Bioactive Ingredients of Helleborus niger L. (Christmas Rose): The Renaissance of an Old Medicinal Herb—A Review

Abstract

The medicinal plant Helleborus niger L. (Ranunculaceae) found in the European Alps has been valued as an effective remedy for numerous diseases for centuries. Recent preclinical evaluations showed pharmacologic potential of the plant extract for cancer treatment. Furthermore, several compounds extracted from H niger exhibited potent pharmacological effects. Thus, the renaissance of this old and proven medicinal herb requires better knowledge of its bioactive ingredients. A key ingredient of H niger is protoanemonin which is responsible for its burning hot taste and vesicant effect. Protoanemonin possesses antibacterial, antifungal, cytotoxic, and antimutagenic activity, and its isolation, synthesis, and preparation as a therapeutically valuable medicinal product are well elaborated. Anemonin, the dimer of protoanemonin, has antimicrobial, anti-inflammatory, and antimalarial activity, whereas (−)-ranunculin, a protoanemonin glycoside, possesses cytotoxicity and antimutagenic activity. (+)-Ranuncoside, another protoanemonin glycoside, has a spiro acetal molecular scaffold, a feature that is responsible for the biological activity of a multitude of natural products. Furthermore, the flavonoids kaempferol glycoside, quercetin glycoside, and glucosyl-phenyllactic acid have been isolated, and other phenolics with complex structures were detected as well. The main active ingredient of H niger rhizomes is the bufadienolide hellebrin. It is a steroidal cardiac diglycoside with utility for heart failure treatment. Hellebrin and its aglycon hellebrigenin are patented as lead compounds to treat cancer. Recently, additional bufadienolides as well as ecdysteroids were isolated from H niger whole plants and showed potent cytotoxicity on human cancer cell lines. Finally, various saponins have been determined by mass spectrometry, of which 4 compounds isolated from the rhizome are awaiting pharmacological assessment. This review gives an overview of the current knowledge of the isolation, synthesis, structure elucidation, derivatization, and biological activities of known H niger constituents. Furthermore, recent advances concerning the development of pharmaceuticals from these constituents and their derivatives are described.

Introduction

Helleborus niger L. (synonyms: Black Hellebore, Christmas Rose) is a perennial, 15 to 30 cm high plant of the Ranunculaceae family and the genus Helleborus (see Figure 1). The subspecies Helleborus niger subsp. macranthus (Freyn) Schiffn. is also known (worldfloraonline.org). The characteristic brown-to-black rootstock of H niger, which gave the plant its species name, is short, strong, and abundantly rooted (see Figure 1a). Its overwintering, leathery, dark green foliage leaves are stalked and 7- to 9-parted. The terminal white flowers open around late December which led to its common name Christmas Rose (see Figure 1b). This late flowering period in winter requires self-pollination because visitation by pollinating insects is unlikely at this time. Due to its pungent burning taste, which is typical of the Ranunculaceae, this plant is usually not eaten by livestock and remains in the pasture.

Figure 1.

The medicinal plant Helleborus niger L. (Christmas Rose). Photos were taken by the author from specimens in his garden in Seeheim, Germany; photo (A) June 2022, photo (B) December 2019.

H niger has survived the Last Glacial Maximum in refugia situated along the southern and also along the northern or northeastern periphery of the European Alps. Being a slow migrator, the species has likely survived repeated glacial–interglacial circles in distributional stasis while the composition and abundance of trees in the habitat changed considerably. ¹ Today, its main distribution area is the forested region of the eastern limestone Alps, where H niger grows up to an altitude of 1800 m mostly on stony, bushy slopes with calcareous subsoil. A map showing the main distribution area of H niger as well as the Helleborus taxa occurring in Europe and Asia Minor has been published. ²

While H niger is rarely found in its natural habitat, it is a popular ornamental flower, often planted in gardens and cemeteries. ³ Gardeners have been able to broaden the color range and vary the shape of the leaves and blossoms by careful selection and cultivation. ⁴ In view of the reproductive development of H niger, the role of its plant hormones has also been described. ⁵

A detailed historical overview of the long tradition from antiquity to the present of H niger in folk medicine, together with the related species H purpurascens Waldst. & Kit. and H odorus Waldst. & Kit., has been published by Balázs et al. ⁶ In this context, Barroso ⁷ surveyed the characteristics and therapeutic use of black and white hellebore, the beloved plants of the ancient Greeks. He emphasized that instead of H niger, other subspecies of hellebore and even other plants like Digitalis might also have been used in this time and named “hellebore.” Paracelsus, a seminal physician and alchemist of the 16th century, valued the leaves and roots of H niger as an effective remedy for numerous diseases.^8–10 In the late 19th and early 20th century, several medico-pharmaceutical practical handbooks presented recipes for remedies containing H niger, but these are now partially obsolete. A typical example from 1913 for the treatment of epilepsy is shown in Figure 2. It describes that the pulverized drug Radix Hellebori nigri is to be given together with other herbal ingredients in purified honey, and it is recommended to take the electuarium in teaspoonful quantities. ¹¹

Figure 2.

(A) Two-volume Hager's Handbook of Pharmaceutical Chemistry ¹¹ from 1913 and (B) a recipe from it for the treatment of epilepsy which includes Radix Hellebori nigri. Photos were taken by the author.

Today, the commercial drug Rhizoma (Radix) Hellebori nigri is rarely used due to its inconsistent effect. Among its limited uses today is the use as a cardiac agent when strophantin is contraindicated and digitalis failed in its effect. Formerly, H niger was also used as a drasticum, purgativum, emeticum, and anthelminticum. It was considered a drug for acute nephritis. In homeopathy, H niger is still very commonly used for meningitis, encephalitis, nephritis, epilepsy, heart failure, occipital pain, and dementia praecox. ³ A clear pharmacognostic identification of the drug Rhizoma (Radix) Hellebori nigri, which excludes other Helleborus species, is difficult. ¹² Thus, the supplement of the 6th edition of the German Pharmacopoeia ¹³ describes that the drug Rhizoma Hellebori originates from H niger L. and H viridis L. The largest single dose prescribed is 0.2 g, and the largest daily dose is 1.0 g. The average single dose as ingestion is 0.05 g, whereas the average content as snuff powder is 10%. In homeopathy, D2 to D6 dilutions are common. Even to this date, extracts of the rhizome and other subterranean parts of this plant are widely used in traditional medicine for a diverse range of symptoms and diseases.^8,12,14 H niger extracts are also applied in anthroposophic medicine, ¹⁵ and clinical uses include the concomitant treatment of oncological diseases. ¹⁶

Of the 22 species in the genus Helleborus, ¹⁷ especially H niger is pharmaceutically becoming more important, as recent preclinical evaluations showed that its extracts are safe and have potential for cancer treatment. Thus, in 2019, Felenda et al ¹⁸ published that H niger aqueous-fermented extract of the fresh whole flowering plant exerted antiangiogenetic effects in human umbilical vein endothelial cells (HUVECs) and antiproliferative effects in 5 human cancer cell lines: renal cancer cell (Caki-2), colon adenocarcinoma (DLD-1), gastric cancer (MKN-1), glioblastoma (LN229), and neuroblastoma (SK-N-SH). The mean IC₅₀ values amounted to 202-549 μg/mL in the Alamar Blue and to 176-584 μg/mL in the Soft Agar assays. The gastric cancer cell line MKN-1 responded most sensitively, and the IC₅₀ of brain tumor cell lines was approximately 2-fold higher. ¹⁸

This finding was supported by 2 other studies, which both found that the proliferation of tumor cells can be inhibited by H niger root and whole plant extracts.^19,20

Considering these promising findings and their importance regarding the development of new drugs, a precise knowledge of the constituents in the individual parts of the plant is essential. This review will summarize the constituents found in H niger by providing the historical background of their discovery, describing their synthesis and structure in detail, and outlining their current status of pharmacological testing.

Protoanemonin 1

In 1987, a high-performance liquid chromatography (HPLC) screening of H niger together with 28 representative species of the Ranunculaceae family confirmed that protoanemonin 1 was a constituent in all 29 species tested. The results indicated that 1 is a useful chemical marker in elucidating systematic relationships within this large plant family. ²¹ A quarter century later, a study of Clematis species (Ranunculaceae) showed that leaves contain more protoanemonin 1 than roots and stems. ²²

Indeed, protoanemonin 1 is the most important active natural ingredient in leaves, stems, and flowers of H niger, together with its dimer anemonin 2 and glycosides (-)-ranunculin 3 and (+)-ranuncoside 4. A common feature of these natural products is their γ-lactone ring (Figure 3, red).

Figure 3.

Chemical structures of protoanemonin 1, anemonin 2 (only 1 enantiomer shown), (−)-ranunculin 3, and (+)-ranuncoside 4. The common γ-lactone ring is highlighted in red color.

Historical Background

Freshly harvested herbs of the genus Anemone, Ranunculus, and Helleborus have a burning hot taste and vesicant effect caused by protoanemonin 1 (ranunculol, anemonol), the lactone of γ-hydroxyvinylacrylic acid. The history of the specific search of this biologically active substance was well documented by Beckurts in 1892. ²³ This comprehensive paper, entitled “Contribution to the knowledge of the anemonin,” particularly mentions that the steam distillate of Anemone pratensis L. loses its spicy taste after letting it sit for a long time and that crystals and a white amorphous powder form during this time. It was correctly suspected that the 2 substances, named “Anemonin” (anemonin) and “Anemoninsäure” (anemoninic acid), were not originally present in the plant but instead originated from a nonisolated pungent substance, a volatile oil. In 1914, Asahina ²⁴ isolated anemonin 2 from the fresh material of Ranunculus japonicus in crystalline form and found that it lacked the characteristic pungent-tasting properties. Six years later, Asahina and Fujita ²⁵ isolated the pungent-tasting oily substance from Ranunculus scleratus L. Because 2 molecules of this compound rapidly bind together and form the nontoxic anemonin 2, they named it protoanemonin 1. Two years later (1922), they published a summary on their entire work on anemonins. ²⁶

Isolation and Stabilization of Protoanemonin 1

In 1985, Bonora et al ²⁷ described the separation and quantification of protoanemonin 1 in the leaves of 10 taxa of the Ranunculaceae family. With a content of 5820.5 μg/g (w.w.) of protoanemonin 1, H niger had the second highest value after Ranunculus bulbosus (7765.6 μg/g) (see Table 1).

Table 1.

Content of Protoanemonin 1 in 10 Taxa of the Ranunculaceae Family. ²⁷

Taxa	Protoanemonina
Anemone nemorosa	333.3
Anemone trifolia albida	169.4
Clematis montana	417.7
Clematis recta	95.6
Clematis vitalba	150.0
Helleborus foetidus	672.0
Helleborus niger	5820.5
Helleborus viridis	28.4
Ranunculus bulbosus	7765.6
Ranunculus repens	125.7

The value for H niger is emphasized in bold.

^aAll data are referred in μg/g of w.w.

For the preparative extraction of protanemonin 1 from H niger, the method described for Ranunculus acer in the patent “Process for obtaining therapeutically useful, stable protoanemonine preparations” by Gaebelein ²⁸ in 1955 should be well suited, because this patent explicitly points out that it is generally applicable for preparative extractions from freshly harvested plants (see Supplemetal information). The procedure is also summarized in Figure 4.

Figure 4.

Flow diagram of the procedure for the isolation of protoanemonin 1 from plants developed and patented by Gaebelein. ²⁸

To prevent dimerization to anemonin 2, the obtained oily protoanemonin 1 was diluted with ethanol and stabilized by the addition of formic acid. The therapeutic efficacy of these alcoholic solutions hardly diminished even after 6 months of storage. Furthermore, the patent reports that often it may be appropriate to evaporate the stabilized alcoholic protoanemonin solutions to dryness and use the resulting dry product therapeutically. Gaebelein ²⁸ stated that protoanemonin solutions are only stable in the presence of certain acids such as aliphatic monobasic carboxylic acids (such as formic acid, acetic acid, and propionic acid) but preferably by polybasic hydroxyacids (malic acid, tartaric acid, and citric acid) and at certain acid concentrations.

Already 10 years earlier (1946), Shaw ²⁹ stated that the main source of potency loss of protoanemonin 1 is polymerization and that it can be inhibited by the addition of small amounts of hydroquinone at a pH range of 4.5 to 8. Under these conditions, protoanemonin solutions retained 80% of their potency after standing 3 weeks at room temperature.

Another protocol recommended a 0.2% solution of protoanemonin 1 in ethylene glycol and the addition of 0.005% of hydroquinone or propyl gallate for stabilization. ³⁰ This solution was stable for a few months at 5 °C, and the pH and antibacterial activities remained unchanged. Similarly, Grundmann et al ³¹ described that protoanemonin 1 was unchanged for several days after the addition of a small amount of hydroquinone and when stored in a refrigerator. Another similar protocol by Goldberg et al ³² recommended the addition of 1% to 10% of hydroquinone in dilute solutions of protoanemonin 1 in water, chloroform, or dichloromethane. A variety of further antioxidants to stabilize protoanemonin solutions have been patented. ³³

Mueller et al ³⁴ studied the stability of protoanemonin 1 in whole plant extracts of H niger and Pulsatilla vulgaris. They found that P vulgaris extracts contain protoanemonin in a more than 5 times higher concentration than H niger extracts (0.38575 vs 0.0662 mg/g). Due to the tendency to become ineffective by dimerization, their content of protoanemonin 1 in the fermented and filtered solutions decreased rapidly over time. Only low levels remained fairly constant over the years.

In summary, protoanemonin-derived antibiotic alternatives for human and veterinary treatment are possible as long as they are kept at nearly neutral pH and small amounts of a radical scavenger are added to prevent dimerization.

Syntheses and Structure of Protoanemonin 1

Asahina and Fujita ²⁵ published the first synthesis of protoanemonin 1 starting from levulinic acid. Subsequent syntheses proceeded also from levulinic acid ³⁵ or from acetylacrylic acid, ²⁹ α-angelicalactone,^30,31 and 2-deoxy-d-ribose ³⁶ or by oxidation reactions of furan derivatives.^32,37 A patent for a process to produce protoanemonin starting from ranunculin 3 exists as well. ³³

Of all the different approaches, the procedure of Kotera et al ³⁶ (see Scheme 1) is the simplest while also having a good yield. The last intermediate of this synthesis, lactone 7, was obtained in 58% yield as a crystalline and stable compound and is thus an ideal synthetic entry to protoanemonin 1. The 4 simple steps of the synthesis start with the glycosylation of commercially available 2-deoxy-d-ribose with methanol to glycoside 5, followed by acylation (5 → 6), oxidation (6 → 7), and elimination to protoanemonin 1 (overall yield 46%).

Scheme 1.

Practicable synthesis of protoanemonin 1 from 2-deoxy-D-ribose. ³⁶

Protoanemonin 1 is a viscous yellow liquid with the b. p. 89 °C to 90 °C, ²⁸ b. p. 40 °C to 43 °C/1 mm Hg, ³² b. p. 32 °C to 36 °C/0.1 mm Hg, and needles with melting point −5 °C. ³⁰ It should be handled with care because of its vesicant properties. ³¹

IR and 300 MHz ¹H and ¹³C NMR spectra of protoanemonin 1 have also been reported.^35,36

Biological Activities of Protoanemonin 1

H niger plants are avoided by grazing cattles because of the bitter taste of protanemonin 1 which is released in greater quantities from glycosidic precursors when the tissue is injured. Furthermore, it is very likely that in the Ranunculaceae family, protoanemonin 1 serves as protection against other organisms, especially fungi and bacteria.

Indeed, 1 has been described to possess antibiotic.^38–43 cytotoxic, ³⁵ and antimutagenic activity. ⁴⁴

In 1993, Didry et al ⁴¹ found promising antibacterial properties in microbiological studies. The minimum inhibitory concentration (MIC) values for aerobic bacteria (28 strains) ranged from 8 to 62.5 μg/mL. While the Gram-negative bacteria Citrobacter freudii, Providencia stuartii, and Pseudomonas aeruginosa required the highest concentrations, the lowest MIC value (8 μg/mL) was observed for a strain of Serratia marcescens isolated from a human clinical sample. With anaerobes (18 strains), MIC values ranged from 15.625 to 125 μg/mL. The lowest values (15.625 μg/mL) were obtained for Bacteroides oralis, Bifidobacterium bifidum, and Bifidobacterium longum. Twenty out of 22 combinations of protoanemonin 1 and antibiotics led to a partial synergism. A total synergism was observed with a combination of protoanemonin and cefamandole against a pathogenic strain of Staphylococcus aureus. The authors concluded that the ability of protoanemonin 1 to penetrate the microbial cell wall could explain the synergistic effect obtained by the combination of 1 and the antibiotic.

Shortly prior to the aforementioned study, Martin et al ⁴³ published MICs for Bacillus subtilis (10 μg/mL) and for Escherichia coli, Saccharomyces cerevisiae, Candida albicans, Yarrowia lipolytica, and Aspergillus niger (15 μg/mL) by an in vitro agar solution method. They suggested that RNA inhibition is the primary mode of activity of protanemonin 1 and, in support of this hypothesis, determined a median lethal dose (LD₅₀) value of 190 mg/kg in male albino mice for 1.

In 1920, Pañeda and Apan ³⁵ evaluated the antiproliferative activities of protoanemonin 1 against prostate adenocarcinoma (PC-3) and glioblastoma (U-251) cell lines, which were determined by using the protein-binding dye sulforhodamine B (SRB) in a microculture method. This assay is efficient and highly sensitive to measure drug-induced cytotoxicity. They found that, compared to cisplatin, synthesized protoanemonin 1 possessed higher cytotoxicity in the human cancer cell lines PC-3 and U-251 with determined IC₅₀ values of 4.1 and 5.3 µM, respectively. The observed significant antiproliferative effect of protoanemonin 1 is in support of an ethnomedical use of H niger and other Ranunculaceae plants as potential anticancer agents, and it may also be a lead for further studies on its mechanism of action in cancer cells. ³⁵

The biosynthesis of protoanemonin 1 is discussed at the end of section (+)-ranuncoside 4.

Anemonin 2

At room temperature, characteristic spontaneous dimerization of protoanemonin 1 to anemonin 2 occurs. The stereoselective formation of dimer 2 results from the stability of diradical 8 by head-to-head arrangement of 2 molecules of 1 (see Scheme 2). ⁴⁵ The underlying racemate possesses RR- and SS-configuration. Font et al ⁴⁶ tried to resolve the enantiomers, but their attempts failed.

Scheme 2.

Preparation of anemonin 2 from protoanemonin 1 by dimerization at room temperature. The radical intermediate 8 (see red dashed line) determines the trans-stereochemistry of 2 (only 1 enantiomer shown). ⁴⁵

Isolation and Synthesis of Anemonin 2

The procedure, described by Zechner and Wohlmuth ⁴⁷ for the isolation of anemonin 2 from Ranunculus acer, should also be applicable to H niger, as the content of protoanemonin 1 in H niger is relatively high and because protoanemonin 1 spontaneously dimerizes to anemonin 2 (see above).

For the synthesis of anemonin 2, all isolation and synthetic procedures of protoanemonin 1 (see above) can be used and allow keeping 1 at room temperature. A synthesis of anemonin 2 on a 3g scale, starting from α-angelicalactone, was described by Stamos et al. ⁴⁸ The photochemical dimerization ⁴⁹ and oligomerization ⁵⁰ of protoanenonin 1 have also been described.

In a recent paper, 400 MHz ¹H, ¹³C NMR, and HR-MS spectra of anemonin 2 were reported. ⁵¹ The crystal structure analysis showed that the molecule is in trans-configuration and that the cyclobutane ring is not planar but instead has a bent configuration. ⁵²

Reduction Products of Anemonin 2

Reduction reactions of anemonin 2 (see Scheme 3) were reported by Stamos et al. ⁴⁸ Hydrogenation of the 2 double bonds of 2 over Pd/C yielded tetrahydroanemonine 9 while sodium amalgam treatment furnished isotetrahydroanemonin 10, confirmed by X-ray structure analysis. Sodium borohydride reduction of anemonin 2 in methanol afforded diketo diester 12 and dilactone 13. ⁴⁸ Reduction of both lactone functions in 9 with lithium aluminum hydride gave the trans-configurated tetraol 11. ⁴⁹

Scheme 3.

Anemonin 2 reductions: (A) hydrogenation to tetrahydroanemonin 9 ⁴⁸ with subsequent hydride reduction to tetraol 11 ⁴⁹ and (B) sodium amalgam treatment to isotetrahydroanemonin 10 and boron hydride reduction to diester 12 and dilactone 13. ⁴⁸ Only 1 enantiomer is shown for 2, 9, 10, and 11.

Anemoninic Acid 15

Anemoninic acid 15 is the hydrolysis product of anemonin 2 and was first obtained by Beckurts ²³ after boiling anemonin 2 with semiconcentrated hydrochloric acid (20%). The reaction passes through the cyclobutane derivative 14 by splitting off both lactone rings (see Scheme 4).

Scheme 4.

Synthesis of anemoninic acid 15 by (A) acid hydrolysis of anemonin 2 via cyclobutane dicarbonic acid 14 ²³ and (B) aldol addition of furfural 16 and levulinic acid 17 to 5-furfurylidenelevulinic acid 18 followed by reduction of the enone double bond (→ 19) and bromine oxidation in water to aldehyde 20. The latter was directly oxidized in the presence of silver oxide to anemoninic acid 15. ⁵³

The structure of anemoninic acid 15 was confirmed by an independent synthesis performed by Fujita. ⁵³ Starting from 5-furfurylidenelevulinic acid 18, prepared by aldol addition of furfural 16 and levulinic acid 17, ⁵⁴ gave after reduction of the enone double bond the furanyl ketocarbonic acid 19. Subsequent bromine oxidation yielded the open-chain aldehyde 20 and further oxidation with silver oxide the anemoninic acid 15 (see Scheme 4). The obtained crystalline product was identical with that of Beckurts. ²³

Biological Activities of Anemonin 2

The first report on the biological activity of anemonin 2 was published in 1942 by Schmidt ⁵⁵ who reported that aqueous solutions of 0.2% anemonin 2, and even further dilutions, up to 1:12 500 and 1:25 000, were bacteriostatic and bacteriocidal for various pathogenic organisms, including diphtheria bacilli, staphylococci, and streptococci. Only much later the antimicrobial activities ⁵⁶ of anemonin 2 against Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Enterobacter cloacae, Staphylococcus aureus, and Micrococcus luteus were tested. Compound 2 had inhibitory activity against these bacteria; however, MICs were not disclosed.

The anti-inflammatory effects of anemonin 2 were confirmed in several papers^57–60 and one patent. ⁶¹ Thus, in 2008, Lee et al ⁵⁸ tested anemonin 2 with regard to its effect on NO production in lipopolysaccharide (LPS) production-activated macrophages. The data indicated that 2 was markedly potent which completely reversed NO production induced by LPS. The inhibitory effect was concentration-dependent, with an IC₅₀ value of 5.37 ± 0.39 μM/NO production (% of vehicle).

In view of diabetic nephropathy, a chronic complication of diabetes, a recent study showed that anemonin 2 significantly reduced blood glucose levels, evidenced by testing serum and urine of rats. ⁶² The inhibition of pigmentation synthesis in human melanocytes ⁶³ as well as in vivo antimalarial activity ⁵¹ was also described.

(−)-Ranunculin 3

Isolation of (−)-Ranunculin 3

In 1951, Hill and van Heyningen ⁶⁴ reported on the isolation of (−)-ranunculin 3 from 4 Ranunculaceae taxa by extraction in diluted nitric or hydrochloric acid. Besides determining the melting point, levorotation, acetylation, properties, and structure, they also proposed the name ranunculin, due to the presence of this compound in the Ranunculaceae family. Furthermore, they concluded that (−)-ranunculin 3 must be the precursor of protoanemonin 1.

Fifteen years later, the occurrence of (−)-ranunculin 3 in H niger was firstly described by Ruijgrok, ⁶⁵ concluding that (−)-ranunculin 3 is the most characteristic compound of the Ranunculaceae family. A ranunculin content of about 3% was calculated for fresh plants of H niger, which is a remarkably high concentration among this species-rich plant family.

The isolation of (−)-ranunculin 3 in crystalline form was later achieved by extraction from several Ranunculaceae species in diluted hydrochloric acid, ⁶⁶ ethanol, ⁶⁷ aqueous acetone, ⁶⁷ and methanol. ⁶⁸

Syntheses of (−)-Ranunculin 3

For the preparation of (−)-ranunculin 3 on a 200 mg scale, 2 protocols are described (see Scheme 5). Both syntheses start with a carbohydrate derivative and proceed via (S)-configurated hydroxymethylfuranon 28 in the last glycosylation step.

Scheme 5.

Chemical synthesis of (−)-ranunculin 3 by Camps et al ⁶⁹ and Fang et al. ⁷⁰

Camps et al ⁶⁹ used d-ribonolactone 21 as the starting material whose reaction with triethyl orthoformate gave 24, and the succeeding thermal elimination yielded 28. Fang et al ⁷⁰ based their synthesis on 2,3-O-isopropylidene-d-glyceraldehyde 23, which is readily accessible in 2 steps from d-mannitol 22. ⁷¹ Stereoselective Wittig reaction of 23 yielded the cis-configured ester 25. Acidic cleavage of the isopropylidene group provided the open-chain ester 26, which directly cyclized into the lactone 28. Koenigs–Knorr reaction conditions of 28 with acetobromoglucose 27 gave ranunculin tetraacetate 29, whose acidic saponification provided the natural product (−)-ranunculin 3. Diastereomeric mixtures of tetracetate 29 have also been published. ⁷²

Chemical Properties and Derivatives of (−)-Ranunculin 3

(−)-Ranunculin 3 is very stable in an acidic solution but is rapidly broken down to d-glucose and protoanemonin 1 in an alkaline solution. ⁶⁴ Distillation of 3 with aqueous sodium acetate gave a nearly quantitative yield of protoanemonin 1 and is thus a convenient stable starting material for the latter. ⁶⁴ Enzymatic cleavage of (−)-ranunculin 3 with almond emulsion (β-glucosidase) gave the aglycon (S)-5-(hydroxymethyl)furan-2(5H)-one 28.^73,74 Hydrogenation of 3 over palladium–charcoal resulted in dihydroranunculin 30, which crystallized as tetraacetate 31 ⁷³ (see Scheme 6).

Scheme 6.

Glycosidic cleavage of (−)-ranunculin 3 with the weak base sodium acetate to protoanemonin 1, ⁶⁴ enzymatic liberation of aglycon 28 with almond emulsion,^73,74 and hydrogenation to dihydroranunculin 30 and tetraacetate 31. ⁷³

The 4S-configuration of the aglycon was established by Benn and Yelland ⁷³ by chemical degradation reactions and in parallel by Boll ⁷⁴ using circular dichroism data. Later, 400 MHz ¹H and ¹³C NMR spectra as well as an X-ray structure analysis of (−)-ranuculin 3 were also published. ⁷⁵

Biological Activities of (−)-Ranunculin 3

In 1993, Li et al ⁷⁶ described the cytotoxicity and mechanism of activity of (−)-ranunculin 3 and suggested that its cytotoxicity in vitro may be due to inhibition of DNA polymerases and increase of oxygen free radicals. In a succeeding paper, ⁷⁷ they also published antimutagenic activity and metabolic transformation of 3 by rat liver microsomes. In 2004, biochemical data of the cytotoxic activity of (−)-ranunculin 3 against A549 (ED₅₀= 7.53 μg/mL), NIH3T (ED₅₀= 13.6 μg/mL), and SK-OV-3 (ED₅₀= 17.5 μg/mL) cell lines were also published. ⁷⁸

(+)-Ranuncoside 4

Isolation of (+)-Ranuncoside 4

In 1972, Tschesche et al ⁶⁶ described that the 2 toxic plants Ranunculus repens and Helleborus foetidus contain glycosides with lactone forming aglycon which displays considerable antibiotic properties. They extracted a novel crystalline compound with positive rotation from a pool of active substances and named it ranuncoside due to its origin from Ranunculaceae species. Parallel to this work and 1 year later, Martinek ⁷⁹ isolated a crystalline compound from the ethanol extract of dried stems, leaves, and flowers of H niger which had been collected in the Austrian Alps (South Carinthia). In a subsequent paper, ⁸⁰ the ingredient was identified as (+)-ranuncoside 4, due to its identical melting point and rotation with a sample provided by Tschesche. Cuny and Klingler ⁸¹ recently investigated the cultivated variant of H niger (Christmas Rose) for the first time, given it is cheap and easily accessible in large quantities from plant nurseries. Their laboratory extraction process is simple and efficient and can be scaled up for the provision of even larger amounts of (+)-ranuncoside 4. The further development to a “green” extraction process ⁸² should also be easily possible because the only required solvents are ethanol and water.

Confusingly, after the first naming by Tschesche et al, the name ranuncoside was later also used for 2 entirely different structures: (a) pentacyclic triterpenoid glycosides from Hydrocotyle ranuncoloides (Apiaceae) ⁸³ and (b) an olefinic glycoside from Ranunculus muricatus (Ranunculaceae) with unsolved stereochemistry in the hexose moiety. ⁸⁴

Synthesis of (+)-Ranuncoside 4

In 2022, Cuny et al ⁸⁵ described a concise synthesis of (+)-ranuncoside 4 (see Scheme 7), which is biomimetic, as it matches the biosynthetic studies by Tschesche et al. ⁸⁶

Scheme 7.

Synthesis of (+)-ranuncoside 4 by β-glycosylation of 5-hydroxylevulinic acid methyl ester 32 with acetobromo-α-D-glucose 33 to glycoside tetraacetate 34. Further deacetylation (34→35) followed by methyl ester saponification resulted, via 36 and ketalization, in the dioxane derivative 37. Finally, lactonization with p-toluenesulfonic acid gave (+)-ranuncoside 4 (overall yield 22%). ⁸⁵

The key step of their synthesis was the glycosylation of 5-hydroxylevulinic acid methyl ester 32 with acetobromo glucose 33 under Koenigs–Knorr conditions to the β-glycoside 34. Subsequent cleavage of the acetyl-protecting groups under Zémplen conditions yielded the free glucosyl ester 35. Its saponification resulted, via the intermediate 36, in the more stable dioxane derivative 37. The generation of the 5-membered butanolide ring and formation of the natural product (+)-ranuncoside 4 succeeded with p-toluenesulfonic acid (see Scheme 7).

Structure of (+)-Ranuncoside 4

(+)-Ranuncoside 4 is a spiro furan–dioxane–pyran tricycle with a complex substitution pattern and stereochemistry. The central dioxane ring is fused 2-fold with the pyranose moiety in trans-manner and is additionally linked to a saturated γ-lactone ring in a spiroacetal manner. In total, 6 stereogenic centers are present in the molecule. The spiro carbon has (R)-configuration, while the other configurations in the pyran portion originate from d-glucose.

Tschesche et al ⁶⁶ elucidated the structure of (+)-ranuncoside 4 by acetylation, trimethylsilylation, and subsequent analysis of the 90 MHz ¹H NMR spectrum of the triacetate. Recently, the structure was unambiguously confirmed by 500 MHz ¹H and ¹³C NMR spectra of underivatized natural product. ⁸¹

X-ray structure analysis clarified beyond any doubt the (R)-spiro configuration of (+)-ranuncoside 4.^81,87,88 As seen in Figure 5 by a side and top view, 2 hydrogen bond bridges in the crystal structure are present: (a) between the carbonyl oxygen atom of the 5-membered ring and 1 molecule of crystal water and (b) between the hydroxyl group at C-8′ and the O-1′-dioxan ring oxygen atom. Another remarkable feature is the spiroacetal attachment of the tetrahydrofuran ring with the axial arrangement of the oxygen atom to the trioxa decaline ring system (see structure 4a). Anomeric stabilization effects are responsible for this configuration and cause the (R)-configuration of (+)-ranuncoside 4 at the spiro center. Both 6-membered dioxan and pyran rings of the trioxa trans-decaline framework possess cyclohexan-type chair conformation. The β-d-glucose unit at the terminal right side has an all-equatorial arrangement of the substituents, as expected. The 5-membered furanoid ring is nearly planar and only slightly angled from the plane (see structure 4b).

Figure 5.

Perspective drawings of (+)-ranuncoside hydrate 4a and 4b as determined by X-ray crystallography analysis. The key characteristics are the 2 hydrogen bonds and the all-chair conformation of the trioxa trans-decaline ring system with a β-D-glucose unit (structure 4a). The 5-membered furanoid ring is only slightly angled from the plane (structure 4b). ⁸¹

(+)-Ranuncoside 4 has the same tricyclic spiro furan scaffold as the algal toxins (+)-okadaic acid 38 and the (+)-dinophysistoxins-1 and -2 (39 and 40). However, distinctive differences to the latter are the reversed stereochemistry at the spiro center and the additional oxygen atom in the central dioxane ring (see Figure 6).

Figure 6.

The plant glycoside (+)-ranuncoside 4, the marine algal toxins (+)-okadaic acid 38, and the (+)-dinophysistoxins-1 and -2 (39 and 40) are characterized by a tricyclic spiroactal domain (highlighted in red color).

The spiro acetal ring structure of (+)-ranuncoside 4 is particularly noteworthy because this molecular scaffold is responsible for the biological activity of a multitude of natural products.^89–91 Thus, efficient and stereo-controlled synthetic routes to analogous spiro domains have been published by Cuny.^92–94 Together with the synthesis of (+)-ranuncoside 4, these procedures enable the generation of candidates for structure–activity relationship studies which could lead to the discovery of possible novel antibiotics and potential selective anticancer agents.

Biosynthesis of Protoanemonin 1, (−)-Ranunculin 3 and (+)-Ranuncoside 4

The biosynthesis of protoanemonin 1, (−)-ranunculin 3, and (+)-ranuncoside 4 in Helleborus foetidus was described by Tschesche et al ⁸⁶ by in vivo incorporation experiments with radioactively labeled 5-hydroxy[1-¹⁴C] and [4-¹⁴C]levulinic acid (Scheme 8, highlighted in blue). The incorporation of radioactivity occurred in all 3 compounds. Acid hydrolysis of (+)-ranuncoside 4 led to labeled 5-hydroxylevulinic acid. These findings suggest that glucoside 36 which is formed from levulinic acid 41 could be the genuine precursor of all 3 compounds studied (see Scheme 8). Remarkably, the incorporation value of (+)-ranunculin 4 is 10 times and 5 times higher than that of (−)-ranunculin 3 and protoanemonin 1, respectively (see Table 2).

Scheme 8.

Presumed biosynthesis of protoanemonin 1, (−)-ranunculin 3, and (+)-ranuncoside 4 in H niger and other Ranunculaceae species as elaborated by Tschesche et al ⁸⁶ in Helleborus foetidus. The radioactively labeled 5-hydroxy[1-14C] and [4-14C]levulinic acid 41 is marked in blue color.

Table 2.

Incorporation Rate of 5-Hydroxy[1-¹⁴C]levulinic Acid into Protoanemonin 1, (−)-Ranunculin 3, and (+)-Ranuncoside 4. ⁸⁶

Metabolite	Incorporation %
Protoanemonin 1	0.66
(−)-Ranunculin 3	0.11
(+)-Ranuncoside 4	2.02

Since protoanemonin 1, (−)-ranunculin 3, and (+)-ranuncoside 4 also occur in H niger, it is very likely that the same biosynthetic pathway is also active in this plant species. To my knowledge, 2 other glucosides of the aforementioned study, ⁸⁶ named isoranunculin and ranunculoside, have not yet been found in any member of the plant kingdom.

Flavonoids (Phenolic Ingredients)

Flavonoids act as antioxidants and provide UV protection in the plant kingdom and are also found in H niger. Vitalini et al ⁹⁵ isolated 2 phenolic compounds from the leaves, the kaempferol 3-O-α-L-arabinopyranosyl-(1→2)-β-D-galactopyranoside-7-O-β-D-glucopyranoside 42 and quercetin 3-O-2-(E-caffeoyl)-α-L-arabinopyranosyl-(1→2)-β-D-glucopyranoside-7-O-β-D-glucopyranoside 43 together with glucosyl-phenyllactic acid 44 with undefined stereochemistry (see Figure 7). Isomeric compounds of 42 and 43 were also found in Ranunculus chinensis BGE. ⁹⁶

Figure 7.

Phenolic natural kaempferol and quercetin glycosides 42 and 43 and glucosyl–phenyllactic acid 44 isolated from the methanol extract of dried leaves of H niger. ⁹⁵

A comprehensive study of the phenolics and saponins from H niger leaves and stems by high-performance liquid chromatography trandem mass spectrometry (HPLC-MS/MS) characterized 7 quercetin and 8 kaempferol derivatives with a complex structure and unknown configuration in the attached sugar moieties (see Figure 8). ⁹⁷

Figure 8.

Quercetin and kaempferol derivatives in leaves and stems of H niger identified by HPLC-MS/MS measurements.

Furthermore, 3 quercetin and 5 kaempferol flavonols have been tentatively identified by the same strategy in the course of determining the sepal phenolic profile during H niger flower development. ⁹⁸

Bufadienolides

Bufadienolides are chemically and biologically related to the cardenolides and differ from them by the presence of a δ-lactone instead of a γ-lactone ring at C-atom 17 of the steroidal ring system (see, for example, structures 47 and 49 in Scheme 9). They have been found in 2 plant families, the Ranunculaceae (Helleborus species) and the Asparagaceae (Scilla and Bowiea species of the Scilloideae subfamily), as well as in the animal kingdom in the skin glands of toads (Bufonidae). The name bufadienolide was derived from the latter. ⁹⁹

Scheme 9.

Structure of hellebrin 45 ¹⁰⁷ and conversion to hellebrigenin 47 via deglucohellebrin 46. ¹⁰⁴ The acetate of hellebrigenin 48 was isolated by Nicolescu et al. ¹⁰³ The different lactone moieties of hellebrigenin 47 and strophanthidin 49 are emphasized in red color.

Isolation and Structure of Hellebrin 45

In 1943, the steroidal bufadienolide hellebrin 45 ((3β,5β)-3-[(6-deoxy-4-O-β-D-glucopyranosyl-α-L-mannopyranosyl)oxy]-5,14-dihydroxy-19-oxo-bufa-20,22-dienolide) (see Scheme 9), a potent cardiac diglycoside, was extracted by Karrer ¹⁰⁰ from the commercial drug Radix Hellebori nigri on a technical scale in the Scientific Laboratories of F. Hoffmann-La Roche & Co. AG at Basel, Switzerland. Interestingly, 30 years later, Wiessner and Kating ¹⁰¹ concluded that neither hellebrin 45 nor other bufadienolides could be detected in H niger by means of thin-layer chromatographic analysis. In contrast to those findings, pharmacological studies of Glombitza et al ¹⁰² not only revealed a positive inotropic effect of the ethanolic root extract of H niger but also found the 2 hellebrin degredation products deglucohellebrin 46 and hellebrigenin 47. Nicolescu et al ¹⁰³ addressed these contradictory results and also disproved Wiessner and Kating by publishing the extraction of hellebrigenin-3-acetate 48 from the rhizomes of H niger. To the best of our knowledge, Karrer's isolation protocol of hellebrin 45 has not been replicated and published, not even on a laboratory scale. The reason for this could be the rather elaborate extraction protocol.

The constitution of the aglycon, hellebrigenin (bufatolidin) 47 ((3β,5β)-3,5,14-trihydroxy-19-oxo-bufa-20,22-dienolide) was elucidated by Schmutz ¹⁰⁴ by enzymatic glycolysis of 45 with strophanthobiose to deglucohellebrin 46 ((3β,5β)-3-[(6-deoxy-α-L-mannopyranosyl)oxy]-5,14-dihydroxy-19-oxo-bufa-20,22-dienolide) and further acid-catalyzed cleavage of rhamnose to 47. An improved enzymatic 1-step hydrolysis of 45 to 47 was later published as well.^105,106 The complete structure and conformation of hellebrigenin 47 was solved by Muhr et al ¹⁰⁷ by means of 2D NMR spectra. The structural similarity of hellebrigenin 47 and strophanthidin 49, the aglycon of strophanthin cardenolide glycosides, is evidenced by the 5- and 6-membered lactone rings (see Scheme 9, highlighted in red color).

In the course of structurally elucidating hellebrigenin 47, Schmutz ¹⁰⁴ described some relevant hellebrigenin 47 derivatives (see Scheme 10). Acetylation of 47 with acetic anhydride in pyridine yielded the 3-acetate 48. As described above, 48 could also be isolated from the rhizome of H niger. Meerwein–Ponndorf–Verley reduction of acetate 48 with aluminum triisopropylate gave alcohol 50, and succeeding acetylation resulted in the diacetate 51. Standard oxidation of 48 with chromium(VI)-oxide furnished the corresponding carbonic acid 52 which could easily be transformed with etheric diazomethane solution to methyl ester 53.

Scheme 10.

Acetylation of hellebrigenin 47 to 3-acetate 48 and further reduction to the alcohol derivatives 50 and 51. Oxidation of 47 gave the carbonic acid 52 with its methyl ester 53. ¹⁰⁴

Cardiac Activity of Hellebrin Derivatives

The strong cardiac activity of hellebrin 45 (1 mg = 2500-3200 frog dose [FD]) is very similar to strophanthin and the digitalis glycosides, but it is less toxic. It has a positive inotropic effect and can be used to treat heart failure as a second-line agent when the usual cardiac glycosides are not effective. ³

The deglucohellebrin 46 is significantly more toxic than the diglycoside hellebrin 45 but less toxic than the aglycon hellebrigenin 47 and its monoacetate 48. This is particularly noteworthy because the aglycon has more potent cardiac activity than the corresponding glycoside. Strophanthidin 49, the aglycon of k-strophanthin, which differs from hellebrigenin 47 in the lactone ring, is comparatively low in its toxicity. The acetate 48, also isolated from H niger, ¹⁰³ is the most toxic one with an LD₅₀ of 0.064 mg/kg (cat) (see Table 3).^104,108

Table 3.

Median Lethal Dose of Hellebrin Derivatives.^104,108

Substance	LD50 (mg/kg) (cat)
Hellebrin 45	0.104
Deglucohellebrin 46	0.087
Hellebrigenin 47	0.077
Hellebrigenin-3-acetate 48	0.064
Strophanthidin 49	0.325

Recently Isolated Bufadienolides

In a recent paper, ¹⁰⁹ the 3 new bufadienolides 54 (3β-[(β-D-glucopyranosyl)oxy]-14β,16β-dihydroxy-19-oxo-5β-bufa-20,22-dienolide), 55 (14β,16β,19-trihydroxy-3-oxobufa-4,20,22-trienolide), and 56 (19-[(apiofuranosyl)oxy-14β,16β-dihydroxy-3-oxobufa-4,20,22-trienolide) were isolated from whole plants of H niger by extraction (4.5 kg) with methanol and succeeding 2-fold column chromatography (Diaion HP-20 and silica gel). The complex structures were elucidated by means of 600 MHz ¹H and ¹³C NMR spectroscopy including 2D spectra and nuclear Overhauser effect measurements. Their structural relationship with hellebrigenin 47 is marked in red color (see Figure 9), while their cytotoxic activities are summarized in Table 4.

Figure 9.

Structure of the bufadenolides 54, 55, and 56. ¹⁰⁹ Their structural difference compared to hellebrigenin 47 is marked in red color.

Table 4.

Cytotoxic Activities of Hellebrin and Hellebrigenin (45 and 47), ¹²¹ Bufadienolides 54-56, Ecdysteroids 57-61, Cisplatin, and Etoposide Against HL-60, A549, and SBC-3 Cancer Cells. ¹⁰⁹

	IC50 (μM)
	HL-60	A549	SBC-3
Hellebrin 45	- -	6	- -
Hellebrigenin 47	- -	3	- -
Bufadienolide glucoside 54	0.013	0.0055	0.0064
Bufadienolide 55	0.61	0.45	0.20
Bufadienolide 56	1.9	1.4	0.68
β-Ecdysone 57	1.7	1.3	1.7
β-Ecdysone isomer 58	0.40	0.27	0.46
Taxisterone 59	0.34	0.19	0.09
Stachysterone B 60	>20	>20	>20
Shidasterone 61	>20	>20	>20
Cisplatin	1.4	2.5	0.58
Etoposide	0.82	- -	0.22

Helleborëin and Helleborin

In 1865, Husemann and Marmé ¹¹⁰ detected the glucosidic constituents helleborëin and helleborin in the roots of H niger. They reported that helleborëin, a crystalline but not uniform substance, had a digitalis-like cardiac effect whereas helleborin was a crystalline strong narcoticum. Pure products were not obtained, and in order to avoid confusion, Karrer ¹⁰⁰ had introduced the new name hellebrin for the crystalline and highly cardioactive substance which he had isolated from Radix Hellebori nigri.

Ecdysteroids

Phytoecdysteroids occur in a wide variety of plants, and over 200 structures have been deduced so far. The biological significance of this extensive structural variety among phytoecdysteroids is currently not clear, but the numerous structural analogs are an ideal source for biochemical and physiological structure–activity studies.^111,112

From the roots and rhizome of H niger, Liedtke et al ¹¹³ isolated β-ecdysone (20-hydroxyecdysone) 57 (see Figure 10) on a 25 mg scale and confirmed the structure by ¹H and ¹³C NMR spectroscopy. The presence of 57 in the leaves and stems of H niger was confirmed by Duckstein and Stintzing ⁹⁷ by LC-MS measurements.

Figure 10.

Ecdysteroids isolated from H niger: β-ecdysone 57, its positional isomer 58, taxisterone 59, stachysterone B 60, and shidasterone 61. ¹⁰⁹ The structural difference of latter compounds to the parent compound β-ecdysone 57 is marked in red color.

Besides bufadienolides, Yokusaka et al ¹⁰⁹ also described the isolation and characterization of 5 ecdysteroids from H niger whole plants by the same procedures: (a) the new ecdysteroid 58 ((2β,3β,5β,16α)-2,3,14,16,20,25-hexahydroxycholest-7-en-6-one) and (b) the 4 known compounds 57 (β-ecdysone), 59 (taxisterone), 60 (stachysterone B), and 61 (shidasterone).

Their structural difference, compared to the parent compound β-ecdysone 57, is highlighted in Figure 10.

The cytotoxicities of the above ecdysteroids are listed in Table 4.

Steroid Saponins

Saponins are a group of phytochemicals found in more than 100 different plant families. They have a fungicidal effect which protects the plants from fungal infections. Chemically, they are glycosides with unpolar steroids or triterpenes as aglycones (sapogenins) and polar 1-3 sugar chains. Thus, they are well water-soluble and very surface-active foaming agents. Most studies support a role for saponins as phytoprotectants, but their function in regulating plant growth and development is also discussed. ¹¹⁴ The medical application of saponins as potential anticancer agents has been reviewed. ¹¹⁵

In 1971, Linde et al ¹¹⁶ first reported on the isolation of a steroidal sapogenin 62 (see Scheme 11) from the rhizome of H niger. They solved the structure by ¹H NMR spectroscopy and derivatization. Thus, acetylation of sapogenin 62 gave the triacetate 63 and isopropylidination with acetone and in the presence of an acid gave the acetonid 64, which was characterized as acetate 65. Oxidation of the hydroxyl group at C-3 of 64 with Kiliani mixture (CrO₃ in aqueous H₂SO₄) yielded the more stable α,β-unsaturated ketone 67 by double bond isomerization of 66. The latter compound could not be obtained in pure form and was not isolated (see Scheme 11). The described compounds, 62, 63, 64, 65, and 67, arose in crystalline form and had clearly defined melting points and negative rotational values.

Scheme 11.

Acetylation and diisopropylation of sapogenin 62 afforded the derivatives 63, 64, and 65. Oxidation of acetonid 64 furnished via 66 the enone 67. ¹¹⁶

More than 2 decades later, Liedke et al ¹¹³ isolated the saponin macranthoside I 68 (see Figure 11) on a 16 mg scale from the roots and rhizome of H niger. They determined the structure by ¹H and ¹³C NMR spectroscopic measurements. Duckstein and Stintzing ⁹⁷ also identified macranthoside I 68 together with a variety of saponins (steroidal glycosides) in leaves and stems using LC-MS/MS technology. After extraction of the leaves with aqueous acetone and removal of the acetone, chromatographic and mass spectrometric analysis was performed in the positive- and negative-ionization modes. With regard to the roots of H niger, they ¹¹⁷ determined additional 39 compounds, including 9 diosgenyl-type saponins, 5 diosgenyl-type saponins possessing an additional OH group, and 24 acetylated polyhydroxy saponins.

Figure 11.

Steroid saponins from H niger: (A) macranthoside I 68 ¹¹³ and (B) the hellebosaponins A and D (69 and 70). ¹¹⁸

The preparative isolation of the acetylated polyhydroxy saponins hellebosaponin A 69 (380 mg) and hellebosaponin D 70 (780 mg) by extraction of cut fresh roots (2.2 kg) of H niger and succeeding column chromatography have also been described by them ¹¹⁸ (for structures, see Figure 11). The structure elucidation was performed by 2D 500 MHz ¹H and ¹³C NMR spectra.

The steroid saponins, sapogenin 62, macranthoside I 68, hellebosaponin A 69, and hellebosaponin 70, which were isolated in pure form from the rhizome and roots of H niger are of particular interest for broad-based biological studies.

Comparative Cytotoxicity of H niger Steroids

The antitumor activities of bufadienolides and their possible role in cancer treatment have been described and reviewed.^119,120 Indeed, H niger extracts have been shown to possess in vitro cytotoxic activity against different tumor cells. Dose-dependent growth inhibition was found in lymphoblastic leukemia (MOLT4, IC₅₀ 171 μg/mL), myosarcoma (SK-UT-1b, IC₅₀ 304 μg/mL), and melanoma (HT-144, IC₅₀ 569 μg/mL) cells. ²⁰

Very recently, the cytotoxic properties of H niger extracts were compared to those from H foetidus and related to the specific saponin and protoanemonin concentrations. It was found that the extract of H foetidus was about 8 to 10 times more effective than the extract of H niger. ¹²² Specifically, MOLT4 proved to be the most sensitive tumor cell line, followed by SK-UT-1b and HT-144 (IC₅₀ 19.2, 33.4, and 72.1 μg/mL, respectively, for H foetidus extracts and IC₅₀ 186, 288, and 587 μg/mL, respectively, for H niger extracts).^122,123

Historically, hellebrigenin 3-acetate 48 isolated in 1968 from the bark of Bersama abyssinica (Melianthaceae)^105,106 and later from H niger ¹⁰³ was the first cardiotonic steroid recognized to show significant and reproducible inhibitory activity against an in vivo tumor system.^105,106

In 2010, Dewelle et al ¹²⁴ patented an invention for the synthesis of novel hellebrin and hellebrigenin derivatives (see Figure 12) for the development of novel medicals to treat cancer and tested a number of developed compounds which exhibited a pronounced cytotoxicity. The observed median IC₅₀ values are shown in Tables 5 and 6.

Figure 12.

Patented steroidal bufadienolides of Tables 5 and 6. ¹²⁴

Table 5.

Hellebrin Derivatives: Median IC₅₀ (nM) of Tumor Cell Lines U373 (Glioma), PC-3 (Prostata), A549 (Lung), MCF-7 (Breast), LoVo (Colon), and BxPC3 (Pancreas). ¹²⁴

Comp.	R1	R2	Median IC50 (nM)
45	-L-rha-D-glc	-CHO	30
71	-L-rha-D-glc	-CH=NOH	34
72	-L-rha-D-glc	-CH=N-NH-C6H5	254
73	-L-rha-D-glc	-CH=N-NH-C6H4-Me	87
74	-L-rha-D-glc	-CH=N-NH-C6H4-OMe	43
75	-L-rha-D-glc	-CH=N-NH-C6H4-F	182

Table 6.

Hellebrigenin Derivatives: Median IC₅₀ (nM) of Tumor Cell Lines U373 (Glioma), PC-3 (Prostata), A549 (Lung), MCF-7 (Breast), LoVo (Colon), and BxPC3 (Pancreas). ¹²⁴

Comp.	R1	R2	Median IC50 (nM)
47	-OH	-CHO	16
76	-OH	-CH=NOH	9
77	-OOC-(CH2)2-COOH	-CHO	126
78	-OOC-(CH2)3-COOH	-CHO	165
79	-OOC-CH2-CMe2-CH2-COOH	-CHO	246
80	-OOC-CH2-NH2	-CHO	9

Synthetic key steps of the described examples are the derivatization of hellebrin 45 (Table 5) and hellebrigenin 47 (Table 6) by oximation and hydrazonation of the aldehyde function at C-10 and by the esterification of the 3-OH group. The biological tests to determine the cytotoxic potency were carried out using the tumor cell lines U373 (glioma), PC-3 (prostata), A549 (lung), MCF-7 (breast), LoVo (colon), and BxPC3 (pancreas).

In the case of hellebrin 45 as the starting material, the cytotoxicity could not be improved, because the oxims and hydrozones (compounds 71-75) possessed slightly higher median IC₅₀ values than the parent compound (see Table 5). However, a noticeable improvement was achieved with hellebrigenin 47 as the parent compound, given that its oxim 76 and amino acid derivative 80 possessed half of the median IC₅₀ value. Other derivatives, compounds 77-79, possessed several-fold higher median IC₅₀ values than 47 (see Table 6).

In 2013, Banuls et al ¹²¹ reported that hellebrin 45 and its aglycon hellebrigenin 47 display similar in vitro growth inhibitory effects when compared to digoxin and other cardiotonic steroids. The IC₅₀ of 45 and 47 to inhibit growth of the human lung adenocarcinoma cell line A549 were reported to be 6 and 3 μM, respectively (see Table 4). Consistent with the studies by Dewelle et al^,123 the aglycon was twice as active than the underlying glycoside (Tables 5 and 6).

Yosuoka et al ¹⁰⁹ recently published that (a) the bufadenolides (hellebrin derivatives) 54, 55, and 56 and (b) the ecdysteroids 57, 58, and 59 showed cytotoxicity against HL-60 human leukemia cells, A549 human lung adenocarcinoma cells, and SBC-3 human small-cell lung cancer cells, with IC₅₀ values ranging from 0.0055 to 1.9 μM (see Table 4). Of these, bufadienolide monoglycoside 54 had the highest cytotoxic potency against all 3 cell lines (Table 4, highlighted in bold). All other H niger ingredients possessed IC₅₀ values comparable to cisplatin and etoposide. The 2 ecdysteroids stachysterone 60 and shidasterone 61 did not show any cytotoxicity effect at concentrations up to 20 μM.

Together, these studies show that using naturally occurring H niger steroids as parent compounds to create synthetic derivatives with improved bioactivity is a very promising approach toward the development of possible highly effective anticancer drugs. Furthermore, even natural compounds themselves, such as bufadienolide glucoside 54, are highly potent, underscoring the value of searching for bioactive compounds in medicinal plants.

Concluding Remarks

Today, over 36 000 medicinal plants have been pharmacologically tested. Of these, H niger, a cultivated and therefore cheap and readily available plant, recently moved into the focus of pharmacological and medical research. This is because recent preclinical evaluations showed that whole plant extracts are safe for medicinal use and might provide promising candidates for cancer treatment once the anticancer activity of such compounds has been clearly confirmed. Therefore, there is an increased necessity to improve our understanding of the biologically active ingredients in this old and proven medicinal herb and its pharmacological potential.

The active ingredients identified in H niger so far include glycosides and their aglycones belonging to (a) γ-lactone, (b) phenol, and (c) steroid groups.

The key compound of the aerial parts of H niger is the γ-lactone protoanemonin 1, accompanied by its dimer anemonin 2, glucoside precursor (−)-ranunculin 3, and spiroacetal (+)-ranuncoside 4. Protoanemonin 1 has antimicrobial and cytotoxic activity, and its extraction, synthesis, and stabilization are well documented. Anemonin 2 has antimicrobial, anti-inflammatory, and antimalarial activities. (−)-Ranunculin 3 possesses cytotoxic and antimutagenic activity and is an ideal source for the preparation of protoanemonin 1. (+)-Ranuncoside 4 has a spiroacetal ring framework, a moiety responsible for the biological activity of numerous natural products. For this reason, (+)-ranuncoside 4 is of particular interest for the generation of candidates for structure–activity relationship studies which could lead to the discovery of potential novel antibiotics and selective anticancer agents.

Phenolic substances found in the leaves of H niger are quercetin and kaempferol glycosides of the flavonoid group which act as antioxidants and provide UV protection.

Steroidal constituents of the whole plant are bufadienolides, ecdysteroids, and saponins. A key compound of the rhizome and roots is hellebrin 45, a potent cardiac diglycoside with cytotoxic activity. A number of recently isolated bufadienolides and ecdysteroids have cytotoxic activity comparable to cisplatin and etoposide. One of them, bufadienolide glucoside 54, significantly exceeds all others in its cytotoxic potency. Thus, 54 is of particular interest for structure–activity relationship studies and the development of novel cytostatica.

A large structural diversity of steroid saponins is found in H niger. Typical representatives from rhizome and roots are sapogenin 62, macranthoside I 68, and hellebosaponin A and D (69 and 70).

This review summarizes the current knowledge of the isolation, synthesis, structure elucidation, derivatization, and biological activities of the constituents of H niger. In addition, the targeted search and development of novel drugs, especially those for the treatment of cancer, are reviewed for the first time.

Methodology for the Literature Search

The literature described and cited in this article was compiled using the internet search engine SciFinder with the search terms “Helleborus niger,” “Black Hellebore,” “Christmas Rose,” “Protoanemonin,” “Anemonin,” “Ranunculin,” and “Hellebrin.” The cited literature covers a period from the year 1865 (reference 110) to February 2023 (reference 122). All cited books are from the author's personal library. Japanese publications from 1920 to 1953 (references 25, 26, and 30) were provided by Professors Toshio Nakagawa (formerly Yokohama City University) and Seiichiro Ogawa (formerly Keio University Yokohama), for which I express my sincere thanks.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X231201053 - Supplemental material for Bioactive Ingredients of Helleborus niger L. (Christmas Rose): The Renaissance of an Old Medicinal Herb—A Review

Supplemental material, sj-docx-1-npx-10.1177_1934578X231201053 for Bioactive Ingredients of Helleborus niger L. (Christmas Rose): The Renaissance of an Old Medicinal Herb—A Review by Eckehard Cuny in Natural Product Communications