The diterpenoid glycoside rubusoside (1) has been isolated from R. chingii and R. suavissimus leaves, and it is 115 times sweeter than sugar (Fig. 1). The aforementioned compound is a major diterpenoid glycoside in the R. suavissimus leaves, along with other kaurane-type diterpenoid glycosides (10–20) (Chaturvedula et al. 2012). The rubusoside’s aglycone is the diterpenoid ent-13-hydroxykaur-16-ene-19-oic acid, known as steviol. More than 15 diterpenoid glycosides have been isolated from the R. chingii leaves, mainly labdane-type goshonosides (2–8) (Yu et al. 2019) and kaurane-type (9–20) (Zhang et al. 2017).

Figure 1. 

Structure of diterpenoids and triterpenoids from the Rubus leaves.

Pentacyclic triterpenoids of oleanane- and ursane-types have been reported (Table 1, Fig. 1). Exclusively in R. moluccanus aerial parts, the oleanane-type analogs rubonic (21) and rubusic acids (22) have been isolated (Patel et al. 2004). Ursolic acid (29) was accompanied by its various derivatives: 3-oxo analog 7 α-hydroxy-3-oxo-12-ursen-28-oic (rubinic) acid (25) as well as 3, 19-dihydroxy and 2, 3, 19-trihydroxy analogs of usrolic acid: pomolic (31), euscaphic (30), and tormentic acid (28) (Patel et al. 2004; Haq et al. 2014; Yu et al. 2019). Taking into consideration the stereoisomerism (α/β mainly at C-3), pairs of isomers were observed among the triterpenoid acids.

Triterpenoid saponins have been isolated from the aerial parts of Rubus taxa, including 28-glucosyl esters of triterpenoid acids, mainly of the ursane-type mono-, di-, tri-, and tetrahydroxyurs-12-ene-28 acids (Table 1). Their analogs with an additional double bond at C-18 (di- and trihydroxyurs-12, 18-diene-28-oic acid) were found, as well as derivatives with a supplementary carboxyl group (2α, 3β, 19α-trihydroxyurs-12-ene-23, 28-dicarboxylic acid). Among them were suavissimoside R1 (35) and niga-ichinoside F1 (36) (Table 1). An ursane-type triterpenoid saponin 2α, 3α, 19α, 24 tetrahydroxyurs-12-ene-28-carboxylic acid-28-O-β-glucopyranosyl ester (37) was isolated from R. sanctus (Tuzlacı and Gürkan 2004). A series of triterpenoid acids along with their hexosyl esters were tentatively identified in R. sanctus and R. ibericus leaves by LC-Orbitrap-HRMS (Zengin et al. 2019). Thus, triterpenoid acids were ascribed to trihydroxyurs-12-en-28 carboxylic acid and tetrahydroxyurs-12-en-28 carboxylic acid, accompanied by analogs with an additional carboxylic group: dihydroxyurs-12-en-23, 28 dicarboxylic acid, and trihydroxyurs-12-en-23, 28 dicarboxylic acid. It should be noted the presence of isobaric pairs assigned as either urs-12-en or olean-12-en possessing α/β С-3 hydroxyl group (Zengin et al. 2019), as was observed in R. crataegifolius and R. aleaefolius leaves (Nam et al. 2007; Guo et al. 2013).

Rubus species are especially rich sources of ellagitannins (ET) and ellagic acid (57) conjugates (Macierzynski et al. 2020). ET are hydrolysable tannins, esters of hexahydroxydiphenic acid (HHDP) with a polyol, usually glucose (Pouysegu et al. 2011). According to Okuda’s classification, ET from the Rubus genus refers to the type II hydrolysable tannins containing HHDP (Okuda et al. 2000). The characteristic monomer of Rubus ET is galloyl-bis-HHDP glucoside (61). Oligomeric ET sanguine H-6 (56) (with 2 galloyl-bis-HHDP-glucose units) and lambertianin C (51) (with 3 galloyl-bis-HHDP-glucose units) were among the first ETs to be thoroughly characterized (Tanaka et al. 1993). ET, together with ellagitannin monomers and EA (57), were commonly found in the leaves of Rubus species (Table 1). The sugar esters 3, 6-di-O-caffeoyl-glucose, and 1-O-caffeoyl-hexose, together with the ET 2,3-HHDP-glucose, bis-2,3,4,6-HHDP-glucose (pedunculagin) (53) and 2,3-O-HHDP-4,6-O-sanguisorbyl-glucose, were isolated from an hydroalcoholic extract of R. sanctus aerial parts (Hussein et al. 2003). In addition, sanguine-10 isomers were annotated by LC-HRMS in both R. sanctus and R. ibericus leaves for the first time, along with bis-HHDP-hexose (unpublished data of the authors, R.G.).

The study of R. grandifolius leaves showed that they consist mainly of ellagitannins (44–49%) (Spinola et al. 2019), which correlates with the LC-QTOF/MS analysis of 26 wild Rubus species (Oszmiański et al. 2015). In the latter study, the total content of phenolic compounds varied from 83 mg/g dry weight (dw) (R. austroslovacus) to 334.24 mg/g (R. perrobustus). The highest amount was found for the ET, from 51.59 mg/g dw (R. austroslovacus) to 255.01 mg/g in R. wimmerianus (the average value in the studied species was 165.84 mg/g) (Oszmiański et al. 2015). Thus, ETs were the predominant compounds in the leaf extracts. Raspberry leaves are particularly rich in ET and gallotannins, ranging from 2.6% to 6.9% (w/w), and the main compound is ellagic acid (up to 2.53%) (Gudej and Tomczyk 2004).

Among the ET, the dimers sanguine H-6 (56), H-10, and the trimers lambertianin D (52) and C (51), as well as methyl gallate, have been identified (Ferlemi and Lamari 2016). Brevifolin derivatives, including brevifolincarboxilic acid and methylbrevifolin, have been annotated in the R. sanctus and R. ibericus leaves (unpublished data of the authors, R.G.). Brevifolin analogs have been rarely discovered in Rubus taxa, i.e., Rubus idaeus var. reveille leaves (Yang et al. 2020), R. caesius (Grochowski et al. 2020), and R. saxatilis (Gudej et al. 1998). The biogenesis of brevifolincarboxilic acid was related to the transformation of DHHDP (dehydrohexahydoxydiphenoyl) residue (Pouysegu et al. 2011).

Raspberry and blackberry leaves contain a variety of phenolic compounds, including hydroxybenzoic and hydroxycinnamic acids and their derivatives, flavonols, flavanols, anthocyanins, ET, and proanthocyanidins (Ferlemi and Lamari 2016).

Raspberry leaves are an especially rich source of flavonols such as kaempferol (Km) (62), quercetin (Qu) (63), and their respective glycosides, achieving up to 1.05% (Gudej and Tomczyk 2004). R. grandifolius leaves contained flavonols (12–16%), flavanols (2–10%), and flavones (0.28–0.32%) (Spinola et al. 2019). The content of flavonols and flavones including Qu (63), Km (62), luteolin (Lu) (108) and apigenin (Api) (105) derivatives has been estimated from 8.68 mg/g in the leaves of R. macrophyllus to 61.27 mg/g in R. crispomarginatus (average value in the studied species as 35.17 mg/g) (Oszmiański et al. 2015). The distribution of flavonoids showed that only 7 flavonoids (out of 17) were found in all assayed species in the aforementioned study. Rarely occur Km-3-O-rhamnosylglucoside-7-O-rhamnoside (in 9 of 26 species); Qu-3-[6’’-(3-hydroxy-3-methylglutaryl)-galactoside] (89) (13 species); and Qu 3-O-pentoside (84) in 14 species. Km 3-O-glucuronide (71) (average content 9.23 mg/g) and Qu 3-O-glucuronide (miquelianin) (82) (7.00 mg/g) are in the greatest quantity.

Gudej and Tomczyk (2004) established the highest content of aglycones after hydrolysis in the R. nessensis leaves (1.06% dw) and the lowest in the R. fruticosus leaves (0.34% dw) (HPLC). Generally, the quantity of flavonoids in the R. idaeus leaves is significantly higher than that in the fruits, where flavonoids present a small extent of the bioactive compounds; leaf flavonoids range from 0.46% to 1.05% (w/w) (Ferlemi and Lamari 2016).

Rutin (83) was the most abundant flavonol, ranging between 157.25 and 585.89 mg/kg in the leaves of raspberry and blackberry cultivars (Pavlovic et al. 2016). Among the flavan-3-ols, a higher content of catechin (98) has been assessed in the raspberry leaves (up to 1913 mg/kg) compared to the blackberry leaves (739 mg/kg). The results from the aforementioned study suggest that the cultivar “Willamette” leaves, especially rich in flavan-3-ols, could be used as a substitute for Camelia sinensis tea (Pavlovic et al. 2016).

Galloyl esters of flavonols were isolated from the hydroalcoholic extract of R. sanctus leaves: Km-, Qu-, and myricetin-3-O-(6”-galloyl)-β-D-galactopyranoside (Badr et al. 2009). Coumaroylhexoside derivatives of flavonols are present in Rubus leaves, including Qu and Km 3-O-(6”-O-p-coumaroyl)-glucoside (tiliroside) (69) (Pavlovic et al. 2016). In the study of 22 accessions of Bulgarian raspberry germplasm collection, the highest content of isoquercitrin (88), tiliroside (69), and hyperoside (81) was assessed in the Bulgarian elite clones: 1.70, 0.60, and 0.97 mg/kg, respectively (Gevrenova et al. 2013). Despite a great variety of flavonoids annotated in R. sanctus and R. ibericus leaves by LC-HRMS, the profiles are dominated by Qu- and Km-hexuronide and tiliroside (69) (Zengin et al. 2019), as was seen in wild species and cultivars (Guo et al. 2013; Oszmianski et al. 2015; Pavlovic et al. 2016; Grochowski et al. 2018, 2020). Both flavonols, miquelianin (82) and tiliroside (69), are considered chemophenetic markers in the Rubus genus (Grochowski et al. 2020).

Derivatives of caffeic, p-coumaric, and ellagic acid were typical for the Rubus leaves, with an average content of 28.74 mg/g (Ferlemi and Lamari 2016). EA (57) was found to be the main phenolic acid in the leaves of raspberry and blackberry cultivars, ranging between 1574 and 2875 mg/kg dw (Pavlovic et al 2016). The leaves of the wild Rubus species were especially rich in p-coumaric acid (132) and neochlorogenic acid (140) (Oszmiański et al. 2015). The leaves of raspberry cultivars possessed a higher quantity of chlorogenic (139) and p-hydroxybenzoic acid (117) (up to 169.74 and 63.73 mg/kg dw, respectively) in comparison with the blackberry accessions (Pavlovic et al. 2016). Caffeic acid (126) was the predominant phenolic acid in the leaves of a Bulgarian raspberry germplasm collection, being up to 1.43 mg/g dw in a Bulgarian elite clone (Gevrenova et al. 2013), while in other studies of raspberry leaves, a significantly lower content of caffeic acid (126) was reported (0.55 mg/g dw) (Ferlemi and Lamari 2016). In-depth studies of the phenolic compounds by LC-HRMS revealed a variety of phenolic acids and derivatives in the Rubus leaf extracts (Pavlovic et al. 2016; Zengin et al. 2019). Thus, numerous hydroxycinnamic acid esters with hexaric, quinic, tartaric, and treonic acids were annotated. Accordingly, a series of acylquinic acids were tentatively identified in R. sanctus and R. ibericus leaves, dominated by monoacylquinic acids (neochlorogenic (140), 4-caffeoylquinic (146), and 3-p-coumaroylquinic acid (151), and diacylquinic acids (3,5- and 4,5-dicaffeoylquinic acid (149 and 150)) (Zengin et al. 2019). In this study, the feruloylquinic and feruloylcaffeoylquinic acids were minor compounds.

The waxes form a microcrystalline layer on the leaf surface and include long-chain esters (R. idaeus); in the young leaves, there are more C23 and C25 homologues and less C29. The main components are primary alcohols, monounsaturated alcohols C24-C34, their acetates, long-chain saturated esters C36-C54, and fatty acids C12-C32 (Patel et al. 2004).

A comparison of the antibacterial activity of different plant parts of R. fruticosus on a panel of bacterial strains (Table 3) shows the following order of activity based on the minimum inhibitory concentration: stem > root > leaves > fruits (Riaz et al. 2011). No activity was found on strains of pathogenic fungi. The preparations obtained from the leaves of R. idaeus L. and R. fruticosus L. could complement classical antibiotics.

In the model of insulin-independent diabetes, water and butanol fractions of 70% alcoholic extract of R. fruticosus leaves restored blood glucose levels (Zia-Ul-Haq et al. 2014). A hypoglycemic effect was observed in normal rats, indicating that regulatory mechanisms cannot rapidly normalize glucose levels (Jouad et al. 2002). Aqueous extracts of the leaves also have an anti-hyperglycemic effect, which can be associated to a certain extent with the content of chromium and zinc. At 5 g/kg daily, the aqueous extract reduced hyperglycemia by 50% in an alloxan-induced diabetes model in rabbits (Zia-Ul-Haq et al. 2014).

In a comparative study of the inhibitory effects of methanol extracts of R. grandifolius leaves and fruits towards α-glucosidase and α-amylase, the leaves showed higher activity with an IC₅₀ of 0.11-0.15 mg/ml compared to that of fruits (0.61–0.68 mg/ml) (positive control acarbose) (Spínola et al. 2017). The assays were performed with α-glucosidase from brewer’s yeast and rats, and the extracts were less active on the mammalian enzyme; the IC₅₀ was about 9 times higher. The trend toward higher activity of the leaf extracts was also observed in the inhibition of α-amylase and pancreatic lipase. Blood sugar control with plant polyphenols is associated with their ability to bind to proteins and therefore modulate glucose breakdown by inhibiting enzymes (Edirisinghe and Burton-Freeman 2016). Although inhibition of digestive enzymes is a therapeutic approach in the treatment of diabetes and obesity, strong inhibition of α-amylase is not recommended. Based on that, extracts with a weak inhibitory effect on α-amylase and a strong effect on α-glucosidase are most suitable to avoid excessive breakdown of undigested starch in the colon, which is associated with abdominal pain and flatulence. Another study revealed R. sanctus aqueous extract to be a promising candidate, showing low α-amylase inhibition and prominent α-glucosidase inhibitory activity (Zengin et al. 2019). R. grandifolius leaf extracts also revealed higher inhibitory activity than fruits on aldose reductase (the first enzyme of the polyol pathway), as well as more pronounced anti-glycation activity (Spínola et al. 2017).

The antioxidant potential of Rubus species fruits and leaves has been evaluated (Oszmiański et al. 2015; Pavlovic et al. 2016; Spínola et al. 2017; Zengin et al. 2019). Generally, leaves have higher activity than fruits in many of the assays, and the results could be related to the phenolic composition patterns (Spínola et al. 2017). Accordingly, the total content of phenolic compounds in R. grandifolius (HPLC-DAD) was 92.96–97.47 mg/g dry extract (berries) and 118.01–137.41 mg/g (leaves). Antioxidant activity was correlated with the total phenols (r ≥ 0.929), as it was found for different blackberry species (Oszmiański et al. 2015). Flavonols have the main contribution to the antioxidant effect (r ≥ 0.947), followed by hydroxycinnamic acids (r ≥ 0.943) and ellagitannins (r ≥ 0.884). The content of ellagitannins and anthocyanins (in the fruits) is thought to play a role in the observed effects.

The in vitro antioxidant activity of ethanolic and aqueous extracts of leaves of R. ibericus was investigated by different assays (DPPH, ABTS, H₂O₂, O², metal chelating activity, reducing power, and inhibition of lipid peroxidation) (Keser et al. 2015). Despite the lower total flavonoids in R. ibericus leaves originating from Serbia (35.63 and 24.49 mg QE/g extract in aqueous and ethanol extracts, respectively, versus 74.54 and 92.75 QE/g in the aforementioned study), high radical scavenging activity and reducing power were observed (Velickovic et al. 2015).

Another study revealed the importance of solvent selection in the sample preparation of R. sanctus and R. ibericus extracts (Zengin et al. 2019). Thus, the aqueous extracts manifested the highest radical scavenging activity (DPPH and ABTS), reducing power (FRAP and CUPRAC), and metal chelating capacity. According to sPLS-DA analysis, the main parameters for the extracts differentiation were total flavonoids, metal chelating activity, and antioxidant potential in the phospho-molibdenum assay. It should be noted that ethylacetate and methanol extracts from R. sanctus and R. ibericus leaves inhibited, to the highest extent, acetyl- and butyrylcholinesterase, tyrosinase, α-glucosidase, and α-amylase.

In a model of CCl₄-induced hepatotoxicity in isolated hepatocytes, the methanol-aqueous extract of R. sanctus (100 mg/ml) showed hepatoprotective activity: it restored the levels of the antioxidant marker GSH, the enzymes lactate dehydrogenase (LDH) by 40%, alanine aminotransferase (ALAT) by 30%, and aspartate aminotransferase (ASAT) by 20% compared to the CCl₄-treated group (Badr et al. 2009). Antioxidant effects were found regarding lipid peroxidation and TBARS (thiobarbituric acid reactive substances) formation in isolated hepatocytes.

In a comparative neuropharmacological study of methanol extracts from different plant parts of R. fruticosus, the following order of activity on the CNS was found: fruits > roots > leaves > stems (Riaz et al. 2014). All extracts have an anxiolytic effect without observing sedative muscle relaxing effects.

The flavonol glucoside tiliroside (R. chingii) at a concentration of 100 μg/ml revealed the highest inhibitory activity of the assayed flavonoids on NO formation in LPS-stimulated RAW 264.7 macrophages, which was very close to the effect of dexamethasone (50 μg/ml). Leaf and fruit polysaccharides also produced a dose-dependent inhibition of NO uptake (2–400 μg/ml) in the same macrophage cell line by suppressing TNF-α, iNOS, and IL-6 gene expression (Zhang et al. 2015).

Polar and butanol fractions of an extract from R. sanctus aerial parts exerted anti-inflammatory activity in a carrageenan-induced inflammation model. Side effects related to irritation of the gastric mucosa have also been reported (Akcos et al. 1998). In a further study, ethanol and aqueous extracts of R. sanctus (aerial parts) revealed antinociceptive activity against p-benzoquinone-induced abdominal contraction in mice. The extracts have anti-inflammatory activity in a carrageenan-induced inflammation model (Erdemoglu et al. 2003).

Another study on R. sanctus methanol extract showed anti-inflammatory activity in an experimental model of ulcerative colitis, revealing significant blunting effects on LPS-induced levels of markers of oxidative stress and tissue damage such as nitrites, MDA, and LDH. Besides, R. sanctus methanol extract displayed a significant inhibition of spontaneous migration of human colon cancer cells HCT116, thus suggesting a potential protective effect against migration and invasion capacities of human colon cancer (Zengin et al. 2019). Ethyl acetate extracts from R. sanctus reduced the LPS-induced serotonin (5-hydroxytriptamine) steady state levels, which play a pro-inflammatory role in ulcerative colitis.

Different polarity extracts of R. sanctus aerial parts (hexane, chloroform, ethyl acetate, and methanol extract) were assayed in two wound healing models in rats: an incisional and an excisional wound model (Suntar et al. 2015). The 1% methanol extract has the highest activity. The mechanism of action included the formation of fibroblasts and collagen fibers in the granulation tissue and epithelization. The effects could be attributed to flavonoids and ellagitannins. The viability of collagen fibrils was increased by inhibiting lipid peroxidation (Suntar et al. 2015).

Few data exist on the safety of Rubus extracts. In an acute toxicity test of R. chingii leaves, a dose of 20 mg/kg/day did not induce toxicity for 2 weeks (Yu et al. 2019). Over 90 days, oral doses of 2.5, 5, and 10 g/kg produced no significant differences in body weight, blood biochemical parameters, pathology, or histopathology. Mutagenic and genotoxic effects have not been established.

Based on the traditional use of raspberry leaves to relieve labor pains, Jing et al. (2010) studied the effects in rats and reported that leaf extracts did not affect oxytocin-induced contacts and lacked a direct effect on uterine contact. In a study of 150 pregnant women, no significant differences were found between groups in terms of blood loss, blood pressure, length of pregnancy, ease of labor, or relief of labor pains (Simpson 2001). A recent review on using raspberry leaf in pregnancy to facilitate labor revealed that there is a lack of evidence to inform the practice (Bowman et al. 2021).

The discussed Rubus species afford a rich source of ellagitannins and ellagic acid conjugates, flavan-3-ols and flavonols, di- and triterpenoids, phenolics, and acylquinic acids. Accordingly, their health-promoting benefits, including an astringent effect, oxidative stress prevention, and inhibition of key enzymes in neurodegenerative and metabolic conditions, have been attributed to the ellagitannin monomers and oligomers, catechin, miquelianin, tiliroside, oleanane-, and ursane-type triterpenoids.