**1. Introduction**

Plants synthesize a large number of small molecules for use in defence against biotic and abiotic stresses [1–3]. These secondary phytochemicals have commercial applications in pharmaceuticals, flavourings and fragrances, insecticides, etc. [4,5]. They are divided into three main groups: phenolic compounds, terpenoids and nitrogen-containing alkaloids [6]. Usually, alkaloids contain basic nitrogen, derived from an amino acid or purine/pyrimidine, while in some pseudoalkaloids the source of nitrogen is a transamination reaction [7]. Among alkaloids, the chromone alkaloids are a unique group with many biological activities, structurally consisting of a nitrogen system (pyridine, piperidine, pyrrolidine) linked to the A-ring of chromone [8].

**Citation:** Ahmed, S.; Asgher, M.; Kumar, A.; Gandhi, S.G. Exogenously Applied Rohitukine Inhibits Photosynthetic Processes, Growth and Induces Antioxidant Defense System in *Arabidopsis thaliana*. *Antioxidants* **2022**, *11*, 1512. https:// doi.org/10.3390/antiox11081512

Academic Editors: Stanley Omaye and Filomena Nazzaro

Received: 1 June 2022 Accepted: 19 July 2022 Published: 3 August 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

The chromone alkaloid rohitukine was isolated for the first time from *A. rohituka* [9] and its structure has been shown to be that of a highly polar molecule [9–11]. It is restricted to only two plant families: Rubiaceae (*Schumanniophyton magnificum* and *S. problematicum*) and Meliaceae (*Dysoxylum gotadhora, D. acutangulum* and *Amoora rohituka*) [12]. Both families are mainly distributed in tropical areas of the world [13]. *D. gotadhora* (Indian white cedar) has been in use in Ayurvedic and other traditional systems of indigenous medicine for many years to treat diabetes, jaundice, leucorrhoea, piles, leprosy, osteomyelitis, etc. [14]. *D. gotadhora* is known as the main source of rohitukine. Within the plant body, rohitukine is found to accumulate in leaves, bark, seeds and fruits [15]. The pharmaceutical potential of rohitukine has been deeply assessed in breast, ovarian and lung cancer cell lines, where it inhibits cyclin-dependent kinase (CDK) CDK2/A and CDK9/T1 complexes by blocking their ATP binding sites [10]. In budding yeast, rohitukine induces ROS generation and apoptosis [16]. P-276-00, IIIM-290 and flavopiridol are semisynthetic derivatives of rohitukine.

The rohitukine-inspired molecule flavopiridol has gained considerable attention in the last two decades for its potent cytotoxic activity against a wide range of cancer cell lines [17] and it has now been approved by the European Medicines Agency for the treatment of chronic lymphocytic leukemia (CLL) [18]. Flavopiridol potently inhibits CDKs 1, 2 and 4, causing cell cycle arrest in G1 and G2 phases in mammalian cells [19]. P-276-00 (Piramal Healthcare Limited, Mumbai, India) is another derivative of rohitukine that has advanced into clinical trials for cancer treatment. P-276-00 selectively inhibits CDK4/D1, CDK1/B and CDK9/T1, and its antiproliferative effect has been observed against a wide range of cancer cell lines [20]. IIIM-290, an orally bioavailable anticancer drug that is already being examined in apre-clinical study, is also derived from rohitukine [21,22].

Similar to many other medicinal plants, the presence of rohitukine in *D. gotadhora* may likely exert allelopathic effects on neighbouring plants. It has been observed that plants release chemical compounds into their surrounding environment which influence their growth and also contribute to restricting invasion by exotic plant species [23,24]. These phytotoxic compounds are usually biosynthesized in plants as secondary products and many of them have been explored for their pharmacological activities [25,26]. To address the phytotoxic effects of secondary metabolites, many studies have been published which show that secondary metabolites have an impact mainly through damage to photosynthetic machinery and frequent decomposition of photosynthetic pigments, the decrease in photosynthetic pigments leading to blockage of energy/electron transfer and inhibition of ATP synthesis [27–29]. Several alkaloids isolated from the medicinal plant *Ruta graveolens* have also been investigated for their photosynthetic inhibitory activities in recipient plants [30]. Antidesmone is a plant secondary metabolite that causes disruption to photosynthetic machinery [31]. Additionally, the secondary compounds of *Satureja hortensis* affect seed germination, morphology and bleach out chlorophyll content in *Amaranthus retroflexus* and *Chenopodium album* [32]. It is quite evident that the targets of toxic plant secondary metabolites are achieved through perturbations to PSII activity [33,34]. In plants, photosynthetic damage is also linked to increased levels of reactive oxygen species (ROS),which indicates oxidative stress [35–37]. Increased ROS levels lead to oxidative damage to cells. Similarly, exposure to toxic secondary metabolites also triggers the ROS pathway in recipient plants [38,39]. To resist oxidative stress, plants, upon exposure to such chemicals, alter the activities of antioxidant enzymes, such as SOD, POD, APX and CAT [27,40,41].

The above-mentioned findings indicate that rohitukine as well as its semisynthetic derivatives have been explored for their remarkable biological activities in animal cells, wherein rohitukine has been reported to trigger ROS generation and apoptosis [16,42]. However, the phytotoxic effect of rohitukine on the growth and development of plants has not been explored. Therefore, here we tried to understand the morphological, physiological and biochemical changes in *A. thaliana* (a model system) treated with appropriate rohitukine concentrations isolated from *D. gotadhora*. The main objectives of the study were to understand the interference of rohitukine with the antioxidant system of *A. thaliana* and

its impacts on photosynthesis. Furthermore, we sought to gain insights into photosynthetic pigments, the phytotoxicity attributed to ROS generation and changes in levels of metabolites, such as amino acids, sugars and other organic acids, in *A. thaliana*.

#### **2. Material and Methods**

## *2.1. Source Plant Material, Extraction, Fractionation and Isolation of Pure Compounds*

Rohitukine was extracted from dry leaf powder of *D. gotadhora*, as described previously by Mahajan et al. (2015) [15]. Briefly, shade-dried leaves were extracted thrice with 50% ethanol by sonication for one hour each at 45 ◦C, and the extract was dried using a rotary evaporator. The crude extract was then subjected to acid–base fractionation to obtain a fraction enriched with the alkaloid rohitukine and purified by repeated column chromatography over a silica gel mesh. The purity of the isolated rohitukine was confirmed by liquid chromatography–mass spectrometry (LC–MS) and high-performance liquid chromatography (HPLC). The purity of compounds was checked using HPLC by following the protocol of Kumar et al. (2016) [43]. An RP-C18 column was used (Neo Sphere, 250 mm × 4.6 mm, 5 μm). The mobile phase consisted of methanol–water (75:25 *v*/*v*) at flow rate of 1.0 mL/min. The temperature of the column oven was 40 ◦C and the injection volume was 4 μL. The detector used was a diode array detector, and the detection wavelength was 254 nm. Solutions of pure rohitukine were prepared by dissolving the alkaloid in sterile double-distilled water, followed by filter sterilization using a syringe filtration unit fitted with a 0.22 μm pore size (Millex-GV, Durapore). The filtered stock solution of rohitukine was then diluted appropriately in autoclaved double-distilled water for foliar treatment.
