**1. Introduction**

Medicinal plants or herbs are enriched in various phytochemicals, which have been shown to have various biological effects and should be researched further [1,2]. The World Health Organization (WHO) recognized the importance of medicinal plants in millions of people's primary care. It is estimated that more than 80% of the world's population relies on these resources as their primary source of health-related problems [3]. One of the medicinal plants that has currently gain much attention because of its therapeutic constituents is *Bougainvillea glabra* [4]. The genus of *Bougainvillea* belongs to the family Nyctaginaceae, which is one of the utmost valuable ornamental and medicinal plants native to South America (Brazil, Peru, and northern Argentina). The name derives from the French navigator Louis Antoine de Bougainville, who was the first to discover this plant in Brazil in 1786 [5,6]. *B. glabra* is a perennial and evergreen shrub that is widely growing in

**Citation:** Nasrat, M.N.; Sakimin, S.Z.; Hakiman, M. Phytochemicals and Antioxidant Activities of Conventionally Propagated Nodal Segment and In Vitro-Induced Callus of *Bougainvillea glabra* Choisy Using Different Solvents. *Horticulturae* **2022**, *8*, 712. https://doi.org/10.3390/ horticulturae8080712

Academic Editor: Luigi De Bellis

Received: 24 July 2022 Accepted: 1 August 2022 Published: 8 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/).

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warm climates like Indonesia, Ethiopia, Philippines, Thailand, Malaysia, Vietnam, Taiwan, India, Australia, Mexico, South Africa, United States, Central America, Caribbean, and the Mediterranean [7].

Species of the *Bougainvillea* genus are frequently explored for their nutritional and therapeutic properties. The plants of the *Bougainvillea* species are considered to be utilized in traditional medicine for a variety of ailments, as phytopharmacological studies have reported, including anti-inflammatory and antipyretic [8], antidiarrheal, pain ailments and antiulcer [9], antimicrobial [10], antidiabetic [11], immunomodulatory [12], hypoglycaemic [13], antihyperlipidemic [14], anti-cough, sore throat, blood vessel troubles, leucorrhoea, hepatitis [15], anti-skin problems (tyrosinase) [16], analgesic [17], antiviral [18], antifungal [19], neuroprotective [4], and anthelmintic [20]. Various parts of the *Bougainvillea* plants are used to treat various ailments. In Panama, the flowers are used for treating hypotension. In India, leaves, flowers, and stem barks treat many illnesses, including stomach acidity, diarrhea, cough, blood vessel problems, sore throat, and hepatitis. In Thailand, the flowers treat stomachache, nausea, and diarrhea [1].

According to the report by Abarca-Vargas and Petricevich [15] on *Bougainvillea glabra*, 35 volatile compounds, 4 phenolic compounds, and 21 flavonoids compounds were extracted from leaves, bracts, and branched. Thus, the demand for raw materials is increased in tropical areas with temperate and cool weather. *B. glabra* is vegetatively propagated by stem cuttings. However, in the traditional propagation method, the production of secondary metabolites is known to be unsuitable due to external factors in the environment such as climate, plant pests and diseases, and fertilizer application. Therefore, the plant tissue culture technique is the most suitable technique for the consistent production of secondary metabolites under a controlled condition. A massive amount of raw materials is needed to extract the high amount of secondary metabolites which is not possible to be produced through conventional methods due to land availability. This problem can be overcome by producing the secondary metabolites in the laboratory by plant tissue culture technique [21]. Plant tissue culture is the most effective way to propagate rare, endangered, and valuable medicinal and commercial plant species on a wide scale in a short time while also protecting them [22]. Furthermore, callus formation investigations are helpful in understanding the metabolic pathways of secondary metabolites. On the other hand, the selection of plant growth regulators (PGRs) and environmental (cultural) conditions have an impact on callus growth in culture. Specific PGRs at appropriate concentrations can play an important role during callogenesis. Auxin and cytokinin type and concentration are essential determinants of in vitro callogenesis and regeneration. The ratio of auxin to cytokinin is the most crucial factor in this case [23]. Because the extraction of secondary metabolites requires a considerable amount of biomass, tissue culture using multiple shoots and callus induction is one of the methods used.

Extraction is the first and vital step in analyzing secondary metabolite constituents from plant materials [24]. Due to the presence of diverse molecules with varying chemical features, the polarity of the solvent used for extraction and the method of extraction play critical roles in both the efficiency and efficacy of plant secondary metabolites [24–28]. Moreover, extraction from plant products is complicated and difficult due to the vast range of structures and polarity of chemical compounds. Solvent, time, solid-to-solvent ratio, number of extractions, temperature, and partial size of the sample material are all important extraction parameters [29].

As far as we know, there are not enough studies on the influence of white light and dark on callus induction and phytochemical/antioxidant activity in *B. glabra.* Hence, we studied the effect of light quality on morphological and biochemical components of in vitro grown node-derived callus cultures of *B. glabra*. This research will help understand the effect of light on the production of commercially essential secondary metabolites and their optimization in the in vitro cultures of *B. glabra*.

#### **2. Materials and Methods**

#### *2.1. In Vitro Callus Induction*

2.1.1. Plant Materials and Sterilization

The nodal segments of conventionally propagated plants of *Bougainvillea glabra* Choisy were collected from the campus, Faculty of Agriculture, Universiti Putra Malaysia, Serdang, Selangor, Malaysia. The samples of *B. glabra* plants used in the study were deposited at Biodiversity Unit, Institute of BioScience, Universiti Putra Malaysia with a plant confirmation voucher number MFI 0197/21.

For the sterilization process, after removing the leaves from cut branches, nodal segments were cut into pieces (0.5–1 cm) and the explants were washed under running tap water containing a few drops of detergent for 30 min to remove dust particles. After that, the explants were washed with sterile distilled water once and put in a glass jar containing 500 mg/250 mL (*w/v*) streptomycin + 500 mg/250 mL (*w/v*) bavistin for pre-treatment of the explants for one hour [30]. Then, the explants were disinfected by immersion in 70% ethanol for 25 s and washed two times with autoclaved distilled water. Then, the explants' surface was sterilized with a bleaching agent with a concentration of 30% of Clorox® (5.25% (*w/v*) of sodium hypochlorite and a few drops of tween-20 as an emulsifier per 100 mL solution for 15 min by using a shaker with 300 rpm [31]. After that, the explants were washed three times with autoclaved distilled water and prepared for culturing.
