Salvia is the largest genus of the Lamiaceae family, with over 900 species widely distributed throughout the world (Hu et al., 2018). The genus is known for its long-standing use in traditional medicine and its diterpene richness (Wu et al., 2012; Wei et al., 2017). One of the most important species is Salvia miltiorrhiza Bunge, which has been part of traditional Chinese medicine since ancient times (Chun-Yan et al., 2015). It has been officially recorded in the Chinese pharmacopoeia since 1963 (Guo et al., 2014) and is used for the treatment of various diseases, such as Alzheimer’s disease (Wong et al., 2010), diabetes (Jia et al., 2019), cerebrovascular disease (Zhou et al., 2011), coronary heart disease (Lam et al., 2006), cancer (Zhang and Wang, 2006), hepatocirrhosis (Lin et al., 2006), and Parkinson’s disease (Zhang et al., 2010). In Chinese traditional medicine there were two primary types of S. miltiorrhiza preparations used by patients: directly prepared herbal raw materials (decoctions, extracts, tinctures) and Chinese patent medicines (water pills, honey pills). However, the use of other products such as injection and ultrafine granular powder is increasing significantly (Shen et al., 2017). One of the preparations of modern Chinese medicine, namely compound Danshen dripping pills, which consist of S. miltiorrhiza, Panax notoginseng, and borneol, is now widely used in China for the prevention and treatment of angina pectoris, hyperlipidemia and coronary heart disease (Chu et al., 2011). In addition, danshen is used as a healing tea to boost immunity and prevent diseases (Wei et al., 2017).

S. miltiorrhiza has attracted much attention due to its content on tanshinones (Jiang et al., 2019), a class of phenolic abietane-type nor-diterpenoids possessing a 14,16-ether D-ring (Ma et al., 2021). Tanshinones were repeatedly isolated and investigated because of their bioactive properties such as anti-inflammatory (Ma et al., 2016), antitumor (Jiang et al., 2019), antibacterial and antiviral (Lai et al., 2021), and antioxidant activities (Li et al., 2013; Sun et al., 2014). Thereby, the cytotoxic activity of selected tanshinones, e.g., tanshinone I and IIA, cryptotanshinone, and dihydrotanshinone I, is of special interest. Tanshinone IIA has been shown to inhibit proliferation and to induce apoptosis of human hepatocellular carcinoma BEL-7402 and SMMC-7721 cells (Yuan et al., 2004) as well as human promyelocytic leukemic cells (HL-60) and human erythroleukemic cells (K562) (Sung et al., 1999; Yoon et al., 1999). It was, furthermore, reported that cryptotanshinone inhibits the growth of H446 lung tumor cells (Man et al., 2016), suppresses cell viability and induces apoptosis of the breast cancer cell line MCF-7 (Park et al., 2012), and induces apoptosis and inhibits movement of CEM/ADR5000 and CCRF-CEM lymphoblastic leukemia cells (Wu et al., 2016). In addition, tanshinone I and dihydrotanshinone I were found to induce apoptosis in human gastric cancer cell lines SGC-7901 and MGC-803 (Cheng et al., 2016; Jing et al., 2016) and breast cancer cell lines MDA-MB-231 and MCF-7 (Tsai et al., 2007; Nizamutdinova et al., 2008).

The compound class of tanshinones, however, is not exclusive to S. miltiorrhiza. There have been reports from more than twenty different species containing these particular compounds, such as S. digitaloides, S. przewalskii, or S. yunnanensis, with most of them occurring in East Asia (Zhang et al., 2012). In addition, S. glutinosa, a plant species widely distributed in Europe but with similar floral morphology to S. miltiorrhiza is known to contain tanshinone derivatives (Nagy et al., 1998; Hu et al., 2018). To date, many phylogenetic studies have been conducted that have divided Salvia into several clades or subgenera placing S. miltiorrhiza in the same clade or subgenus as S. glutinosa (Drew et al., 2017; Will and Claßen-Bockhoff, 2017; Hu et al., 2018; Kriebel et al., 2019; Rose et al., 2021; Wu et al., 2021; Moein et al., 2023). However, S. glutinosa differs from other species of the same subgenus or clade in that it is the only one distributed not only in Asia but also in Europe, while the other species are mainly represented by Chinese species (Will and Claßen-Bockhoff, 2017).

S. glutinosa, which is referred to as glutinous sage or Jupiter’s sage, is an aromatic perennial plant that grows in moist forests from Central and Eastern Europe to Western Asia (Kaya et al., 2003). There is plenty of evidence of the traditional use of glutinous sage in folk medicine in many countries. In Romania, a decoction of the roots was used against dizziness and an infusion of the flowers and leaves was administered against gastroenteritis and cough pains and to treat injuries and animal bites (Nicolescu et al., 2022). The leaves of S. glutinosa are also used to prepare an infusion for digestion and to treat headaches and sore throats (Idolo et al., 2010) or for culinary purposes as an aromatic herb (Motti, 2021).

After the first reports for tanshinones in S. glutinosa (Nagy et al., 1998), additional derivatives were isolated from this species, inter alia the well-known tanshinone I, tanshinone IIA, cryptotanshinone, or dihydrotanshinone I (Nagy et al., 1999; Senol et al., 2017; Ayvazyan et al., 2023). However, no reports on the content of tanshinones in S. glutinosa have been made so far. Because of the long maturation time of S. miltiorrhiza and growing pharmaceutical demand for tanshinones, alternative sources for this particular compound class are becoming of increased interest. Thus, the aim of our study was to determine the amount of the major tanshinones in S. glutinosa roots and to identify eventual cytotoxic constituents, which are less abundant in danshen and therefore not yet biologically characterized.

Diterpenoids of the genus Salvia and in particular the group of tanshinones have been extensively studied for their pharmacological properties in the last decades. Especially their potential use in cancer therapy has been of interest, as several tanshinones were reported to exhibit pronounced apoptotic and cytotoxic effects. Tanshinone IIA (7), the major tanshinone in S. miltiorrhiza induced apoptosis in human leukemia cells by activation of caspase-3 (Sung et al., 1999). These effects were observed in K562 erythroleukemic cells and HL-60 promyelocytic cells, which were also investigated in our study. The compound, furthermore, inhibited growth and proliferation of human hepatocellular carcinoma cells by apoptosis induction (Yuan et al., 2004) and showed additive effects with taxol against Tau protein-related resistance in MCF-7 cells (Lin et al., 2017). Cryptotanshinone, the second major tanshinone in the S. miltiorrhiza samples of our study, was also studied in MCF-7 cells, thereby showing antiproliferative and apoptotic effects via the PI3K/AKT signalling pathway (Shi et al., 2022). The compound, moreover, affected leukemia cells, namely acute lymphoblastic leukemia cells, via inhibition of NFκB (Wu et al., 2016). In addition, cryptotanshinone showed apoptotic and antiproliferative effects against hepatic stellate cells (Hou et al., 2023) and inhibited lung tumor growth via activation of the JAK2/STAT4 pathway and increased CD4⁺ T cell toxicity (Man et al., 2016). In another study on lung cancer cells, which also included tanshinone IIA (7) and cryptotanshinone, tanshinone I was found to inhibit proliferation of the human lung adenocarcinoma cell line CL1-5 by 73% at a concentration of 10 µg/ml after 48 hours of incubation, whereas the other two tanshinones showed an inhibition of 50% (Lee et al., 2008). Subsequent in vivo studies demonstrated that the compound also reduced tumorigenesis and metastasis in mice. Here, the aromatic A-ring of tanshinone I (and a thus missing methyl group at position C-4) must be the reason for the stronger effects compared to tanshinone IIA (7) and cryptotanshinone, which both show an aliphatic A-ring and two methyl groups instead. Tanshinone I was, furthermore, found to induce apoptosis in gastric cancer cells (Jing et al., 2016) as well as in MCF-7 and MDA-MB-231 breast cancer cells (Nizamutdinova et al., 2008). The same two breast cancer cell lines were also used in a study by Tsai et al. (2007), who compared 15,16-dihydrotanshinone I (4), cryptotanshinone, and tanshinone I for their cytotoxic activity. After dihydrotanshinone I (4) showed the strongest effects, further studies found the compound to inhibit cancer cell proliferation and tumor growth in MDA-MB-231 cells. Same as tanshinone I, dihydrotanshinone I (4) bears an aromatic A-ring but also a dihydrofuran D-ring, which seems to be responsible for the increased activity compared to tanshinone I (bearing a furan D-ring instead). The stronger cytotoxic activity of dihydrotanshinone I (4) compared to other tanshinones was also emphasized by Cheng et al. (2016), who reported the compound to induce growth inhibition and apoptosis in gastric cancer cells.

The stronger antiproliferative effects of dihydrotanshinone I (4) compared to e.g., tanshinone IIA (7) was also corroborated by the results our study. While tanshinone IIA (7) reduced the proliferation of both MDA-MB-231 and HL-60 cells to 80% at a concentration of 5 µM ( Figure 2 ), dihydrotanshinone I (4) exhibited the same effect at a concentration of 1 µM ( Figure 3 ). At 5 µM instead, dihydrotanshinone I (4) reduced proliferation to less than 20% ( Figure 3 ) and metabolic activity of both cell lines to approximately 40% ( Figure 4 ), thus demonstrating the abovementioned cytotoxic potential. Another fact that was evident by our results is the lower proliferative activity of non-tanshinone abietane type diterpenoids, such as compounds 1 to 3 ( Figure 2 ). Also, the only p-quinoid tanshinone (compound 6), did not exhibit noteworthy effects. However, dihydrotanshinone I (4) was not the most active compound observed in our study, but the structurally closely related danshenol A (5, Figure 1 ). Same as dihydrotanshinone I (4), danshenol A (5) bears an aromatic A-ring and a dihydrofuran D-ring and thus two of the features relevant for the pronounced effects in some of the aforementioned studies. However, in contrast to the keto function in position 11, which appear in the four major tanshinones (tanshinone IIA (7), cryptotanshinone, tanshinone I, and dihydrotanshinone I (4)), danshenol A (5) is substituted with an acetonyl group and a hydroxy group. Not possessing an o-quinone structure anymore, danshenol A (5) thus is to be regarded as a tanshinone derivative rather than a real tanshinone. However, with regard to its biological effects the compound surpassed the tanshinones (o- and p-quinones) and other diterpenoids investigated in our study including the highly active dihydrotanshinone I (4, Figures 4 , 5 ). Thereby, the effect on MDA-MB-231 cells are certainly of great interest, as both tanshinone I and dihydrotanshinone I (4) were reported to induce pronounced antiproliferative and apoptotic effects, with the latter compound also demonstrating anti-tumor potential in vivo. The antiproliferative effects against MDA-MB-231 cells in our study revealed a complete inhibition of proliferation at a concentration of 5 µM (which was achieved at 10 µM of compound 4) and a reduction to around 28% at 2.5 µM. At the same concentration dihydrotanshinone I (4) reduced proliferation to 70% and thus to a much lesser extent ( Figure 4A ). A similar picture was observed in the cytotoxicity assays ( Figure 5A ), where danshenol A (5) was approximately twice as active as dihydrotanshinone I (4) in the investigations on the HL-60 cell line ( Figures 4B , 5B ). The strong cytotoxic potential of danshenol A (5), which was also evident in the wound healing assay ( Figure 7 ), together with the missing toxicity against healthy cells ( Figure 6 ) renders the compound a more than interesting compound for future studies. Even more so, as structurally related but probably less cytotoxic diterpenoids were repeatedly investigated and proven to exhibit various anticancer activities.

Of course, the amount of tanshinones found in S. miltiorrhiza is by far exceeding the concentrations in S. glutinosa ( Table 1 ), which is an explanation for its long use in traditional medicine in contrast to the rather scarce mentions of the widely occurring glutinous sage. However, it also highlights a problem in modern phytopharmacology, where not seldom studies are accomplished with a few readily available compounds. This is most obvious for the tanshinones, where the majority of investigations were conducted with tanshinone IIA (7), cryptotanshinone, tanshinone I, and dihydrotanshinone I (4), thereby omitting eventual more promising candidates. Danshenol A (5) could be such a candidate, which became of interest in the last years due to its positive anti-atherosclerotic and anti-inflammatory effects (Zhao et al., 2017). Though the compound was first isolated from S. miltiorrhiza, it is not among the principal components in danshen and was also not detected in our study. Therefore, it was probably not considered for pharmacological studies. In contrast, it was one of the first two compounds isolated from S. glutinosa (Nagy et al., 1998) and was found in both of our collections ( Table 1 ). Thus, phytochemical studies on taxonomically related species of well-known medicinal plants are a valuable strategy to discover potent bioactives, even though the investigated species itself is of only limited medicinal interest.

In conclusion, the present study demonstrated promising antiproliferative and cytotoxic effects for danshenol A (5) isolated from the glutinous sage (Salvia glutinosa). Though our analysis of S. glutinosa did not reveal the species as a potent source for tanshinones in terms of quantity, its particular tanshinone derivative danshenol A (5) was surpassing the effects of dihydrotanshinone I (4), which itself is regarded as one of the most cytotoxic tanshinones. Hence, further studies on the anticancer properties of danshenol A (5) are certainly warranted.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

Ethical approval was not required for the studies on humans in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used.

AA: Data curation, Investigation, Writing – original draft. LD: Data curation, Investigation, Writing – original draft. CZ: Writing – original draft, Resources. BK: Supervision, Writing – original draft, Data curation, Investigation. SÇ: Conceptualization, Funding acquisition, Writing – original draft, Writing – review & editing.