Constituents of Picrorhiza with immunomodulatory and related activities

4.1 Cucurbitacins

4.2 Iridoid glucosides

4.3 Phenylethanoids

4.4 Phenolics

Chapter 4 46 | Chapter 4

Introduction

Most of the publications on the biological activities and constituents of Picrorhiza deal with the species P. kurrooa, while P. scrophulariiflora is scarcely described. However, the identification of the raw material has usually not been elaborated on in these publications. Quite often, plant material is obtained through a chain of buyers and sellers, and ultimately there is no information on the origin of the material. Generally, no distinction is made between samples from Nepal (P. scrophulariiflora) and India (P. kurrooa). A proper identification can only be made when selective criteria are found to distinguish between rhizomes from both species, e.g. microscopically, chemically, or by DNA analysis. However, these criteria have not been met so far. The difficulty in distinguishing both species from each other based on rhizomes only makes interpretation of experimental results on Picrorhiza species ambiguous. This uncertainty has to be taken into consideration when referring to publications on P. kurrooa. In cases where the origin of the material in not clearly described, there is a possibility that the material investigated is erroneously substituted for P. scrophulariiflora. These observations provide ample arguments to introduce publications on both species in this chapter.

4.1 Cucurbitacins

Introduction The cucurbitacins constitute a group of triterpenic substances, especially known by their bitterness and toxicity. They are characterized by the cucurbitane skeleton, are mainly tetracyclic, and oxygenated at different sites. The basic structure is 9β-methyl-19- norlanosta-5-ene (figure 1), also known as 10α-cucurbit-5-ene (Lavie and Glotter, 1971). Cucurbitacins are usually present within plants as β-glucosides and have wide-ranging pharmacological activities. Several authors have reviewed this group of compounds, from a chemical (Lavie and Glotter, 1971), biological (Rehm, 1960, Guha and Sen, 1975), or pharmacological perspective (Miró, 1995).

21 H 22 24 26 18 20 23 25 12 17 H 11 27 13 H H 16 1 9 14 2 10 8 15 19 30 3 4 5 7 6

28 29 Figure 1. Basic structure of the 10α-cucurbit-5-ene skeleton (9β-methyl-19-norlanosta-5- ene) Constituents of Picrorhiza with immunomodulatory and related activities | 47

Cucurbitacins from Picrorhiza So far, 23 different cucurbitacin glucosides and 1 aglucone have been isolated from Picrorhiza species, mainly from P. kurrooa (table 1).

Table 1. Cucurbitacins isolated from P. kurrooa and P. scrophulariiflora. R

OH HO O O HO HO OH HO

R = O OH O 20 HO HO 20 20

Stuppner et al., 1991† Wang et al., 1993‡ Stuppner et al., 1991† Li et al., 1998‡ O O HO OH HO O 20 20 O Stuppner and Wagner, 1989a† Stuppner et al., 1991† Li et al., 1998‡ O O O HO OH HO O O 20 20 HO O O 20 Stuppner and Wagner, 1989a† Laurie et al., 1985†, Stuppner et al., 1991† Wang et al., 1993‡ O H O 20 O Stuppner et al., 1991† O HO OH

OH R

HO

R =

HO O

O O O HO HO OH Stuppner et al., 1991† Stuppner et al., 1991† 48 | Chapter 4

Table 1. Continued. R O OH HO O O HO HO OH HO

R = HO 20

OH Stuppner and Müller, 1993† O O HO OH HO O 20 20 O Stuppner et al., 1990† Stuppner and Wagner, 1989a†

20 20 OH O O OH OH Stuppner and Müller, 1993† Stuppner and Müller, 1993† R O OH HO O O HO HO OH O

R = OH O HO HO HO 20 20 20 OH Stuppner and Wagner, 1989a† Stuppner et al., 1990† Stuppner and Müller, 1993† O O O HO OH HO O HO O 20 20 20 O O arvenin III arvenin I arvenin II Stuppner et al., 1990† Stuppner and Wagner, 1989a† Stuppner and Wagner, 1989a†

20 20 OH O O OH OH Stuppner and Müller, 1993† Stuppner and Müller, 1993† † Constituent of P. kurrooa; ‡ constituent of P. scrophulariiflora.

Cytotoxicity Strong cytotoxic effects have been described for several cucurbitacins, using KB cell cultures, derived from human nasopharyngeal carcinoma, or HeLa cells (Miró, 1995). It was suggested that the cytotoxic mechanism involves selective interference with DNA synthesis, as seen by the inhibition of [3H]-thymidine incorporation in cultured Constituents of Picrorhiza with immunomodulatory and related activities | 49 lymphocytes incubated for 30 min with cucurbitacin B, D, or dihydrocucurbitacin B. RNA and protein synthesis were not affected (Whitehouse and Doskotch, 1969). Later experiments showed that cucurbitacin I inhibits the incorporation of radioactive precursors into DNA, RNA, as well as protein in HeLa S3 cells. ID50 values of several cucurbitacins were similar to the ED50 values of cucurbitacin-induced growth inhibition, suggesting a relationship between inhibition of intracellular metabolic activity and growth-inhibitory activity of cucurbitacins. Cell lysis due to exposure to cucurbitacins was excluded (Witkowski et al., 1984).

Table 2. Schematic presentations of some relevant cucurbitacins. O O HO O HO OH

O O O H H OH H H OH HO HO

O O cucurbitacin B cucurbitacin D O O HO O HO OH

O O O H H OH H H OH HO HO

O HO cucurbitacin E cucurbitacin F O O HO OH HO OH

O O H H OH H H OH HO HO

O O cucurbitacin I cucurbitacin R

Cucurbitacins were also shown to inhibit the binding of cortisol to glucocorticoid receptor (GCR) in HeLa cells at 37 °C in a dose-dependent manner. At 0-4 °C and in cell- free HeLa systems, however, inhibition was decreased or completely abrogated, suggesting a metabolic transformation of cucurbitacins in functionally HeLa cells into derivatives with a higher affinity to GRC than the parent compounds. Moreover, the inhibition of cortisol binding to GCR by cucurbitacins in intact cells at 37 °C showed a strong correlation with cytotoxic activity, suggesting that cytotoxicity of cucurbitacins involves binding to the glucocorticoid receptor (Witkowski and Konopa, 1981). Incorporation experiments confirmed the suggestion of an active metabolite, because cucurbitacin I suppressed the 50 | Chapter 4 incorporation of radio-labeled precursors when cells were permeabilized upon exposure, but did not suppress incorporation when cells were exposed to cucurbitacin I after permeabilization (Witkowski et al., 1984). Later experiments, however, have shown that glucocorticoids alone do not affect the incorporation of radio-labeled precursors into HeLa S3 cells and, in combination with cucurbitacin I, could not reverse its inhibitory activity, while the concentrations of steroids used were large enough to induce complete inhibition. These data suggest that the inhibition of macromolecule synthesis is not mediated by the GCR, but instead might involve immediate extranuclear effects of glucocorticoids, which are not necessarily receptor-mediated (Witkowski et al., 1984).

Effects on cytoskeleton Cucurbitacins D, I, E, and 2-methoxy-cucurbitacin E were shown to induce morphological changes to Ehrlich ascites tumor cells at low concentrations. Only at higher concentrations, respiration, permeability, and viability of these cells were affected, without an increase in morphological changes. Similar changes were observed in samples that were taken 1 hour after i.p. injection of cucurbitacin D into ascites-bearing mice. In samples taken 24 h after injection, however, no deformed or necrotized tumor cells were found (Gitter et al., 1961). Furthermore, it was shown that lymphocytes of chronic lymphatic leukemia patients as well as those of leukocytotic lymphosarcoma patients were much more sensitive to cucurbitacin D (elatericin A) than those isolated from normal individuals (Shohat et al., 1967). Exposure of prostrate carcinoma cells to cucurbitacin E also led to the development of morphologically abnormal, multinucleated cells, which presented a change in vimentin distribution. Furthermore, cucurbitacin E induced cell growth inhibition of prostrate carcinoma cells, even after drug removal. It was shown to disrupt the F-actin cytoskeleton, which correlated with antiproliferative effects (Duncan et al., 1996). Moreover, cucurbitacin E was shown to interfere with the actin cytoskeleton of human endothelial cells, preferentially targeting proliferating versus quiescent cells. It was suggested that the condensation of actin microfilaments in quiescent cells, which does not occur in proliferating cells, renders the molecular targets for cucurbitacin E unavailable for interaction (Duncan and Duncan, 1997).

Antitumor activity Cytotoxic and antiproliferative effects, as described above, make cucurbitacins interesting agents in the treatment of tumors. The National Cancer Institute (NCI) has screened several plants containing cucurbitacins and has isolated their active constituents (Fuller et al., 1994). Cucurbitacins have shown in vivo inhibitory activity in several carcinoma, sarcoma, and leukemia models (Gitter et al., 1961, Gallily et al., 1962, Kupchan et al., 1967, Lavie and Glotter, 1971, Kupchan et al., 1972, Konopa et al., 1974, Fang et al., 1984, Reddy et al., 1988).

Immunological and related activities Cucurbitacin B, isolated from Ecballium elaterium (Cucurbitaceae), exhibited anti- inflammatory activity in acetic acid-induced vascular permeability, serotinin-induced paw edema, and bradykinin-induced paw edema in mice (Yesilada et al., 1988, 1989). Furthermore, cucurbitacins B and E, isolated from Wilbrandia ebracteata (Cucurbitaceae), showed anti-inflammatory activity in carrageenan-induced paw edema in rats (Peters et al., Constituents of Picrorhiza with immunomodulatory and related activities | 51

1997), while cucurbitacin B reduced PGE2 levels in exudates of carrageenan-induced pleurisy in mice (Peters et al., 1999). Cucurbitacin E, isolated from Conobea scoparioides (Scrophulariaceae), and several related cucurbitacins were demonstrated to inhibit LFA-1- and ICAM-1-mediated JY/HeLa cell adhesion. These results were in line with the inhibition of actin polymerization in fMLP-stimulated neutrophils, suggesting that the mechanism of inhibiting cell adhesion is by interference with the cytoskeleton (Musza et al., 1994). In addition to modulation of cell-mediated immunity, interference with humoral immune responses has also been described. Picfeltarraenins, cucurbitacin glycosides isolated from Picria fel-terrae (Scrophulariaceae), were shown to inhibit the classical and alternative pathways of the complement system (Huang et al., 1998). Cucurbitacin I showed only minor inhibition of the classical pathway (Knaus and Wagner, 1996). In contrast to cucurbitacin B, cucurbitacin D was shown to enhance capillary permeability. The activity was in a similar order of magnitude as that of histamine, while the activity could not be reversed with antihistamines. The enhanced permeability was associated with a persistent fall in blood pressure and the accumulation of fluid in thoracic and abdominal cavities in mice. The long latent period before the appearance of pharmacological effects, suggests that the compound had to be converted into an active metabolite (Edery et al., 1961).

Adaptogenic activities Cucurbitacin R-diglucoside was shown to moderately stimulate the secretion of corticosterone by the adrenal cortex, and to inhibit stress- or ACTH-induced corticosterone formation in vitro and in vivo, suggesting an adaptogenic effect. Furthermore, preliminary administration of cucurbitacin R reversed stress-induced alterations in eicosanoid levels, increasing PGE2 and decreasing 5-HETE, which supports the suggestion of adaptogenic activity (Panossian et al., 1997, 1999). Cucurbitacin B, isolated from Ecballium elaterium, was shown to have antihepatotoxic activities. It reduced CCl4-induced liver damage as shown by a decrease in sGPT levels, hepatic collagen and β-lipoproteins, and a reduction of pathological liver changes (Han et al., 1979, Miró, 1995, Agil et al., 1999).

In vivo toxicity Due to severe toxicity, the medicinal usage of plants that produce high amounts of cucurbitacins has become obsolete. LD50 values of several cucurbitacins are described in literature, ranging from 0.5 mg/kg for cucurbitacin B (rabbits, i.v.) to 340 mg/kg for cucurbitacin E (mice, p.o.) (David and Vallance, 1955, Edery et al., 1961, Le Men et al., 1969). Particularly, the absence of ∆1, and the presence of ∆23 and 25-acetoxy functionalities seem to enhance toxicity (Le Men et al., 1969).

4.2 Iridoid glucosides

Introduction Iridoids represent a large group of monoterpenes and accumulate in a number of plant families, mainly concentrated in the subclass Asteridae (Cronquist system). They consist of 52 | Chapter 4 a basic cyclopentano-[c]-pyran skeleton, which is formed during ring closure of 8-epi- iridodial (figure 2) and usually occur as glucosides. Several authors have reviewed their occurrence (El-Nagar and Beal, 1980, Boros and Stermitz, 1990), distribution (Hegnauer and Kooiman, 1978), biosynthesis (Inouye, 1978), and biological activities (Ghisalberti, 1998, Mandal et al., 1998).

11 11 H H 6 6 4 4 3 3 5 5 7 7 9 O 9 O 8 1 8 1 H H 10 O 10 OH Figure 2. Diagram of 8-epi-iridodial, precursor in the biosynthesis of catalpol.

Iridoids from Picrorhiza Several iridoid glucosides have been isolated from Picrorhiza species (table 3). Extensive research has been devoted to standardized iridoid fractions of P. kurrooa, e.g. kutkin and Picroliv. Kutkin is obtained by crystallization and consists of the glucosides picroside I and kutkoside in a ratio of 1: 2 and other minor glycosides (Singh and Rastogi, 1972, Ansari et al., 1988). Picroliv is a similar but less purified fraction, containing about 60 % of an equal mixture of picroside I and kutkoside (Dwivedi et al., 1989). More recent publications describe Picroliv as a standardized iridoid fraction containing 60 % of this mixture in a 1: 1.5 ratio (Dhawan, 1995).

Table 3. Iridoid glucosides isolated from P. kurrooa and P. scrophulariiflora. R1

O O R2 O R3 O HO HO OH R1 = OH, R2 = OH, R3 = OH catalpol Wang et al., 1993‡ R2 = OH, R3 = OH, R1 = R2 = OH, R3 = OH, R1 = R1 = OH, R3 = OH, R2 = OH OH

O O O OMe OMe O O O veronicoside picroside II kutkoside Stuppner and Wagner, 1989b† Weinges et al., 1972† Singh and Rastogi, 1972† Wang et al., 1993‡ Constituents of Picrorhiza with immunomodulatory and related activities | 53

Table 3. Continued. R2 = OH, R3 = OH, R1 = OH OH

O O OH O O specioside verminoside Li et al., 1998‡ Li et al., 1998‡ OH OMe O O OH O O OMe OH O OMe O 6-feruloylcatalpol picroside V minecoside Stuppner and Wagner, 1989b† Simons, 1989† Stuppner and Wagner, 1989b† Simons, 1989† Simons, 1989† R1 = OH, R2 = OH, R3 = OH OH

O O O OMe O O O picroside I picroside IV picroside III Kitagawa et al., 1971† Li et al., 1998‡ Weinges and Künstler, 1977† O HO MeO O H O HO HO O HO O O HO O HO O HO HO OH HO O HO HO OH aucubin pikuroside Wang et al., 1993‡ Jia et al., 1999† † Constituent of P. kurrooa; ‡ constituent of P. scrophulariiflora.

Biological activities A wide range of biological activities have been attributed to iridoids, such as cardiovascular, antihepatotoxic, choleretic, hypoglycemic, hypolipidemic, anti- inflammatory, antispasmodic, antitumor, antiviral, purgative, immunomodulatory, antioxidant, anti-phosphodiesterase, neuritogenic, molluscicidal, and leishmanicidal activities (reviewed by Ghisalberti, 1998). In a number of publications referring to the antihepatotoxic, hepatoregenerative, choleretic, and hypolipidemic effects of Picroliv, the preparation was shown to have similar or more potent activities than silymarin, a purified fraction of Silybum marianum (Asteraceae), commonly used in the treatment of liver disorders (reviewed by Dhawan, 1995). Hepatoregenerative effects were associated with an increased recovery of the liver anti-oxidant system after partial hepatectomy or carbon tetrachloride-induced liver damage of Picroliv-treated rats (Rastogi et al., 1995, 1997). Hypolipidemic effects were observed 54 | Chapter 4 in rats with triton- and cholesterol-induced hyperlipidemia (Khanna et al., 1994). Oral administration of Picroliv to mice prior immunized with sheep erythrocytes resulted in an increase in hemagglutinating antibody titers, antibody-forming cells, and DTH responses to sheep erythrocytes. Furthermore, it enhanced non-specific immune responses of peritoneal macrophages and the [3H]-thymidine uptake by lymphocytes isolated from mice treated with Picroliv (Puri et al., 1992). Anti-inflammatory activity was observed for kutkin in experimentally induced arthritis, edema, vascular permeability, and leukocyte migration in rodents (Singh et al., 1993). Aucubin was also shown to potently inhibit phorbolester-induced edema in mice ears, while catalpol and picroside II were not active (100 mg/kg, p.o.). The latter iridoids showed only minor anti-inflammatory effects upon topical administration (Recio et al., 1994). Moderate anti-inflammatory activity of picroside II, when administered topically, was confirmed later, while pikuroside was ineffective (Jia et al., 1999). Picrosides II, III, V, 6-feruloylcatalpol, and minecoside moderately inhibited chemiluminescence generated by activated polymorphonuclear neutrophils (PMNs), while picroside I was not active; scavenging effects of these compounds were excluded (Simons, 1989, p. 102). Picroliv however, as well as picroside I, were shown to be moderate superoxide scavengers, while kutkoside showed only weak activity (Chander et al., 1992). Furthermore, Picroliv protected cells against hypoxia, enhanced the expression of VEGF and HIF-1, selectively inhibited protein tyrosine kinase activity, and reduced PKC (Gaddipati et al., 1999).

4.3 Phenylethanoids

Only recently, another group of compounds have been isolated from Picrorhiza species. P. scrophulariiflora was shown to constitute the phenylethanoid glucosides scroside A-C and plantamajoside (Li et al., 1998). Plantamajoside, isolated from Plantago major (Plantaginaceae), is a potent inhibitor of phosphodiesterase and 5-lipoxygenase (Ravn et al., 1990), as is reported for similar compounds (Zhou et al., 1998). Moreover, phenylethanoids were shown to be antibacterial (Didry et al., 1999) and scavengers of reactive oxygen species (ROS; Çalis et al., 1999).

Table 4. Phenylethanoids isolated from P. scrophulariiflora. OMe

O OH R1 O R2 O OH HO O HO HO R3 R2 = OH, R3 = glucose, R1 = R2 = OH, R3 = OH, R1 = R1 = OH, R3 = glucose, R2 = O O O MeO MeO MeO O O O

HO HO HO scroside A; Li et al., 1998‡ scroside B; Li et al., 1998‡ scroside C; Li et al., 1998‡ Constituents of Picrorhiza with immunomodulatory and related activities | 55

Table 4. Continued. OH

HO O OH HO O OHO OH HO O plantamajoside Li et al., 1998‡ ‡ Constituent of P. scrophulariiflora.

4.4 Phenolics

Introduction Monocyclic phenolic compounds are very common in the plant kingdom. They are precursors and degradation products of lignin, which provides sturdiness to the plant and a physical defense barrier against parasites (Steinegger and Hänsel, 1988, p. 366). Furthermore, they usually act as antifungal agents, providing direct protection to the plant (Kokubun and Harborne, 1995, Aoyama et al., 1997).

Phenolics from Picrorhiza Vanillic acid, apocynin, and their respective glucosides have been isolated from Picrorhiza (table 5).

Table 5. Phenolic compounds isolated from P. kurrooa and P. scrophulariiflora. O OH R = OH R = glucose

OMe vanillic acid picein R Rastogi et al., 1949† Stuppner and Wagner, 1989b† O R = OH R = glucose

apocynin androsin † † OMe Basu et al., 1971 Stuppner and Wagner, 1989b R Wang et al., 1993‡ † Constituent of P. kurrooa; ‡ constituent of P. scrophulariiflora.

Antioxidant activities Apocynin, and to a minor extent vanillic acid, were isolated guided by their inhibition of chemiluminescence by activated PMNs (Simons et al., 1989). Apocynin completely inhibited oxygen uptake by activated neutrophils and human eosinophils, but not by human alveolar macrophages. This effect was mediated by an inhibition of NADPH oxidase activity, which was associated with the translocation of the cytosolic oxidase components p47phox and p67phox to the membrane fraction of neutrophils (Simons et al., 1990, Stolk et 56 | Chapter 4 al., 1994). In order to exhibit these effects, apocynin needs conversion into an active metabolite by MPO and hydrogen peroxide, but so far, the structure of this compound has not been elucidated (’t Hart and Simons, 1992, Stolk et al., 1994, Müller et al., 1999). Furthermore, apocynin, upon prior activation, revealed inhibitory activity towards LTB4-, fMLP-, and PAF-induced chemotaxis of PMNs, which was associated with an inhibition of cytoskeletal rearrangement after stimulation with fMLP (Müller et al., 1999). Another oxygen-related effect was discovered in the form of enhanced intracellular GSH synthesis, which appeared to be mediated by increased expression of γ-GCS mRNA and enzyme activity through activation of transcription factor AP-1 (Lapperre et al., 1999). These effects have led to the testing of apocynin in several experimental models pertaining to a wide range of diseases, especially those in which NADPH oxidase plays a role.

Anti-inflammatory activity Apocynin reduced joint swelling in collagen-induced arthritis in rats (’t Hart et al., 1990). Furthermore, it reversed the inhibition of cartilage matrix proteoglycan synthesis induced by mononuclear cells isolated from rheumatoid arthritis (RA) patients. These effects were accompanied by a reduced production of pro-inflammatory cytokines from monocytes and T cells, without affecting T-cell proliferation. Moreover, apocynin diminished the release of proteoglycan from cartilage matrix (Lafeber et al., 1999). Continuous administration of apocynin prevented the formation of ulcerative skin lesions induced by complete Freund’s adjuvant, without affecting humoral and cellular immune responses, as determined by serum antibodies and DTH responses (’t Hart et al., 1992). Moreover, apocynin concentration-dependently inhibited TXA2 formation, which effect was associated with an inhibition of arachidonic acid-induced aggregation of bovine platelets, whereas the release of PGE2 and PGF2α was stimulated (Engels et al., 1992, Lin et al., 1999).

Other activities In relation to the inhibitory effects of apocynin on NADPH oxidase activity, implications for several diseases have been investigated. It has been shown to be effective in mechanisms underlying, among others, sepsis (Wang et al., 1994, Mattson et al., 1996), atherosclerosis (Aviram et al., 1996, Holland et al., 1997, 1998, Chen et al., 1998), and asthma (Zhou and Lai, 1994, Tsukagoshi et al., 1994, 1995, Iwamae et al., 1998). Apocynin did not demonstrate genotoxic effects, as determined by the Ames test and the sister chromatid exchange assay (Pfuhler et al., 1995).

Conclusion

Picrorhiza species accumulate cucurbitacin glucosides, iridoid glucosides, phenylethanoid glucosides, and acetophenone glucosides. These compounds have a wide range of biological activities. Although hardly any information on the effects of the cucurbitacins from Picrorhiza is available, it seems reasonable that they exhibit effects similar to other cucurbitacins. In chapters 8 and 9, we confirm that some of the activities Constituents of Picrorhiza with immunomodulatory and related activities | 57 described here specifically relate to particular cucurbitacins isolated from Picrorhiza as well. Regarding the activities described for constituents of Picrorhiza, anti-inflammatory effects seem to prevail. As far as iridoid glucosides are concerned, the effects described mainly relate to a protection of the liver against damage and subsequent inflammation. In contrast, phenylethanoid glucosides exhibit anti-inflammatory effects by inhibiting pro- inflammatory responses, e.g. by diminishing phosphodiesterase and 5-lipoxygenase activities. Acetophenones like apocynin contribute to the anti-inflammatory effects by interfering with the formation of reactive oxygen species and enhancing the physiological antioxidant system, which lead to a wide range of secondary effects.

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