###### Abstract  
This work aims to study the qualitative composition of biologically active substance (BAS) extracts in vitro callus, cell suspension, and root cultures of the medicinal plant _Rhaponticum carthamoides_. The research methodology is based on high-performance liquid chromatography, and 1H nuclear magnetic resonance (NMR) spectra, to study the qualitative and quantitative analysis of BAS. The results of the qualitative composition analysis of the dried biomass extracts of in vitro callus, cell suspension and root cultures showed that the main biologically active substances in the medicinal plant _Rhaponticum carthamoides_ are 2-deoxy-5,20,26-trihydrosycodyson (7 mg, yield 0.12%), 5,20,26-trihydroxyedyson 20,22-acetonide (15 mg, yield 0.25%), 2-deoxy-5,20,26-trihydrosycodyson 20,22-acetonide (6 mg, yield 0.10%), 20,26-dihydrosycydyson 20,22-acetonide (20 mg, yield 0.09%), and ecdyson 20,22-acetonide (6 mg, yield 0.10%). In the future, it is planned to study the antimicrobial, antioxidant, and antitumor activity of BAS of extracts of in vitro callus, cell suspension, and root cultures of the medicinal plant _Rhaponticum carthamoides_, for the production of pharmaceuticals and dietary supplements with antitumor, antimicrobial and antioxidant effects. Acticle 2021 11 2555 11  
It is believed that plants have many natural compounds, such as terpenoids (isoprenoids, more than 50,000 different structures are distinguished), the production of which has two pathways (the cytosolic mevalonate or plastid methylerythtitol phosphate) [8,9]. The cytosolic mevalonate pathway is considered to be the main pathway for producing sesquiterpenoids (in particular artemisinin) and triterpenoids (ginsenosides) [7,10,11,12,13,14,14], while the plastid methylerythtitol phosphate pathway is considered to be the main pathway for triterpenoids (such as tanshinones and taxol) and monoterpenoids (such as limonene) [9,13,15]. Phenylpropanoids (anthocyanin, catechchin, salvianic acid, etc.) are derivatives of the shikimate pathway, synthesized from phenylalanine and tyrosine [6,16]. It is known that plants often synthesize biologically active compounds in small amounts [1], and only in some of the tissues [10,17]. It was established that compounds such as tanshinones, ginseng and flavones are formed and accumulate in the roots of _Salvia milliorrhiza_, ginseng (_Panax_), and _Scutellaria bacialensis_[10,18,19]. It is known that catharantin accumulates in all tissues and organs, while vinblastine and vincristine only accumulate in the aerial parts of the _Catharantthus roseus_ plant [20,21]. In _Artemisia annua_, artemisinin is formed in the glandular secretory trichomes of leaves [7]. Natural medicinal plants often require many years of growth before biologically active compounds are synthesized in their parts, for example, in the roots, usually in very small doses [22]. Moreover, the environmental and ecological problems lead to a deterioration in the quality characteristics of both plants and their derivatives [23,24]. The study and research of alternative methods of obtaining compounds with useful and unique characteristics from plant materials is relevant. The biosynthesis of in vitro transformed callus, cell suspension, and root cultures (hairy roots), from the point of view of the controlled production of biologically active substances with desired properties, has potential, and is of interest to both researchers and producers [25]. It is known that hairy roots grow faster than any roots of intact plants [26,27] and accumulate a higher content of valuable compounds in their cells [19,28,29]. The total content of tanshinone in the hairy roots of transgenic _S. miliiorrhiza_ was several times higher than the content in the roots of field plants (15.4 and 1.7-9.7 mg/g dry weight, respectively) [19,28]. The total content of vilorin in the hairy roots of _Tripterysimu wilfordii_ Hook.f. was significantly higher than in the adventitious roots [29].