The genus Scrophularia: a source of iridoids and terpenoids with a diverse biological activity

Abstract Context: Scrophularia genus (Scrophulariaceae) includes about 350 species commonly known as figwort. Many species of this genus grow wild in nature and have not been cultivated yet. However, some species are in danger of extinction. Objective: This paper reviews the chemical compounds, biological activities and the ethnopharmacology of some Scrophularia species. Materials and methods: All information was obtained through reported data on bibliographic database such as Scopus, United States National Agricultural Library, Biological Abstracts, EMBASE, PubMed, MedlinePlus, PubChem and Springer Link (1934–2017). The information in different Pharmacopoeias on this genus was also gathered from 1957 to 2007. Results: The structures of 204 compounds and their biological activity were presented in the manuscript: glycoside esters, iridoid glycosides and triterpenoids are the most common compounds in this genus. Among them, scropolioside like iridoids have shown potential for anti-inflammatory, hepatoprotective and wound healing activity. Among the less frequently isolated compounds, resin glycosides such as crypthophilic acids have shown potent antiprotozoal and antimicrobial activities. Conclusion: The Scrophularia genus seems to be a rich source of iridoids and terpenoids, but isolation and identification of its alkaloids have been a neglected area of scientific study. The diverse chemical compounds and biological activities of this genus will motivate further investigation on Scrophularia genus as a source of new therapeutic medications.


Introduction
The Scrophulariaceae family consists of 220 genera. Scrophularia genus is one of the large genera of the Scrophulariaceae. Distribution of these genera occurs mainly through mountainous regions (e.g., Scrophularia farinosa Boiss. and Scrophularia amplexicaulis Benth.) to rarely in deserts (e.g., Scrophularia deserti Delile). This genus is represented by 60 species in the flora of Iran and can be used as heart stimulant, circulatory stimulant and diuretic. Other traditional uses of this genus include antipyretic, febrifuge, antibacterial, anti-erythema, anticonstipation, antifurunculosis, ulcerous stomatitis and tonsillitis treatment.
Among these traditional uses of the Scrophularia, anti-inflammatory and anti-infections' treatment in different types of diseases is common (Viola 1966;Swiatek and Dombrowicz 1975). The therapeutic potential of the Scrophularia has led researchers to focus on the isolation and determination of their bio-active compounds. Some of these species are characterized mainly by glycoside esters or phenylpropanoid glycosides (Calis et al. 1988b;de Santos et al. 2000;Li et al. , 2009, saponins, and iridoids (Çalis et al. 1993a;Yamamoto et al. 1993;Pachaly et al. 1994;Maksudov et al. 1996;Bhandari et al. 1997;Chen et al. 2007;Chebaki et al. 2011). According to some findings, phenylpropanoid glycosides and iridoids are the major part of Scrophularia genus secondary metabolites, which showed apparent therapeutic potential in numerous investigations ( Figure 1). Several biological effects of phenylpropanoid such as antioxidants, hepatoprotective, antitumor, anti-inflammatory and other useful effects have been studied over the past few years (Garrido et al. 2004;Korkina et al. 2007). Another main class of secondary metabolites is iridoids compounds which constitute the most chemical and biological diversity in Scrophularia genus. The several reported biological activities of these compounds have led to increased inclination for the isolation of these classes of chemical compounds (Garg et al. 1994;Giner et al. 2000;Kim and Kim 2000;Kim et al. 2002a;Lee et al. 2002;Stevenson et al. 2002;Kim et al. 2003a;Tasdemir et al. 2005;Valiyari et al. 2012). Based on data extracted from different studies, most biological activities of iridoids include anti-inflammatory, anticancer and antiprotozoal (Dinda et al. 2009). This review presents a brief case for the medicinal uses and the phytochemical and pharmacological properties of the Scrophularia genus.

Materials and methods
All information regarding the chemical and biological activity of the plants were obtained through reported data from 1934 to 2017 on bibliographic database such as Scopus, United States

Biology and ethnopharmacology
Most Scrophularia species are annual or perennial herbaceous plants, with woody base and rarely suffruticose, and can also be spinose in rare cases. However, a few of this genus are subshrubs. Flowers are urceolate or tubulose. The length of corolla ranges from 3 to 20 mm. Lips are equal or unequal, which is one of the important characteristics for distinguishing species. With thyrse inflorescence or in rare cases, racemose with one or two flower in each cyme, mostly have four-angled stems and opposite leaves. Some Scrophularia species are widely used as traditional medicine. Several countries, including China, Korea and Japan, have used these species as traditional therapeutics as anti-inflammatory and anticancer remedies. Roots of S. ningpoensis Hemsl. "Xuan Shen", S. buergeriana Miquel, Ann. Mus. Bot. Lugduno-Batavi. "Beixuan Shen" and S. nodosa L. (common figwort) have been used as therapeutic remedies in fever, swelling, constipation, pharyngitis, neuritis and laryngitis. In Europe, other species, such as S. aquatica L. (water figwort), are used as laxatives, heart stimulants, circulatory stimulants and diuretics. In ancient Iranian medicine, roots and aerial parts of S. lucida L. "Sinderitis" and S. chryasanthemifolia Bory & Chaub. "Heterasinderitis" are used as heart and circulatory stimulants. Table 1 shows Scrophularia species which are used traditionally as therapeutic remedy.

Phytochemistry
From the genus Scrophularia, chemical compounds such as flavonoids, phenylethanoids and glycoside esters, phenolic acids, C 9 iridoid, glycosides, resin glycosides and fatty acids derivatives, triterpenes, triterpenoid glycosides, alkaloids, diterpenoids and essential oils can be isolated (Tables 2 and 3 and Figure 1). As mentioned above, some of these chemical substances produce bioactivities in various models (Table 4).
E-p-Methoxycinnamic acid and E-isoferulic acid isolated from S. buregeriana significantly improved memory deficit, induced by scopolamine in mice (Kim et al. 2003a). E-p-Methoxycinnamic acid (Table 2 and Figure 3) also has a protective role against NMDA and glutamate-induced neurotoxicity (Kim et al. 2002b). In another experiment, m-and p-methoxycinnamic acid and ferulic acid showed hepatoprotective activities against carbon tetrachloride (CCl 4 ) in animal tests (Lee et al. 2002a;Kim et al. 2011).

Phenylethanoid glycosides
Phenylethanoid as one of the main phytochemical compounds plays specific role in biological activity of these plants. Many biological activities such as antimicrobial, anti-inflammatory, antitumor, heart function improvement and neuroprotective activities are attributed to these compounds (Zhu 1998;Koo et al. 2005;Deyama et al. 2006;Georgiev et al. 2011). Previous studies revealed that one of the main constituents of Scrophularia plants is phenylethanoid glycosides, and many of the therapeutic potentials can be attributed to them (Zhang and Li 2011).
Sixteen phenylethanoid glycosides compounds (43-59, Figure 4) have been isolated from Scrophularia (Calis et al. 1988b;de Santos et al. 2000;Lee et al. 2002a). Some of these compounds showed cytotoxicity upon investigations, for example, angoroside compounds which are isolated from S. scopolii Hoppe ex Pers. Among these isolated compounds, angoroside A (39) showed most cytotoxic activity compared with angoroside B (40) and angoroside C (41). The relationship between compound structures and their activities were elucidated.
C 9 iridoids isolated from these plants are listed in Table 2 and Figure 6.

S. oxysepala
Methyl benzaldehyde, methyl benzyl alcohol, 1-octen-3-ol, eugenol and phytol S. amplexcaulis Eugenol, 1-cten-3-ol, anethole, caryophyllene oxide and eugenol acetate S. striata 1-octen-3-ol, banzyl banzoat, benzaldehyde, linalool and phytol S. frigida Oxygenated monoterpenes, L-linalool, geraniol, a-terpineol, and 1-octen-3-ol anti-inflammatory activities (Table 4). Among the chemical compounds isolated from S. koelzii Pennell. such as harpagoside (113), koelzioside (132) and scropolioside A (134), scropolioside A demonstrated maximum hepatoprotective activity against thioacetamide-induced hepatotoxicity in animal model (Garg et al. 1994). Research on S. deserti led to the isolation of scropolioside D 2 (133) and harpagoside B (99), which have significant antidiabetic and anti-inflammatory activities (Ahmed et al. 2003). Among the various bioactivities observed of these compounds, anti-inflammatory effect is the most investigated. Zhu et al. (2015), in working on anti-inflammatory activity of isolated iridoid glycosides from S. dentata Royle ex Benth. and comparison between their potentials, reported their anti-inflammatory activities against LPS-induced NF-jB activity, cytokines mRNA expression, IL-1b secretion and cyclooxygenase-2 activity depending on whether the 6-O-substituted cinnamyl moiety was linked to C 00 2-OH, C 00 3-OH or C 00 4-OH, and on the number of moieties linked, which is closely related to the enhancement of anti-inflammatory activity (Pieroni et al. 2004). Structural diversity of iridoid glycosides in this genus can be categorized into three classes including (a) moieties which exist on cyclopentane ring, (b) moieties which exist on different position of glucose attached in [c] pyran ring and (c) moieties which exist on different position of rhamnose that are attached in C6 cyclopentane ring. Among these structural classes, diversity of iridoid glycosides with moieties in rhamnose attached in C6 cyclopentane ring position is more than other classes. Subsequently, structures with moieties are placed in different positions of cyclopentane ring, and finally structures with moieties in different positions of glucose are attached in [c] pyran ring. Table 2 shows various isolated Scrophularia iridoid glycosides.

Diterpenoids
Five new 19(4!3)-abeo-abietane diterpenoids, scrodentoids A-E (195-199) were isolated from S. dentata, which is a famous traditional remedy for the treatment of smallpox, measles, high-heat plague and poisoning (Zhang et al. 2015a). These compounds are isolated from low-polar extract of S. dentata by column chromatography and reversed-phase HPLC techniques. The anti-inflammatory, immunosuppressive, antifertility, anticystogenesis and anticancer activities of 19(4!3)-abeo-abietane diterpenoids have been previously reported (Zhang et al. 2015b). Scrodentoids A-E were investigated for immunosuppressive effect and cytotoxic effects, especially against B16 and MCF-7 cells line. According to this investigation, scrodentoids A (195) and D (198) showed the most potential in this biological test (Table 2 and Figure 10).

Essential oils
The essential oils of a few Scrophularia species have been investigated until now. The essential oil of S. oxysepala, an endemic plant of western and central regions of Iran, was characterized by the presence of high percent of eugenol (200), dehydroeugenol (201) and methyl benzyl alcohol (202) as phenolic compounds. In addition, a high amount of eugenol (200) and eugenol acetate (203) have been reported from the essential oil of S. amplexicaulis Benth, another endemic plant of Iran, which showed antimicrobial activity against S. aureus . According to research on S. oxysepala, S. amplexicaulis, S. striata and S. frigida Boiss, it was indicated that probably, 1-octen-3-ol (204) is a chemical compound marker in Scrophularia species (Table 3 and

Antimicrobial and antiprotozoal
Essential oil of Iranian endemic plant, S. amplexicaulis, showed antibacterial activity against S. aureus in the well diffusion method. The essential oil of this plant is characterized by a high content of eugenol (53.8%) and eugenol acetate (24.5%), and the antibacterial activity of these compounds has been identified previously (Didry et al. 1994;. In another research on methanolic extract and fractions of S. amplexicaulis, 80% and 60% (IC 50 0.827, 0.431 mg/mL) methanol in water of solid-phase extraction (SFE) showed significant activity in haeme biocrystallization assay for potential antimalarial property . Tasdemir et al. (2005Tasdemir et al. ( , 2008 investigated the antiprotozoal and antimycobacterial activities of the chemical compounds of S. cryptophila, tryptophan and buddlejasaponin III (184) which showed growth-inhibitory effect against Trypanosoma brucei (IC 50 4.1 and 9.7 mg/mL). Harpagide (114) and crypthophilic acid C (173) showed the best leishmanicidal activity (IC 50 2.0 and 5.8 mg/mL) in comparison with other isolated compounds. In antimalarial activity against Plasmodium falciparum, crypthophilic acid C (173), tryptophan and buddlejasaponin III (184) showed antimalarial activity with IC 50 values of 4.2, 16.6 and 22.4 mg/mL, respectively (Tasdemir et al. 2008). Investigation on the ethanol extract of S. deserti showed that plant have antibacterial potential against Brucellla melitensis, in other studies related to this plant, three isolated compounds including 3(f)-hydroxy-octadeca-4(E), 6(Z)-dienoic acid (170), ajugoside (89) and scropolioside B (135) exhibited moderate antibacterial activity against multidrug and methicillinresistant S. aureus (MRSA) as well as mycobacteria with minimum inhibitory concentration (MIC) values, ranging from 32 to 128 mg/mL (Stavri et al. 2006;Bahmani et al. 2013). Fernandez et al. investigated the antibacterial and active fraction of S. frutescens and S. sambucifolia L. on several micro-organisms such as Bacillus cereus, Bacillus megaterium, Bacillus subtilis, S. aureus, Escherichia coli, Serratia marcescens, Salmonella typhimurium and Moraxella lacunata. Results of this investigation indicated that the phenolic fractions of both species showed more activity against Gram-positive bacteria, specifically against Bacillus sp. (Fernandez et al. 1996). The 70% ethanol extracts of leaves and scrokoelziside A (175) which were isolated from S. ningpoensis "Xuan Shen" showed anti-bacterial activity against beta-haemolytic streptococci (Figures 11 and 12; Li et al. 2009).

Hepatoprotective and neuroprotective
E-p-Methoxycinnamic acid (12) isolated from S. buergeriana showed anti-amnesic activity and protective effect on cultured neuronal cells against neurotoxicity induced by glutamate (Kim et al. 2003a). Future investigations for finding other active compounds of S. buergeriana in neuroprotection led to the isolation of 10 phenylpropanoid esters from roots of this plant, although all isolated phenylpropanoid esters exerted significant protective effects against glutamate-induced neurodegeneration, but buergeriside A1 (67), buergeriside B1 (66) and (E)-p-methoxycinnamic acid (12) exhibited better protection (Kim and Kim 2000). In the continuous isolation of other neuroprotective compounds, 8-O-E-p-methoxycinnamoyl harpagide (102) and harpagide (114), 8-O-Z-p-methoxycinnamoyl harpagide (103), 6 0 -O-E-p-methoxycinnamoy lharpagide (104), 6 0 -O-Z-p-methoxycinnamoyl harpagide (105) E-harpagoside and Z-harpagoside were isolated from these plants and tested for the reduction of glutamate-induced neurotoxicity in rat. According to the result, these compounds demonstrated protective effect on cultured neurons against glutamateinduced oxidative stress (Kim and Kim 2000;Kim et al. 2002aKim et al. , 2003b. Isolated phenylpropanoids from roots of S. buergeriana exhibit hepatoprotective effect in CCl 4 -induced toxicity (Kim et al. 2002a). Chloroformic fraction of the alcoholic extract of the aerial parts of S. koelzii showed hepatoprotective activity. Further investigation led to the isolation of several iridoid glycosides, and among these compounds, scropolioside A showed maximum hepatoprotective activity in thioacetamide hepatotoxicity model (Figures 11 and 12; Garg et al. 1994).

Conclusion
Recently, the amount of research on metabolites, pharmacological activities and traditional uses of the various Scrophularia species has increased significantly. According to reviewed literatures, several reasons could contribute to the screening of this genus which include (1) some of the species have been used as a traditional or local therapeutic remedy especially in Asia and Europe for long time, and the effectiveness and safety of these species have been established. Therefore, such sources have generated much interest and new field for easier search of potential compounds.
glycosides have been identified as the three main chemical compositions of Scrophularia. Among them, scropoliosides like iridoid structures have shown potential for anti-inflammatory, hepatoprotective and wound healing activity effects. Among the less frequently isolated compounds, resin glycosides such as crypthophilic acids have shown good properties in antiprotozoal and antibacterial assays. Therefore, chemical compounds of this genus will motivate further investigation on Scrophularia, and have great potential as sources of finding new therapeutic medications.
(3) Only 17 of the approx. 350 species have been studied in some detail. Among the isolated metabolites from Scrophularia spp., only a few of them has been investigated for their biological activities. Many of the conducted researches on isolation or biological screening have been conducted on iridoids and phenylethanoids while other classes of phytochemicals such as alkaloids, diterpenoids and flavonoids have been less considered by researchers.
On one hand, most of the studies on the isolated compounds have been carried and in vitro/in vivo and we could not find any clinical trials on biological activities of Scrophularia. Thus, pharmacokinetic and metabolism of these metabolites are unclear in human body. On the other hand, the exact mechanism of the active isolated molecules is still unknown. Considering these issues, there is huge gap between the current situation and the final goal which is developing approved drug from the isolated molecules or even developing supplements from the Scrophularia spp. extracts. Conducting ADME (absorption, distribution, metabolism and excretion) studies on the isolated bioactive compound of the genus seems to be essential.
In most cases, quantitative analysis of bioactive compounds has not been considered which might guide researchers to find other species of Scrophularia with more content of bioactive compounds. Despite the presence of some Scrophularia species in different pharmacopeias and their application in tradition or folk medicine of different societies, lack of analytical investigations on the bioactive compounds of these species resulted in difficulties in quality control and standardizations of these herbs.
Some metabolites, such as iridoids which also demonstrated some biological activities, are common between these plants and it is possible to consider them as biomarkers for Scrophularia spp.
Conducting complementary studies on isolated bioactive compound from this genus, such as Quantitative structure-activity relationship (QSAR) studies on the isolated bioactive compounds  as well as preparing semi-synthetic derivatives, may result in more active metabolites.