resulting samples were then centrifuged and filtered through a 0.22 µM filter, and the clear supernatants were used for high-performance liquid chromatography (HPLC) analysis (Fulzele and Satdive 2005b).

The HPLC was performed on Waters Breeze QS HPLC System equipped with an autosampler injector (Model No. 2707, Waters) with a 25 µL loop and a Waters 2998 Photodiode Array Detector (Model No. 2998, Waters). Separations were performed on Waters Symmetry Column C18 (75 mm × 4.6 I.D.). Data collection and integration were accomplished using Waters Empower QS Software solutions for chromatography. The camptothecin was determined using acetonitrile: water (40:60, v/v) as a mobile phase. The flow rate was 1 mL/min with an injection volume of 10 µL for all the samples. The elution was monitored at 254 nm. Standard solutions were prepared by dissolving camptothecin in methanol (1 mg/10 mL). The identity of the alkaloids was confirmed by co-chromatography with an authentic camptothecin sample. A standard calibration curve of camptothecin was plotted by analyzing fifteen distinct concentrations of the CPT standard. The linear relationship between the peak area and CPT amount (µg) was determined and expressed as Y = 7,211,700.15 x + 1.03884E6 with the correlation coefficient (r = 0.98419). The injection volumes were fixed at 10 µL to analyze plant samples. This standard calibration curve served as the basis for calculating the CPT content in the different organ extracts.

Statistical analyses

All the experiments were carried out with three replicates and repeated at least twice. The results are expressed as means with standard errors. All the statistical analysis was done using Microcal™ Origin 8.1. To analyze significant differences among all the organs, one-way ANOVA was performed at a significance level of p ≤ 0.05. Subsequently, a Tukey test was conducted as a post hoc analysis to determine specific group differences.

Result and discussion

This study investigated the spatial distribution of CPT in various organs of Ophiorrhiza plants (one, four and five-month old). It was found that plant differentiation and age significantly influence CPT accumulation. The results revealed a distinct variation in CPT concentration in different plant parts at different growth stages. Analysis of the shoot and root of one-month-old in-vitro plantlets revealed 0.22% dw and 0.247% dw of CPT, respectively. In the case of a four-month-old plant, young leaves and roots showed the highest CPT content (0.23% dw and 0.315% dw, respectively), while mature leaves showed comparatively less CPT content (0.19%) dry weight. The nodal and inter-nodal regions of the main stem accumulated 0.212% dw and 0.135% dw CPT, respectively (Fig. 2). Our results are in accordance with Yamazaki et al. (2003), who reported that the younger O. pumila parts, such as the youngest leaves and flower buds, had the highest CPT content in a six-month-old plant. Furthermore, numerous investigations on C. acuminata have found that young leaves and stems possessed higher CPT concentrations than older leaves and stems despite CPT accumulating in all regions of the plant (Yan et al. 2003; Wen-Zhe 2004; Sankar-Thomas and Lieberei 2011). Mingzhang et al. (2011) also reported that CPT content decreased with tissue age; CPT in tender leaves of N. nimmoniana was roughly six times higher than that of senescent leaves, and the highest level was found in roots (0.187% dw). The lowest contents were recorded in the bark and the senescent leaves. Namdeo and Sharma (2012), in his findings, observed the maximum CPT in roots, followed by fruits, stems, and the minimum in leaves. The roots showed a threefold higher concentration of CPT than in the leaves and stem.

Fig. 2.

Different tissues of O. rugosa, developed through direct and indirect regeneration methods, and selected for CPT concentration estimation. a Explant, b multiple shoots, c callus, d multiple shoot regeneration from callus, e elongated shoots, f plantlet with rooting, g one-month-old potted plant, h four-months-old potted plant, and i five-months-old plant with inflorescences

Degambada et al. (2023) observed that in contrast to leaves with petioles, twigs, and stem bark, a significantly high concentration of CPT was observed in roots. They also studied the CPT localization in roots, which was found to be localized in xylem elements and idioblast cells of the epidermis and outer cortex in the roots. A few have reported the tissue-specific biosynthesis of camptothecin. Yamazaki et al. (2003) observed the biosynthesis of CPT in stem and roots but not in the leaves of O. pumila. He attributed it to the presence of the enzyme strictosidine synthase in these tissues but not in the leaves, suggesting that these tissues might serve as the primary sites for CPT production and is translocated later. Consistent with camptothecin accumulation, Hao et al. (2023) studied the expression of genes involved in camptothecin biosynthesis through transcriptomics and found it to be maximum in the roots of O. pumila.

The role of differentiation in CPT accumulation has been reported only by a few. Kaplan et al. (2008) reported that with developmental stages, the percentage of secondary metabolites varies within plant organs. The production of plant secondary metabolites in plants remains active in differentiated tissues (Flores et al. 1987) and is not diminished as in callus or cell suspension cultures, which are undifferentiated (Wink 1987). Wetterauer et al. (2018) also observed low CPT in callus and cell suspension cultures in C. acuminata, similar to our findings. These results confirm that a certain extent of differentiation is necessary for CPT biosynthesis, and it increases with further plant tissue differentiation. This may be a reason why callus having traces of CPT when differentiated and gave rise to a complete plant showed CPT similar to the plant obtained through direct regeneration.

After flowering, the accumulation of CPT changed significantly (p ≤ 0.05). The highest CPT was found in inflorescence (0.348% dw), followed by mature leaves with 0.19% dw, and it reduced to 0.18% dw in young leaves located close to the inflorescence (Figs. 3, 4). All other tissues also showed a decline, with 0.19% dw in the roots, 0.05% dw in the nodal, and 0.03% dw in the inter-nodal stem regions. Lee et al. (2022) reported the maximum CPT concentration in the reproductive organ, followed by the root, stem, and leaves in O. pumila, again suggesting that younger organs have greater CPT concentrations than older organs. The findings of this work are also consistent with the observations mentioned above, as it was found that before flowering initiation, younger leaves had 1.17-fold higher CPT accumulation than the mature leaves, which decreased to 0.95-fold after the flowering occurred, and the inflorescence being the youngest organ, displayed the highest CPT accumulation at this stage. Some studies suggest that CPT serves a role in chemical defense, which explains this decrease in CPT. Young, delicate leaves serve as a major source of photosynthates and have high nitrogen content, which attracts insects, diseases, and microbial attacks, and their physical defensive system (such as cutin, suberin, and wax) is also underdeveloped (Lorence and Nessler 2004). These could all be the contributing factors. Therefore, these parts and reproductive organs accumulate CPT as a protective mechanism. Montoro et al. (2010) also reported higher CPT in young leaves and leaf buds compared to mature leaves in C. acuminata, reflecting its requirement as a chemical defense mechanism. As the CPT decreases after the onset of flowering, this also suggests that the optimal time to harvest the plant would be before the initiation of flowering, which is between four and four-and-a-half months.

Fig. 3.

The percentage CPT estimated before (four-month-old) and after flowering (five-month-old) to study its effect on CPT accumulation in the various organs of O. rugosa. The scale bar represents standard error. Bars with different letters indicate significantly different values at p ≤ 0.05

Fig. 4.

Chromatograms obtained from HPLC analysis of different tissues in O. rugosa. a CPT standard, b inflorescence, c roots, d mature leaves, e young leaves and f callus

Conclusion

Our studies revealed that differentiation of plant organs significantly influenced CPT concentration in O. rugosa, though CPT accumulation in a mature plant was independent of the method of plant regeneration. Among various plant organs, the highest CPT was accumulated in inflorescence but with a concomitant decrease in all other parts. The results suggest that the optimal harvest age should be 4–4.5 months, just before flowering begins.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

KS: Methodology, Data curation, Formal analysis, Original draft; RKS: Methodology, Formal analysis, Original draft; SS: Conceptualization, Methodology, Data curation, Formal analysis, Supervision, Original draft and Review.

Funding

Work communicated in the present manuscript is funded by Department of Atomic Energy, Government of India.

Data availability

Not applicable.

Code availability

Not applicable.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest in the publication.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed consent

All the authors gave their consent for publication of the results.