Cucurbitacins isolation

Audier and Das [48] published a specific report on the MS fragmentation of cucurbitacins which exemplified how the use of modem MS techniques leads to a better understanding of molecule fragmentation. In a similar vein, Johnson et al. [49] reported the diagnostic fragmentation pathways that occur in cucurbitacins isolated from Fevillea cordifolia after using electron impact (El)-, chemical ionization (Cl)-, and fast-atom bombardment (FAB)-MS. 

Jacobs et al. [1] used two-dimensional NMR spectroscopy to assign the H and 13C chemical shifts of cucurbitacin B, establishing these values as a benchmark for analyzing the spectra of other cucurbitacins isolated from the same source. In fact, the characteristic groups present in all cucurbitacins are easily detectable by means of NMR and typically include the presence of carbonyls at C-ll and C-22, which appear between 212-213 ppm, as well as hydroxyls at C-16(a) and C-20( 3), which appear at approximately 71-73 ppm and 77-79 ppm, respectively. Other characteristic patterns of substitution such as the presence of acetoxyl groups at C-2 and C-25 have also been observed. In this case, the acetylation of the hydroxyl at C-2 modifies both the C-l and C-3 values. Indeed, when a carbonyl is present, this latter change involves a major shift, namely from 213 to 205 ppm. In the case of a second double... 

Using assays involving EBV-EA activation and two-stage carcinogenesis of skin tumors, the same authors [73] had previously studied the inhibitory effects of nine cucurbitacins isolated from... 

Ubiquitous in seeds is phytic acid, the hexaphosphate ester of inositol, which has been isolated from cucurbit seeds (57). Small amounts of free sugars and terpenoid glycosides (cucurbitacins) are also present (58-60), but starch is absent (1, 1). Cellulosic cell wall materials comprise the remaining carbohydrate content. 

Finally, the triterpenoid cucurbitacin B, isolated from L. intrapetiolaris, showed activity in the KB (human oral epidermoid carcinoma) assay with EC50 value of 0.008 pg/ml. The clerodane diterpenes intrapetacin A and intrapetacin B displayed moderate activity vs KB as well [4],... 

Cucurbitacin is a bitter-tasting principle that can be isolated from members of the family Cucurbitaceae, such as cucumber Cucumis sativus) and melon (Cucumis melo L.). In particular, cucurbitacin149 and momordicine,150 which have an intensely bitter taste, are contained abundantly in Momordica charantia (bitter melon in English, go-yaa in Okinawa, Japan), which people enjoy due to its bitterness. There are more than 18 kinds of cucurbitacin, and among them cucurbitacin B is a typical component. It has been reported that cucurbitacin exhibits anticancer activity.1 1 In addition, it is used for the treatment of hepatic disease in traditional Chinese medicine. It is also found in some herbal teas. 

D-Glucopyranosylcucurbitacin I (25) and 2-0-/3-D-glucopyrano-sylcucurbitacin E (26) have been isolated from Citrullus lanatus.30 Several new cucurbitacins from Ecballium elaterium have been reported.31 They include cucur-bitacin R (dihydrocucurbitacin D) (27), anhydro-22-deoxo-3-epi-isocucurbitacin D (28), hexanorcucurbitacin (29), and 16-deoxy-A16-hexanorcucurbitacin O (30). 

Arvenins III (36) and IV (37) are two further cucurbitacin glucosides from Anagallis arvensis. The former was reported last year from Ecballium elaterium (see Vol. 9, p. 193) while the latter has been isolated, together with the related glucosides (38) and (39) and iso-23,24-dihydrocucurbitacin D (40), from the roots of Bryonia alba. °... 

Litsomentol (32), the simplest member of the cucurbitacin group, has been isolated from Litsea tomentosa. The structural proof was based on chemical and spectroscopic evidence and correlation with dihydroagnosterol via the epoxide (33). Kupchan and his colleagues have described the isolation and... 

Isocucurbitacin D (41) and 3-epi-isocucurbitacin D (42) are new tumour-inhibitory compounds from the leaves of Phormium tenax. They may be artefacts of isolation since they are both formed by isomerization of cucurbitacin D (43) on silica gel. Dihydroisocucurbitacin B (44) has been isolated from Marah... 

A partially purified enzyme preparation which catalyses the hydroxylation of the C-19 methyl group of cucurbitacins has been isolated from the ripe fruit of Cucurbita maxima. Cucurbitacins E and I act as feeding inhibitors for the flea beetle in the green parts of Iberis amara. ... 

The main kind of anti-inflammatory triterpenes isolated have oleanane, ursane, taraxastane, lupane and lanostane skeletons (Table 1). Some minor compounds such as hopane are included in other structural groups. Other anti-inflammatory triterpenes like the different cucurbitacins are not included in this review because of their high toxicity. 

In general, cucurbitacins are distinguished by use of correlative letters, e.g. cucurbitacin B however, some of these compounds are described by evoking a chemical modification, e.g. 23-dihydrocucurbitacin B or 3-epi-cucurbitacin F [2]. In still other cases, the name of the compound comes from the genus or species in which it was originally isolated, e.g. briogenin from Bryonia [9] or cayaponosides from Cayaponia tayuya [32-36],... 

Purification can be accomplished with different chromatographic protocols, but in most cases, open-column chromatography is used. For example, during the separation process of cucurbitacins from Cucurbita andreana, Jayaprakasam et al. [12] employed a medium pressure liquid chromatography (MPLC) system, using silica gel and mixtures of chloroform-acetone as eluent. A similar protocol was used by Peters et al. [27] during the isolation of the cucurbitacins from Wilbrandia ebracteata, but in this case the elution was performed first with mixtures of petroleum ether / ethyl acetate and then with ethyl acetate / i-propanol. 

Delporte et al. [13] isolated two cucurbitacins from Kageneckia oblonga using a complex method involving column chromatography on Amberlite, Sephadex LH-20, and MPLC. However, to separate the two major components of Cayaponia tayuya, Recio et al. [43] used a simple method based on gel-filtration through Sephadex LH-20 with dichloromethane as the mobile phase. 

Some species containing cucurbitacins are used in traditional medicine as anti-inflammatories in different pathologies. In general, they are used in topical applications as they have a certain level of toxicity when applied per os (p.o.). In one paper, Miro [2] compiled the data on this subject that had been published up to 1995. The review cited only the antiinflammatory activity of cucurbitacin B isolated from the juice of Ecballium elaterium, as well as its possible mechanism of action by means of a modification in leukotriene B4 (LTB4) production. 

Several of the compounds cited above, e.g. cucurbitacins B, D, and E, together with cucurbitacin I, Fig. (6), were isolated from the fruits of Cucurbita andreana and their activity against COX-1 and COX-2 enzymes was studied. All of the compounds studied exerted a moderate, but significant inhibition of the induced form COX-2, but none of them inhibited the constitutive form COX-1. The range of activity at 100 pg/ml was 32% (cucurbitacin B), 29% (cucurbitacin D), 35% (cucurbitacin E), and 27% (cucurbitacin I), all of which are clearly inferior to the values obtained with specific inhibitors of COX, such as ibuprofen, naproxen, or rofecoxib. Whereas cucurbitacin B was found to inhibit lipid peroxidation by 59% at 100 pg/ml, cucurbitacin I did so only by 23% at the same concentration while the other two compounds were inactive [12]. This mechanism may well be a complementary pathway of action of cucurbitacins in inflammation however, other mechanisms such as the inhibition of integrin-mediated cell adhesion [51] should also be given further consideration. 

Some of the pharmacological data reported for cucurbitacin B have been corroborated in different studies. In one paper, Yesilada et al. [52] reported on the isolation of cucurbitacin B from the fruit juice of Ecballium elaterium. They further showed how the compound significantly reduced the vascular permeability induced by acetic acid in mice, giving an effective dose-50 (ED50) of 6.1 mg/kg however, the... 

In another paper, Rios et al. [10] reported the anti-inflammatory activity of the chloroform extract from Cayaponia tayuya, which exhibited a high potency against carrageenan-induced mouse paw edema, with an ED50 of 122.5 mg/kg (p.o.) and 27.8 (i.p.). From the active extract, Recio et al. [43] isolated two cucurbitacins which were identified as cucurbitacin R and 23,24-dihydrocucurbitacin B, Fig. (7). 

Ukiya et al. [9] isolated twelve cucurbitacins from Bryonia dioica roots and studied the anti-inflammatory activity of six of them in a 12-0-tetradecanoylphorbol 13-acetate (TPA)-induced mouse ear edema. All six of the compounds showed anti-inflammatory activity with an ID50 range from 0.2 to 0.7 mg/ear however, no relationship was established between chemical structure and anti-inflammatory activity. The most potent compounds were bryoniosides B, E, and G, Fig. (9), which inhibited the edema by 94%, 94%, and 90%, respectively, with ID50 values of 0.2 mg/ear each. 

Different extracts obtained from species of Wilbrandia exhibited antiinflammatory activity when assayed in different experimental models of inflammation, such as carrageenan-induced rat hind paw edema, carrageenan-induced granuloma in rats, and acetic acid-induced vascular permeability in mice. The authors attributed the anti-inflammatory effect to the two nor-cucurbitacin glucosides isolated previously from the same source, namely cucurbitacins WGi and WG2, Fig. (10) [6]. 

From the anti-inflammatory, antipyretic, analgesic, and antioxidant extract of Kageneckia oblonga, Delporte et al. [13] isolated two cucurbitacins which were assayed as potential antioxidants and also as inhibitors of enzymes implicated in inflammatory reactions. Isolated compounds, 23,24-dihydrocucurbitacin F and 3P-( 3-D-glucosyloxy)-16a,23a-epoxy-cucurbitan-5,24-diene-ll-one, Fig. (11), inhibited the production of superoxide anion as well as elastase release in stimulated human neutrophils. In addition, the compounds inhibited both nitrite and... 

Both the extracts containing cucurbitacins and the isolated compounds themselves have been reported as analgesic agents. The dichloromethane extract from Wilbrandia ebracteata roots administered i.p., for example, had an analgesic effect in the acetic acid writhing test in mice, reducing... 

In a similar screening, Konoshima et al. [72] studied the inhibitory effects of twenty-four 29-nor-cucurbitacin glucosides isolated from the roots of Cayaponia tayuya and found that five of them, cayaponosides B, B3, D, D3b, and C2, exhibited significant inhibitory effects on EBV activation induced by the tumor promoter TPA. Moreover, two of the cucurbitacins shown to be active in vitro, cayaponosides B and C2, Fig. (14), inhibited mouse skin tumor promotion in a two-stage in vivo carcinogenesis test. 

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