Genuine and Sequestered Natural Products from the Genus Orobanche (Orobanchaceae, Lamiales)

The present review gives an overview about natural products from the holoparasitic genus Orobanche (Orobanchaceae). We cover both genuine natural products as well as compounds sequestered by Orobanche taxa from their host plants. However, the distinction between these two categories is not always easy. In cases where the respective authors had not indicated the opposite, all compounds detected in Orobanche taxa were regarded as genuine Orobanche natural products. From the about 200 species of Orobanche s.l. (i.e., including Phelipanche) known worldwide, only 26 species have so far been investigated phytochemically (22 Orobanche and four Phelipanche species), from 17 Orobanche and three Phelipanche species defined natural products (and not only natural product classes) have been reported. For two species of Orobanche and one of Phelipanche dedicated studies have been performed to analyze the phenomenon of natural product sequestration by parasitic plants from their host plants. In total, 70 presumably genuine natural products and 19 sequestered natural products have been described from Orobanche s.l.; these form the basis of 140 chemosystematic records (natural product reports per taxon). Bioactivities described for Orobanche s.l. extracts and natural products isolated from Orobanche species include in addition to antioxidative and anti-inflammatory effects, e.g., analgesic, antifungal and antibacterial activities, inhibition of amyloid β aggregation, memory enhancing effects as well as anti-hypertensive effects, inhibition of blood platelet aggregation, and diuretic effects. Moreover, muscle relaxant and anti-spasmodic effects as well as anti-photoaging effects have been described.

Orobanche caryophyllacea Sm.-The tropone derivative orobanone (45) was isolated from O. caryophyllacea (using the synonym O. major L.) by Fruchier et al. [whole plant; water, chloroform; IR, UV, MS, CI-MS, 1 H and 13 C NMR] [no information about the host] [12].  whole plant] [host species not mentioned] but no further specification of structures nor of any analytical methods were indicated in the report [15].
Orobanche pubescens dÚrv.-Aynilian et al. screened several Orobanche species for their contents of alkaloids, tannins, and saponins, without investigating any particular structures of the metabolites. Tannins were found in O. pubescens (using the synonym O. versicolor F.W.Schultz) [plant material obtained from The Post Herbarium of the American University of Beirut, Lebanon; petroleum benzine (defatting), ethanol 95%, ethanol 80%] [40]. Orobanche owerinii Beck-Dzhumyrko and Sergeeva detected several carotenoids in O. owerinii [epigeal parts; n-hexane, petrol ether; co-chromatography with reference compounds and UV spectroscopy] growing on the hypogeal organs of Fraxinus [47]. Violaxanthin, auroxanthin, the ester of violaxanthin and palmitic acid, as well as αand βcarotenes were detected.

Reports Describing the Investigation of more than One Species
Besides investigating the phytochemical composition of one species there are also several reports dealing with more than one species. Serafini et al. investigated the secondary metabolite contents of several Orobanche species [Sardinia, Italy; flowering samples; alcoholic extract; 1 H and 13 C NMR, HPLC, co-elution of extracts with isolated and identified phenylpropanoid glycosides] [host species not mentioned] [14].

Reports Describing the Investigation of more than One Species
Besides investigating the phytochemical composition of one species there are also several reports dealing with more than one species. Serafini et al. investigated the secondary metabolite contents of several Orobanche species [Sardinia, Italy; flowering samples; alcoholic extract; 1 H and 13 C NMR, HPLC, co-elution of extracts with isolated and identified phenylpropanoid glycosides] [host species not mentioned] [14].

Reports Describing the Investigation of more than One Species
Besides investigating the phytochemical composition of one species there are also several reports dealing with more than one species. Serafini et al. investigated the secondary metabolite contents of several Orobanche species [Sardinia, Italy; flowering samples; alcoholic extract; 1 H and 13 C NMR, HPLC, co-elution of extracts with isolated and identified phenylpropanoid glycosides] [host species not mentioned] [14].

Reports Describing the Investigation of more than One Species
Besides investigating the phytochemical composition of one species there are also several reports dealing with more than one species. Serafini et al. investigated the secondary metabolite contents of several Orobanche species [Sardinia, Italy; flowering samples; alcoholic extract; 1 H and 13 C NMR, HPLC, co-elution of extracts with isolated and identified phenylpropanoid glycosides] [host species not mentioned] [14].

Reports Describing the Investigation of more than One Species
Besides investigating the phytochemical composition of one species there are also several reports dealing with more than one species. Serafini et al. investigated the secondary metabolite contents of several Orobanche species [Sardinia, Italy; flowering samples; alcoholic extract; 1 H and 13 C NMR, HPLC, co-elution of extracts with isolated and identified phenylpropanoid glycosides] [host species not mentioned] [14].

Reports Describing the Investigation of more than One Species
Besides investigating the phytochemical composition of one species there are also several reports dealing with more than one species. Serafini et al. investigated the secondary metabolite contents of several Orobanche species [Sardinia, Italy; flowering samples; alcoholic extract; 1 H and 13 C NMR, HPLC, co-elution of extracts with isolated and identified phenylpropanoid glycosides] [host species not mentioned] [14].

Sequestration of Secondary Metabolites by Orobanche s.l. from Their Host Species
As obligate holoparasites Orobanche species drain water and essential nutrients from their host plants. Also the sequestration bioactive natural products from the hosts seems likely [62], but up to now little is known about the uptake of other substances by holoparasitic Orobanche species from the plants they parasitize on and only very few reports deal with the sequestration of secondary metabolites from hosts. Orobanche hederae Duby-Sequestration of minerals and fatty acids by Orobanche hederae from its host Hedera helix was described by Lotti and Paradossi [Tuscany, Italy; whole plants; petrol ether, soxhlet; GC [63,64]. Sareedenchai and Zidorn reported the uptake of polyacetylenes falcarinol (S15), 11,12-dehydrofalcarinol (S16) and 11,12,16,17-didehydrofalcarinol (S17) from the roots of Hedera helix by Orobanche hederae [Trentino-Alto Adige, Italy; whole plants; dichloromethane; HPLC-DAD, HPLC-MS, comparison with authentic reference compounds and literature data] [65].

Sequestration of Secondary Metabolites by Orobanche s.l. from Their Host Species
As obligate holoparasites Orobanche species drain water and essential nutrients from their host plants. Also the sequestration bioactive natural products from the hosts seems likely [62], but up to now little is known about the uptake of other substances by holoparasitic Orobanche species from the plants they parasitize on and only very few reports deal with the sequestration of secondary metabolites from hosts. Orobanche hederae Duby-Sequestration of minerals and fatty acids by Orobanche hederae from its host Hedera helix was described by Lotti and Paradossi [Tuscany, Italy; whole plants; petrol ether, soxhlet; GC [63,64]. Sareedenchai and Zidorn reported the uptake of polyacetylenes falcarinol (S15), 11,12-dehydrofalcarinol (S16) and 11,12,16,17-didehydrofalcarinol (S17) from the roots of Hedera helix by Orobanche hederae [Trentino-Alto Adige, Italy; whole plants; dichloromethane; HPLC-DAD, HPLC-MS, comparison with authentic reference compounds and literature data] [65].   Figure 23. Quinolizidine alkaloids II, S11-S13. Figure 24. Piperidine alkaloid-Ammodendrine S14.

Antimicrobial Activities
The phenolic composition of O. crenata 80% methanolic extract and its in vivo efficacy against fungal postharvest diseases were studied in an attempt to find new strategies for reducing postharvest diseases in sweet cherry fruit and replacing or integrating the use of synthetic fungicides. Sweet cherry fruit were sprayed with O. crenata extract (different concentrations: 1×, 2×, 4×; the 1× concentration corresponding to 0.170 mg dry matter/mL of buffer), O. crenata extract added with salts (CaCl 2 or NaHCO 3 , 1% w/v), salt solutions, and the same buffer solution used to prepare the plant extracts (0.1 M K-phosphate, pH 5.5) as a positive control few hours after harvesting. Afterwards they were stored under controlled conditions. Rot incidence, expressed as the percentage of rotten fruit with respect to the total number of fruit in each tray, was assessed daily. At a rot incidence of around 50% the inhibition values of different treatments were evaluated. O. crenata extract inhibited postharvest rot in higher extract concentrations. An increase in extract concentration produced an increase in the percentage of inhibition from 64% to 76% for O. crenata. Addition of salt to the most concentrated extract further increased the inhibition of postharvest rot to 82% and 84% for NaHCO 3 and CaCl 2 , respectively, and hereby proved to have a high antifungal efficacy [33]. Antifungal activity of O. aegyptiaca ethanolic and acetone extracts against Fusarium oxysporum Schlechtend., was evaluated in solid media using five different concentrations (0.12, 0.25, 0.50, 1.00, 2.00 mg/mL) of solutions of the tested substances, and for bacteria an agar diffusion method with four different concentrations of solutions of the tested substances (12.5, 25, 50, 100 mg/mL) together with MIC measurement in liquid media with six different concentrations of the tested substances (0.1, 0.5, 1.0, 1.5, 2.0, 2.5 mg/mL) were used. Orobanchoside (29) and caffeic acid (34) showed pronounced antifungal activities with MIC values of 2.00 mg/mL and 0.25 mg/mL, respectively, against S. sclerotiorum in media with pH 5. Orobanchoside (29) furthermore had an MIC value of 0.25 mg/mL against S. sclerotiorum and 1.00 mg/mL against B. cinera in media with pH 7, while caffeic acid (34) showed an MIC of 0.25 mg/mL against B. cinera in pH 7 medium. Verbascoside (10) and poliumoside (11) were both able to reduce growth of S. sclerotiorum and B. cinera in pH 5 and pH 7 media, but complete inhibition was not observed. Ferulic acid (36) showed MIC values of 0.13 mg/mL against S. scleretorium and B. cinerea in pH 5 and pH 7 media. Chlorogenic acid (37) was able to reduce growth of S. sclerotiorum and B. cinerea in pH 5 and pH 7 media, but again, complete inhibition was not observed. Against the tested bacteria MIC values were as follows: MIC of caffeic acid (34) against C. rathayi (1.0 mg/mL), C. fascians (1.0 mg/mL), C. sepedonicum (0.1 mg/mL), A. tumefaciens (1.0 mg/mL), E.carotovora var. carotovora (1.0 mg/mL), X. pelargonii (1.0 mg/mL), P. syringae (not determined), S. aureus (1.5 mg/mL), E. coli (1.5 mg/mL); MIC of ferulic acid (36) against C. rathayi (0.5 mg/mL), C. fascians (0.5 mg/mL), C. sepedonicum (0.5 mg/mL), A. tumefaciens (1.0 mg/mL), E.carotovora var. carotovora (1.0 mg/mL), X. pelargonii (0.5 mg/mL), P. syringae (1.0 mg/mL), S. aureus (1.0 mg/mL), E. coli (1.5 mg/mL); MIC of chlorogenic acid (37) against C. rathayi (1.5 mg/mL), C. fascians (1.0 mg/mL), C. sepedonicum (1.0 mg/mL), A. tumefaciens (2.0 mg/mL), E.carotovora var. carotovora (2.5 mg/mL), X. pelargonii (1.5 mg/mL), P. syringae (2.0 mg/mL), S. aureus (>2.5 mg/mL), E. coli (2.5 mg/mL); MIC values of verbascoside (10), poliumoside (11), and orobanchoside (29) were not investigated. Caffeic acid (34) and its derivatives are potential natural plant protective agents against some plant-pathogenic fungi and bacteria as demonstrated in this work. Streptomycin, tested along with the caffeic acid derivatives, was a much more potent bacterial growth inhibitor than the other tested compounds with MIC values of <0.1 mg/mL (C. rathayi, C. fascians, C. sepedonicum, A. tumefaciens, E.carotovora var. carotovora, S. aureus, E. coli), with an exception for X. pelargonii (>2.5 mg/mL) (P. syringae MIC not determined) [99].

Antioxidant Activities as Food Preservative
O. crenata ethanolic extract total antioxidant activity was tested using the phosphomolybdenum method with ascorbic acid as standard. The antioxidant activity was expressed as ascorbic acid equivalents (AE) (mg/g of extract). The two investigated individual Orobanche plants showed good total antioxidant activity 619 ± 9 mg AE/g extract and 561 ± 9 mg AE/g extract [35].

Antioxidative Effects, Anti-Inflammatory Activity in Human Leucocytes, Effects on Production of Reactive Oxygen Species (ROS)
Phenylpropanoid glycosides isolated from O. coerulescens were tested for their antioxidative effects on human low-density lipoprotein. For evaluation of their antioxidant activity dialyzed LDL obtained from human blood samples was diluted with PBS to 100 µg/mL, pre-incubated with the test compounds at 37 • C for 30 min, and then incubated with CuSO 4 at 37 • C to induce lipid peroxidation. Resveratrol, a natural phenolic antioxidant e.g., from red wine, was used as a positive control. Conjugated diene formation was monitored and prolonged lag phase (min) used as an index of antioxidant activity when an antioxidant was present in LDL oxidation with Cu 2+ . All seven isolated phytochemical compounds, phenylpropanoid glycosides desrhamnosyl acteoside (9), acteoside (10), caerulescenoside (13), campneoside II (17), isoacteoside (23), oraposide (29), and 3 -methyl crenatoside (30)  Intravenous injection of the glycosidic fraction of O. crenata 70% ethanolic extract into rats in doses up to 20 mg/100 g led to a temporary lowering of the arterial blood pressure of the treated animals. Higher doses caused slight, persistent lowering of the arterial blood pressure [29]. Hypotensive activity of O. aegyptiaca 30% aqueous extract and of the alkaloid containing chloroform fraction (further fractionation of the extract with different solvents gave hexane, ether, chloroform, alcohol, and water fractions) after i.v. injection into dogs was also evaluated. The alkaloidal fraction showed strong hypotensive effects. (Hypertension was artificially induced using the Goldblatt technique.) 10 mg i.v. lowered the blood pressure by about 48 mmHg for three hours [57]. A mixture of verbascoside (10) and orobanchoside (29) extracted from O. hederae was tested for its effect on ADP-induced (10-15 µM) blood platelet aggregation and blood pressure in New Zealand male rabbits and Wistar male rats. A dose-dependent inhibition of ADP-induced platelet aggregation of 12.9 ± 4.0%, 43.7 ± 7.8%, 49.4 ± 6.4%, 59.4 ± 6.9%, and 73.7 ± 8.3% at concentrations of 0.2 mg/mL, 0.4 mg/mL, 0.6 mg/mL, 0.8 mg/mL, and 1.0 mg/mL phenylpropanoid glycosides respectively was observed using an aggregometer. Blood pressure was not affected by phenylpropanoid glycosides injected i.v. into the test animals [42].

Contractions of Toad and Rabbit Hearts and Rat Intestines
O. aegyptiaca 30% aqueous extract was further fractionated with different solvents to give hexane, ether, chloroform, alcohol, and water fractions. The extract and fractions were tested for different biological activities. Effects on toad (Bufo regularis Reuss) and rabbit hearts were investigated. Doses of 1, 2, 3, and 4 mL of the aqueous extract were added to 50 mL bath (Ringer´s solution for toad hearts, Lock´s solution for rabbit hearts) and the amplitude or heart rate (toad hearts) as well as the volume of Lock´s solution perfused by the heart (rabbit hearts) were recorded. Contractions of toad hearts and rabbits' hearts perfused by the extract were stimulated. Also contractions of isolated rats intestines were stimulated whereas uterine contractions in rats were inhibited [57].

Diuretic Effects
Oral application of the phenylpropanoid containing fraction of O. crenata extract in rats had strong diuretic effects. O. crenata extract doses of 100 mg/100 g body weight and 200 mg/100 g body weight were orally applied. Rats were put in diuresis cages and the volume of the collected urine was measured after 1, 3, 6, and 24 h. The untreated control group produced 0, 2.65 ± 0.22, 6.25 ± 0.05, and 11.2 ± 0.1 mL urine after 1, 3, 6, and 24 h, respectively. After application of O. crenata extract doses of 100 mg/100 g b.w. 0, 3.27 ± 0.05, 6.95 ± 0.05, and 12.9 ± 0.1 mL urine were collected after 1, 3, 6, and 24 h, respectively. O. crenata extract doses of 200 mg/100 g b.w. led to 0.37 ± 0.05, 3.95 ± 0.05, 8.25 ± 0.12, and 14.3 ± 0.2 mL of urine after 1, 3, 6, and 24 h, respectively, showing increasing diuresis with higher O. cernua extract doses [29]. O. aegyptiaca 30% aqueous extract was further fractionated with different solvents to give hexane, ether, chloroform, alcohol, and water fractions. The extract and fractions were tested for different biological activities. Diuretic effects of the 20% alcoholic extract were observed in rabbits. Urine volumes of treated animals were measured after 0.5, 1, 2, 3, and 24 h and compared to urine volumes of the animals after 24 h without any treatment. The average urine volume of treated animals (average dose of 9.5 mL of 20% extract/kg b.w.) after 24 h was 107 ± 51 in comparison with 73 ± 21.2 mL for the untreated animals [57].

Memory Enhancing Effects
Acteoside (10) showed memory enhancing effects and increased significantly the expression of nerve growth factor (NGF) and tropomycin receptor kinase A (TrkA) mRNA and protein in the hippocampus in mice [13]. NGF and TrkA are closely associated with cognitive function and a decrease thereof is related to Alzheimer´s disease. Acteoside (10) treatment resulted in an improvement of learning and memory deficits via promotion of NGF and TrkA expression in the brain. The authors used a senescent mouse model induced by a combination of chronic intraperitoneal administration of D-gal (60 mg/kg/day) and oral administration of AlCl 3 (5 mg/kg/day) once daily for 90 days. After 60 days mice in three different groups were treated intragastrically with acteoside (10) (30, 60, and 120 mg/kg/day) for 30 days. Learning ability and memory of the mice were tested using the Morris water maze test. Afterwards mice brains were removed and the hippocampus CA1 region studied immunohistochemically. Reverse transcription polymerase chain reactions (RT-PCR) and western blot analyses were performed to investigate the expression of NGF mRNA and TrkA mRNA [13].

Nutrient Source
O. crenata was found to be a good source of nutrients. It contained a low moisture level (<8%), a high amount of protein (7.30%), ash contents of 9.20-10.1%, a crude fiber content ranging from 22.1 to 23.5%, and a nutritive value of 244-247 kcal/100 g plant dry weight [35].
in NHDFs. O. cernua extract and acteoside (10) treatment furthermore led to the inhibition of the UVB-activated MAPK/AP-1 pathway by inhibiting the UVB-induced phosphorylation of ERK, JNK, and p38 and the expression of p-c-fos and p-c-jun. Levels of cytoprotective agents HO-1 and NQO-1 were increased by O. cernua extract and acteoside, hereby increasing protection against UVB-induced oxidative stress through activation of the cutaneous endogenous antioxidant system. UVB-induced enhacement of Smad7 expression and decrease of Smad2/phosphorylation were reversed by O. cernua extract and acteoside (10), and TGF-β1 expression was enhanced, hereby repairing the TGF-β/Smad signaling pathway and enhancing type-I procollagen synthesis [100]. (No standard deviations of the measured values described above were indicated.)

Summary of Bioactivities
In conclusion, Orobanche extracts and isolated Orobanche natural products were positively tested for a variety of biological activities including anti-hypertensive, anti-platelet aggregating, and memory enhancing effects. UV protecting and anti-photoaging effects open an interesting field of study and antioxidant activities on human LDL, inflammation modulating effects in human leucocytes, ROS production and amyloid β-aggregation inhibiting effects make the species containing the responsible substances potential agents for treatment of Alzheimer's disease and oxidation related diseases. Furthermore, Orobanche extracts are active against a wide variety of pathogenic fungi and bacteria and can be potential alternatives to synthetic antibiotics and plant protecting agents. The by far best investigated compound is the phenylpropanoid glycoside acteoside (10) which is responsible for a large part of the observed effects, such as antioxidant, anti-inflammatory, radical scavenging, amyloid β-aggregation inhibiting, memory enhancing, antimicrobial, and photoprotective effects and also oraposide (29) was shown to have several interesting effects. However, the occurrence of acteoside (10) is not restricted to Orobanche or Orobanchaceae but the compound is widely distributed in the plant kingdom. It is found in over 200 species belonging to 23 plant families, most of them belonging to the order Lamiales [101]. Thus, even though Orobanche extracts and substances extracted thereof show the above stated biological activities, there might be better and easier accessible sources for the bioactive compounds than the holoparasitic taxa of the genus Orobanche.

Discussion
Most of the natural products found in the genus Orobanche (n = 70) have so far been reported only from one source (n = 51), and only three compounds from more than four taxa: acteoside 10 (from 13 source taxa), oraposide 29 (from 12 source taxa), and orobanone 45 (also from 12 source taxa). While most of the literature on Orobanche is about strigolactones, seed germination stimulants, parasitic weed management, and host-parasite interaction (SciFinder, last accessed first of October, 2018), publications on secondary metabolism of Orobanche species are relatively rare. Of the more than 200 species belonging to Orobanche s.l. only 27 species have been investigated for secondary metabolites. Compound classes detected in the analyzed species comprise aromatic aldehydes, ketones and phenylmethanoids (Figure 1), phenylethanoids (Figure 2), phenylethanoid glycosides (Figure 3), phenylpropanoid glycosides (Figures 4-8), phenolic acids (Figure 9), lignans ( Figures 10 and 11), flavonoids (Figures 12 and 13), a tropone derivative (Figure 14), and sterols (Figures 15-20). Investigations on biological activities of Orobanche extracts and isolated pure secondary metabolites from Orobanche species show a wide variety of effects, e.g., antibacterial and antifungal activities [35,46,98], inhibition of amyloid-β-aggregation [45,46] or photoprotection against UVB-irradiation [100]. Orobanche are not only destructive weeds, but might also be a source of active agents against several diseases, in particular against fungal and bacterial, and inflammatory diseases, correlated with ROS production. Nevertheless, it has to be considered, that phenylpropanoids in general and e.g., acteoside, one of the best investigated compounds of Orobanche in particular, are not restricted to Orobanche species but are widely distributed in the plant kingdom, possibly making other species more interesting sources of these compounds [101,102]. An aspect that deserves more research and could be a challenging subject for future studies is the idea that natural products sequestered by Orobanche species from their host species could be further metabolized by the parasites. Metabolization of host plant natural products could result in new, formerly undescribed hybrid compounds not synthesized by a single species. To study this phenomenon, more analytical studies of the secondary metabolism of Orobanche species and their host plants are warranted.
Supplementary Materials: The following are available online. Table S1: Overview Natural Products synthesized by Orobanche species. Table S2: Natural Products sequestered by Orobanche species from host species. Text S1: Literature search strategy & key words.