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Stability of the constituents of Calendula, milk-thistle and passionflower tinctures by LC-DAD and LC-MS.

As a part of our investigations on the stability of tinctures, we evaluated 40 and 60% v/v tinctures of Calendula flower, Milk-thistle fruit and Passionflower. These preparations are widely employed in phytotherapy, thus Calendula is used externally for anti-inflammatory properties, Milk-thistle and Passionflower are employed for hepatic injuries and in tenseness with difficulty in falling asleep, respectively. Aim of this work was to assess the chemical stability of their active or marker constituents from accelerated and long-term testing by using HPLC. For Calendula flower and Passionflower active constituents are not known, however, flavonoids seem to have a crucial importance for the activity, and thus are considered the markers of Calendula and of Passionflower. Active constituents of Milk-thistle are represented by silymarin that is a phytocomplex mainly constituted by three flavolignans: silybin, silychristin and silydianin. Our investigation showed a very low thermal stability of the constituents from accelerated and long-term testing and determined by HPLC-DAD and -MS analyses and was related both to the class of flavonoids and water content of the investigated tinctures. Thus, shelf-lives at 25 degrees C of the most stable tincture (Passionflower 60% v/v) was about 6 months and only about 3 months the stability of Milk-thistle tinctures.

Calendula↗

Plant analysis by butterflies: occurrence of cyclopentenylglycines in Passifloraceae, Flacourtiaceae, and Turneraceae and discovery of the novel nonproteinogenic amino acid 2-(3'-cyclopentenyl)glycine in Rinorea.

Following records about feeding habits of nymphalid butterflies, a novel nonproteinogenic L-amino acid, (S)-2-(3'-cyclopentenyl)glycine (11), was discovered in Rinorea ilicifolia, a species where the presence of a cyclopentanoid natural product of this kind was neither known nor anticipated from the taxonomic point of view. Another novel amino acid, (2S,1'S,2'S)-2-(2'-hydroxy-3'-cyclopentenyl)glycine (12), the stereochemistry of which was determined by single-crystal X-ray diffraction, was shown to occur in species belonging to Flacourtiaceae, Passifloraceae, and Turneraceae. These species, many of which serve as hosts for nymphalid butterflies (Acraeinae, Heliconiinae, Argynninae), also produce 2-(2'-cyclopentenyl)glycine. Cyclopentenylglycines are proposed to be novel chemical recognition templates for plant-insect interactions. Ratios between the epimers of (2S)-2-(2'-cyclopentenyl)glycine, which co-occur in plants, were determined by (1)H NMR spectroscopy. Contrary to a previous report, the (2S,1'R) epimer always appears to predominate over the (2S,1'S) epimer. Stereochemical aspects of biosynthesis of natural cyclopentanoid cyanogenic glycosides are discussed in relation to these findings.

Animals↗

Inhibition of neoplastic transformation of benzo[alpha]pyrene-treated BALB/c 3T3 murine cells by a phytochemical extract of passionfruit juice.

The phytochemical composition of passionfruit juice (PFJ) was hypothesized to have valuable anti-cancer activity, and this was tested in a BALB/c 3T3 neoplastic transformation model. A higher concentration of PFJ compared with a lower concentration was effective in reducing the number, size, and invasiveness of transformed foci. When incubated with another mammalian cell line, the MOLT-4, PFJ was unable to alter the cell cycle kinetics while at the same time was successful in inducing the activity of caspase-3, an enzyme that commits the cell to apoptosis. This suggests that phytochemicals found in PFJ were able to produce the changes in transformed foci due to apoptotic mechanisms rather than by a reduction in cell proliferation. These beneficial results were achieved at levels that could theoretically be attained in the plasma after consumption of the juice.

3T3 Cells↗

Passionflower fruit-a "new" source of lycopene?

Many population studies have established a link between dietary intake of the carotenoid antioxidant lycopene and a reduced risk of chronic diseases. Unlike most carotenoids, lycopene occurs in a few places in the diet. Besides tomatoes and tomato products, major sources of lycopene, other lycopene-rich foods include watermelon, pink grapefruit, pink guava, and papaya. Dried apricots and pureed rosehips contain relatively large amounts, too. In our study we found that passionflower fruit (skin and pericarp) contains a great amount of lycopene, whereas the content of other carotenoids is very low, and almost inexistent. This edible fruit could be an alternative source of a potential important nutrient for those people who do not eat tomatoes and tomato products.

Antioxidants↗

Potent inhibition by star fruit of human cytochrome P450 3A (CYP3A) activity.

There has been very limited information on the capacities of tropical fruits to inhibit human cytochrome P450 3A (CYP3A) activity. Thus, the inhibitory effects of tropical fruits on midazolam 1'-hydroxylase activity of CYP3A in human liver microsomes were evaluated. Eight tropical fruits such as common papaw, dragon fruit, kiwi fruit, mango, passion fruit, pomegranate, rambutan, and star fruit were tested. We also examined the inhibition of CYP3A activity by grapefruit (white) and Valencia orange as controls. The juice of star fruit showed the most potent inhibition of CYP3A. The addition of a star fruit juice (5.0%, v/v) resulted in the almost complete inhibition of midazolam 1'-hydroxylase activity (residual activity of 0.1%). In the case of grape-fruit, the residual activity was 14.7%. The inhibition depended on the amount of fruit juice added to the incubation mixture (0.2-6.0%, v/v). The elongation of the preincubation period of a juice from star fruit (1.25 or 2.5%, v/v) with the microsomal fraction did not alter the CYP3A inhibition, suggesting that the star fruit did not contain a mechanism-based inhibitor. Thus, we discovered filtered extracts of star fruit juice to be inhibitors of human CYP3A activity in vitro.

Actinidia↗

The microflora of frozen passionfruit nectar base.

A considerable and varied microflora is introduced into passionfruit nectar base under present methods of production. In spite of the great acidity of the nectar base (pH range: 2.8 to 3.2), the high sucrose concentration (approximately 50%), and storage at -20 C, remnants of the microflora persist for a year or longer. During storage, however, there is a steady and gradual decrease, until after about 18 months the microflora is near to extinction. Sample regression lines show straight-line slopes for this diminution in numbers.A battery of nine media was used to grow a representative aerobic flora. Purified cultures of isolates were identified to genera. Yeasts were the most numerous organisms in all samples, followed by molds, bacteria, and streptomycetes. The bacteria were the first group to disappear during storage. No fecal streptococci or gram-negative bacilli were found in any samples.

Bacteria↗

Survival times of selected enteropathogenic bacteria in frozen passionfruit nectar base.

FIVE TEST ORGANISMS WERE USED: Escherichia coli, Salmonella typhosa, Salmonella schottmuelleri, Salmonella enteritidis, and Shigella paradysenteriae. Even when large inocula of these test cultures were introduced into fresh passionfruit nectar base, all test organisms were killed within 1 to 2 hr, provided the nectar base was held at room temperature for more than 1 hr before freezing. If the nectar base was frozen immediately after inoculation, four of the five test organisms were eliminated almost as quickly. But the fifth, Salmonella enteritidis, proved to be exceptional: it was being recovered after 90 days of storage at -20 C, when the last available sample was analyzed.

Escherichia coli↗

Effect of arbuscular mycorrhizal fungi and soil phosphorus level on expression of protein and activity of peroxidase on passion fruit roots.

The effects of mycorrhizal inoculation and increasing soil P levels on the expression of total proteins and peroxidase activity on passion fruit roots were evaluated. The experimental design was entirely at random, with four treatments of inoculation (a--control; b--Gigaspora albida; c--Scutellospora heterogama; d--mixture of G. albida, G. margarita, S. heterogama, and Glomus clarum) x three levels of soil P (4, 11, and 30 mg/dm3 of soil), each with three replicates. Plants were harvested 70 days after inoculation, when root colonization, shoot P level, protein content, and enzymatic activity of peroxidase (PAGE--7%) on root extract were evaluated. Regarding protein, there was no significant difference among the treatments, except between those roots receiving mixed inoculum and 11 mg P/dm3 of soil. Effect of P on protein concentration, when compared with the inoculation effect was observed. For peroxidase, there was an eletrophoretic band common to all treatments (rf: 0.43) and another that was absent only in noncolonized plants, grown in soil with lower P (rf: 0.46). Mycorrhizal specific bands were not present but a small decrease of intensity of bands in noncolonized plants was observed. Conversely, the control roots presented a single band (rf: 0.33) not observed in the other extracts, that may demonstrate an inhibitory effect of AMF on some host activities. The data showed the influence of P level in soil on the protein expression of roots, suggesting the influence of this nutrient on root genetic expression as well as on the mechanisms of symbiotic control/recognition.

Fungi↗

Stability testing on typical flavonoid containing herbal drugs.

The aim of the presented work was to examine possible changes in the flavonoid pattern of common flavonoid containing herbal drugs during long term and stress testing storage periods. HPLC fingerprint was used to demonstrate the differences in stability of individual flavonoid components. In addition, the total flavonoid content was determined according to the pharmacopoeial photometrical method. Drug material was stored according to the ICH-guidelines at 25 degrees C and 60% rh (relative humidity) for long term testing over a 24 months period or at 40 degrees C and 75% rh under stress conditions for 6 months. Increased temperatures of 80 degrees C and 100 degrees C were chosen to elucidate possible instabilities of selected flavonoids. As an overall result, during long term testing, no significant changes in the flavonoid pattern can be detected. However, some flavonoid containing herbal drugs (e.g. birch leaves), showed a decrease of most flavonoids when stored at high temperature by an increase in the respective aglycones. Similar results were obtained during storage at 40 degrees C/75% rh.

Betula↗