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Supramolecular phospholipids–polyphenolics interactions: The PHYTOSOME® strategy to improve the bioavailability of phytochemicals
Fitoterapia 81 (2010) 306–314
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Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f i t o t e
Review
Supramolecular phospholipids–polyphenolics interactions: The PHYTOSOME® strategy to improve the bioavailability of phytochemicals Ajay Semalty a,⁎, Mona Semalty a, Mohan Singh Maniyari Rawat b, Federico Franceschi c,⁎ a b c
Department of Pharmaceutical Sciences, H.N.B. Garhwal University Srinagar (Garhwal), India Department of Chemistry, H.N.B. Garhwal University Srinagar (Garhwal), India Research and Development Laboratories, Indena S.p.A. (Settala), Italy
a r t i c l e
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Article history: Received 20 October 2009 Accepted in revised form 3 November 2009 Available online 14 November 2009 Keywords: Flavonoid Bioavailability PHYTOSOME® Herbal drugs Phospholipid complex
a b s t r a c t The poor and/or erratic oral bioavailability of polyphenolics can be improved using the PHYTOSOME® 1 delivery system, a strategy that enhances the rate and the extent of solubilization into aqueous intestinal fluids and the capacity to cross biomembranes. Phospholipids show affinity for polyphenolics, and form supramolecular adducts having a definite stoichiometry. This article reviews the preparation and characterization of PHYTOSOME® complexes and their activity in various medicinal (cardiovascular, anti-inflammatory, hepatoprotective, anticancer) and cosmetic (skin aging) realms of application. © 2009 Elsevier B.V. All rights reserved.
Contents 1. 2. 3. 4.
Introduction . . . . . . . . . . . . . . . . . . . . Physical and chemical properties . . . . . . . . . . Methods of preparation . . . . . . . . . . . . . . . Characterization . . . . . . . . . . . . . . . . . . 4.1. Spectroscopy . . . . . . . . . . . . . . . . 4.2. Thermal gravimetric analysis (TGA)/differential 5. Biological profile of PHYTOSOME® adducts . . . . . . 5.1. Cardiovascular properties . . . . . . . . . . 5.2. Anti-inflammatory properties . . . . . . . . . 5.3. Anti-aging properties . . . . . . . . . . . . 5.4. Hepatoprotective properties . . . . . . . . . 5.5. Anticancer properties . . . . . . . . . . . . 5.6. Weight management . . . . . . . . . . . . . 6. Conclusions . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . .
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⁎ Corresponding authors. Semalty is to be contacted at Department of Pharmaceutical Sciences, PB No.-32, H.N.B. Garhwal University Srinagar (Garhwal)-246174, India. Franceschi, Indena S.p.A., via Don Minzoni 6, 20090 Settala (MI), Italy. E-mail addresses: [email protected] (A. Semalty), [email protected] (F. Franceschi). 1 PHYTOSOME® is a registered trademark of Indena S.p.A. Milan, Italy. 0367-326X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2009.11.001
A. Semalty et al. / Fitoterapia 81 (2010) 306–314
1. Introduction For good bioavailability, natural products must have a good balance between hydrophilicity (for dissolving into the gastrointestinal fluids) and lipophilicity (to cross lipidic biomembranes). Many phytoconstituents like polyphenolics have good water solubility, but are, nevertheless, poorly absorbed [1] because of their large size, incompatible with a process of passive diffusion and/or their poor miscibility with oils and other lipids. As a result, the ability of flavonoids to cross the lipid-rich outer membrane of small intestine enterocytes is severely limited. Water-soluble phytoconstituents (mainly polyphenolics) can be converted into a lipid-compatible molecular complex known as PHYTOSOME®. A PHYTOSOME® is generally more bioavailable than a simple herbal extract due to its enhanced capacity to cross the lipid-rich biomembranes and reach circulation [2–5]. Phospholipids are small lipid molecules where glycerol is bonded to two fatty acids, with the third hydroxyl, normally one of the two primary methylenes, bearing a phosphate group [6]. Phospholipids from soy, mainly phosphatidylcholine, are lipophilic substances and readily complex polyphenolics. In this context, phosphatidylcholine, the major molecular building block of cell membranes and a compound miscible in both water and in oil/lipid environments, is well absorbed orally, and has the potential to act as a chaperon for polyphenolics, accompanying them through biological membranes [7]. PHYTOSOME® complexes were developed at Indena (Milan, Italy) in the late Eighties. Many popular standardized herbal extracts [e. g. Ginkgo biloba L., grape (Vitis vinifera L.) seeds, milk thistle (Silybum marianum (L.) Gaertn), green tea (Camellia sinensis (L.) O. Kuntze), ginseng (Panax ginseng C.A. Meyer), licorice (Glycyrrhiza glabra L.), horse chestnut (Aesculus hippocastanum L.), Centella asiatica (L.) Urban, olive (Olea europea L.), Terminalia sericea Burch. Ex. DC, Amni visnaga (L.) Lam, turmeric (Curcuma longa L.) and hawthorn (Crataegus spp.)] are currently commercially available in the PHYTOSOME® form (see Table 1). Flavonoids and terpenoids from herbal extracts undergo different absorption pathways
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when bound to phospholipids, and this article reviews various aspects and the latest trends of PHYTOSOME® research, highlighting recent advances in their therapeutic potential. 2. Physical and chemical properties The first PHYTOSOME® generation was prepared by combining selected polyphenols or polyphenol extracts with phospholipids in non-polar solvents [2], but, more recently, a newer PHYTOSOME® generation was developed using hydro-ethanolic solvents. Products obtained in this way comply with current food specifications [8,9], and expand the PHYTOSOME®' potential from the pharma/cosmetic field to the health-food one. A PHYTOSOME® is an amphiphilic substance with a definite melting point, generally soluble in nonpolar solvents (where its hydrophilic moiety is not), and moderately soluble in fats. The low solubility in aqueous media makes the formation of stable emulsions and creams possible (Fig. 1), improving the biopharmaceutical properties of both highly lipid insoluble and poorly water-soluble phytoconstituents. The PHYTOSOME® formulation increases the absorption of active ingredients when topically applied on the skin [10–19], and improves systemic bioavailability when administered orally [20–24]. In water medium, a PHYTOSOME® will assume a micellar shape, forming a liposome-like structure. Fundamental differences exit, however, between a PHYTOSOME® and a liposome. In liposomes, the active principles are dissolved in the central part of the cavity, with no possibility of molecular interaction between the surrounding lipid and a hydrophilic substance. On the contrary, the PHYTOSOME® complex can somewhat be compared to an integral part of the lipid membrane (Fig. 2), where the polar functionalities of the lipophilic guest interact via hydrogen bonds with the polar head of a phospholipid (i.e. phosphate and ammonium groups), forming a unique arrangement that can be evidenced by spectroscopy [10,11,18,25–28]. Thus, IR and multi-nuclear spectroscopic studies show that a PHYTOSOME® is not a mechanical mixture of two constituents,
Table 1 Available PHYTOSOME® complexes on the market. PHYTOSOME® and all other trademarks are owned by Indena S.p.A. Milan, Italy. Trade name
Phytoconstituent complexed with phospholipid
Indication
Escin ß-sitosterol Phytosome® Siliphos® Silymarin Phytosome® Meriva™ Virtiva® Ginkgoselect® Phytosome® Ginselect® Phytosome® Leucoselect® Phytosome® Centella Phytosome® 18 ß-glycyrrhetinic acid Phytosome® Crataegus Phytosome® Ginkgo biloba Dimeric Flavonoids Phytosome® Ginkgo biloba Terpenes Phytosome® Sericoside Phytosome® Greenselect® Phytosome®
Escin ß-sitosterol from horse chestnut fruit Silybin from milk thistle seed Silymarin from milk thistle seed Curcuminoids from turmeric rhizome Ginkgoflavonglucosides, ginkgolides, bilobalide from Ginkgo biloba leaf Ginkgoflavonglucosides, ginkgolides, bilobalide from Ginkgo biloba leaf Ginsenosides from Panax ginseng rhizome Polyphenols from grape seed Triterpenes from Centella asiatica leaf 18 ß-glycyrrhetinic acid from licorice rhizome Vitexin-2″-O-rhamnoside from Hawthorn flower Dimeric flavonoids from Ginkgo biloba leaf
Anti-oedema Hepatocyte protection Antihepatotoxic
Visnadex® PA2 Phytosome®
Visnadin from Amni visnaga umbel Proanthocyanidin A2 from horse chestnut bark
Ginkgolides and bilobalide from Ginkgo biloba leaf Sericoside from Terminalia sericea bark root Polyphenols from green tea leaf
Vasokinetic Vasokinetic Skin elasticity improver, adaptogenic Antioxidant, capillarotropic Cicatrizing, trophodermic Soothing Antioxidant Lipolytic, vasokinetic Soothing Anti-wrinkles Prevention of free radical-mediated tissue damages and weight management Vasokinetic Anti-wrinkles, UV protectant
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depending on the protocol employed. Thus, the phospholipid complex of curcumin [34] prepared in an aprotic solvent [35] showed remarkable differences from the one prepared in a protic solvent [8], and three different silybin–phospholipid complexes have been reported. Two of them were prepared in aprotic solvent [Silipide (IdB 1016) [30], a pharma-grade PHYTOSOME® which has been extensively characterized [27], and Siliphos®], while a third silybin–phospholipid complex was obtained in protic solvents [32]. Different phospholipids afford different complexes, as found for G. biloba extracts, whose PHYTOSOME® extract with phosphatidylserine complex is known as Virtiva®, while the standard phosphatidylcholine complex is known as Ginkgoselect® PHYTOSOME®) [31]. Fig. 1. Electron-microscopic examination of lyophilized microdispersion of Ginselect® PHYTOSOME® 0.1% in water. Magnification 1 × 4000.
but a specific complex between a hydrophilic guest and a lipophilic host characterized by specific dipolar interactions, in accordance with the spectroscopic differences between a PHYTOSOME and a mechanical mixture of its two constituents. 3. Methods of preparation A PHYTOSOME® is prepared by complexing a polyphenolic phytoconstituent or mixture with a phospholipid. Depending on the product, mass ratios in the 1:1.5–1:4 are observed. Various preparation methods for a PHYTOSOME® [3,8,9,12– 16,19,29–33] and the resulting complex can be different
Fig. 2. Major difference between liposome and PHYTOSOME®.
4. Characterization 4.1. Spectroscopy Complexation and molecular interactions between phytoconstituents and phosphatidylcholine in solution have been studied by 1 H-NMR [2,8,10,25–27], 1 3 C-NMR [2,8,10,27], 31P-NMR, [8–10] as well as by IR spectroscopy [18]. Complex formation is associated with changes of chemical shift and line broadening of some characteristic signals in NMR spectra and with appearance of new bands in IR spectra. 4.2. Thermal gravimetric analysis (TGA)/differential scanning calorimetry (DSC) Detection and measurement of thermal effects such as fusion, solid–solid transitions, glass transitions, loss of solvent, and decomposition, can be used to characterize a solid PHYTOSOME®. The TG/DT analyses of two complexes and the mechanical mixtures of phytoconstituent and phospholipid components are reported in Fig. 3 [36]. The DT profile of Silymarin PHYTOSOME ® (green solid line in Fig. 3a) shows an endothermic peak with a maximum at about 173 °C, corresponding to a ca. 3% weight loss, and a less intense and broader endothermic signal with a maximum at about 148 °C. The TG profile (blue solid line in Fig. 3a) of some PHYTOSOME® adducts shows an initial loss on drying below 100 °C (about 4%), and a massive weight loss starting at about 175 °C due to degradation. The TG/DT profiles of the phytoconstituent/phospholipid mechanical mixture (dashed lines) show two broad endothermic peaks, the first one with a maximum at about 146 °C and corresponding to a weight loss of ca. 2%, and the second one at about 167 °C with a loss of weight of ca. 3% followed by the massive weight loss expected for degradation. Fig. 3b shows the TG/DT profile of Ginkgoselect® PHYTOSOME® and the one of a Ginkgoselect®/phospholipid mechanical mixture. The DT profile of the PHYTOSOME® (green solid line in Fig. 3b) shows only an endothermic peak with a maximum at about 179 °C, linked to a weight loss of about 2%. Conversely, the TG profile (blue solid line) shows an initial loss on drying below 100 °C (about 2%) and a massive weight loss starting at about 180 °C due to degradation.
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Fig. 3. a: TG curves (blue) and DT profiles (green) of Silymarin PHYTOSOME® (solid line) and silymarin/phospholipid mechanical mixture (dashed line). b: TG curves (blue) and DT profiles (green) of Ginkgoselect® PHYTOSOME® (solid line) and Ginkgoselect®/phospholipid mechanical mixture (dashed line).
5. Biological profile of PHYTOSOME® adducts
PHYTOSOME® to treat peripheral vascular disease (e.g., Raynaud's disease and intermittent claudication) was found to be 30–60% higher than that of Ginkgoselect® [37]. Ginkgoselect® PHYTOSOME® was also recently validated as a cardioprotective agent [38]. The two G. biloba L. PHYTOSOME® complexes showed different cognitive effects, with the phosphatidylserine complex (Virtiva® ) being active at lower doses than the standard phosphatidylcholine complex (Ginkgoselect® PHYTOSOME®) [31,39]. The activity of Leucoselect® PHYTOSOME® to decrease low-density lipoprotein susceptibility to oxidation and oxidative stress damage in a group of heavy smokers was also investigated, with positive results [40].
5.1. Cardiovascular properties
5.2. Anti-inflammatory properties
Preliminary studies have shown that the PHYTOSOME® of G. biloba L. and that of grape seed extracts are more potent than their non-complexed forms. The efficacy of Ginkgoselect®
PHYTOSOME® complexes show better anti-inflammatory activity than their uncomplexed herbal extracts. Croton oilinduced dermatitis was used, as an inflammation model
The TG/DT profiles of the mechanical mixture of Ginkgoselect® and a phospholipid (dashed lines) shows two broad endothermic peaks: the first with a maximum at about 135 °C simultaneous with a loss of weight of about 3%, and the second with a maximum at about 171 °C with a loss of weight of about 2% followed by a massive weight due to degradation. In both cases, phytoconstituent–phospholipid molecular interactions in the complex lead to a radically different thermal profile and behavior of the PHYTOSOME® complex compared to its corresponding mechanical mixture.
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involving both tissue and vascular damage, to evaluate the anti-inflammatory activity of PHYTOSOME® complexes in animals [18]. The results obtained with glycyrrhetinic acid, a powerful anti-inflammatory and anti-allergic triterpenoid, and with silymarin, a free radical scavenger flavanolignan endowed with anti-phlogistic activity, were discussed. The products were applied topically as microdispersion in water, and the reduction of edema was used as the end point. Phospholipids are per se devoid of anti-inflammatory activity, but it was found that their complexation with glycyrrhetinic acid prolonged, its activity. Thus, the anti-edematous activity of 18-β-glycyrrhetinic acid gradually waned with time and was almost completely terminated after 24 h, a time at which its phosphatidyl complex still induced an 80% reduction in edema. The silymarin complex showed greater reduction of edema (76% reduction in 6 h) as compared to that with the free form (33% even after 12 h) in the croton oil test in mice (Fig. 4). Therefore, the PHYTOSOME® was about two-fold more effective than the free phytoconstituent. In another study, the 18-β-glycyrrhetinic acid complex exhibited potent activity against the erythema secondary to UVA radiation [28]. 5.3. Anti-aging properties PHYTOSOME® as a delivery system offers interesting applications and opens new opportunities for the use of active ingredients in the cosmetic field. The G. biloba PHYTOSOME® was investigated for the treatment of skin aging connected to superficial capillary blood flow circulation. Extracts from G. biloba are used orally to improve peripheral circulation [41], and their phospholipid complexes were found to improve skin microcirculation after topical application. Activation of microcirculation meliorated skin aging associated to dystrophic alteration of the epidermis and dermis, and the regressive abiotrophic pannicular disease of lower limbs and breast associated to venous stasis and/or chronic venous insufficiency. The role of PHYTOSOME® in
Fig. 4. Reduction of edematous response to croton oil-induced by silymarin and its complex with distearoylphosphatidylcholine at different times (dose 0.5 µmol/ear).
functional cosmetics was reviewed [25], and the use of Silymarin PHYTOSOME® on aging skin was also reported [18,42]. 5.4. Hepatoprotective properties Most studies on PHYTOSOME® are focused on Silybum marianum Gaertner and its liver-protectant flavolignans. The fruit of milk thistle (Silybum marianum) contains flavonoids showing hepatoprotective properties [43,44]. Silymarin has been shown to be effective in the treatment of liver diseases of various kinds, including hepatitis, cirrhosis, fatty infiltration of the liver (chemical and alcohol induced fatty liver), and inflammation of the bile duct [45–48]. The antioxidant properties of silymarin substantially boost the liver resistance to toxic insults [49]. Silymarin has a complex composition, based on three patterns of covalent interaction between its flavonol and lignan constituents (Fig. 5). Silybin, in turn the mixture of two diastereomers, is the major and more potent constituent of silymarin [43]. Silybin protects the liver by conserving glutathione in the parenchymal cells [49], while phosphatidylcholine helps repair and replace cell membranes [7]. These constituents act synergistically, sparing liver cells from destruction. While silybin was found active in many models of human disease, clinical translation of these results was hampered by its extremely poor oral bioavailability. In a pharmacokinetic study [32], the oral bioavailability of silybin was increased remarkably when administered as silybin– phospholipid complex. The increased lipophilicity of the silybin–phospholipid complex, have been claimed to explain the improved bioavailability [7]. AFB1 (aflatoxin B1, a fungal compound produced by various strains of Aspergillus flavus and Aspergillus parasiticus) causes aflatoxicosis in chickens, and is characterized by mortality, listlessness, anorexia, decreased growth rates, negative feed conversions, fatty liver, decreased egg production, poor pigmentation, and increased susceptibility to other diseases. For these reasons, intensive research has been carried out to develop cost-effective and safe methods to reduce the effects of AFB 1 . Remarkably, Silymarin PHYTOSOME® showed better antihepatotoxic activity than silymarin alone, and could provide protection against the toxic effects of aflatoxin B1 on safety and quality performance of broiler chickens [50]. A series of studies on silymarin administered as Silymarin PHYTOSOME® found that the complex could protect the fetus from maternally ingested EtOH [51]. In similar studies, it was reported that Silymarin PHYTOSOME® shows a better fetoprotectant activity against ethanol-induced behavioral deficits than uncomplexed silymarin on fetuses of both sexes [52]. In the first study, gamma glutamyl transpeptidase (GGTP) activity was determined for liver and brain tissue from both the fetus and the dams on day 21 of pregnancy, while the offspring were tested at age 90 days on the social recognition task and at 75 days on the radial arm maze in the later study. Female EtOHexposed offsprings performed more poorly on the radial arm maze than did female EtOH/Silymarin PHYTOSOME® offsprings and that of controls. In a similar way, male EtOH-exposed offsprings were less able to form social memories than the male EtOH/Silymarin PHYTOSOME® offsprings and the male offspring control.
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Fig. 5. Flavolignans of Silybum marianum and hydrogen bond interactions in phosphatidylcholine complex of silybin.
Silymarin PHYTOSOME®, was also found to have a much higher and specific activity and a longer lasting activity than any single silymarin component in terms of oedema, inhibition of myeloperoxidase activity, antioxidant and free radical scavenging properties [42]. When silybin was administered as Silipide (a pharmagrade silybin–phospholipid complex) at a dose of 200 mg per kg body weight, plasma concentration of silybin remained high 70 h after the oral dosing, while, after 25 h, silybin barely rose above the detection threshold [21]. In another study on rats, silybin given as Silipide at 200 mg/kg was detectable in the plasma within minutes, peaked after 1 h, and its plasma levels remained elevated for over 6 h. Silipide was also shown to rapidly reach the liver, cross the liver cells, and appear in the bile within 2 h. The amount of silybin reaching the bile from PHYTOSOME® dosing was at least 6.5 times higher than that from non-complexed silybin (13% versus 2%, over 24 h) [22]. In general, Silipide was found to show higher pharmacological potency in animal models of hepatic injury than uncomplexed silybin, and the oral bioavailability and organ distribution profile of Silipide was much better than that of silybin from silymarin [53]. The pharmacokinetics of Silipide in healthy human subjects showed that complexation with phosphatidylcholine improved the oral bioavailability of silybin 4.6 fold compared with silymarin, probably because
of a facilitated passage across the gastrointestinal mucosa [20]. A pilot study on the liver protective effect of Silipide in chronic active hepatitis (CAH) found that Silipide improved liver function tests related to hepatocellular necrosis and/or increased membrane permeability in patients affected by CAH [54]. The iron chelating properties of Silipide were also investigated during a 12 week study on 37 patients with chronic hepatitis C and Batts–Ludwig fibrosis. Treatment with silybin–phosphatidylcholine complex was clearly associated with a reduction in iron stores in patients with advanced fibrosis [55]. Hepatitis C infection was also improved by a combination of Silipide and vitamin E (SPV) [56]. In another study, the role of SPV complex for the treatment of non alcoholic fatty liver disease (NAFLD) was investigated [57]. Significant correlation among indexes of fibrosis, body mass, insulinemia, plasma levels of transforming growth factor-β, tumor necrosis factor-α, degree of steatosis and γ-glutamyl transpeptidase was observed. Reduction in ultrasonographic scores for liver steatosis and improvement of liver enzyme levels, hyperinsulinemia and indexes of liver fibrosis suggest that SPV complex could be used as a complementary approach to the treatment of patients with chronic liver damage.
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The addition of increasing concentrations of Silipide to isolated rat hepatocytes caused a dose-dependent inhibition of lipid peroxidation induced by cumene hydroperoxide, concluding that Silipide acted as potentially useful protective agent against free radical mediated toxic liver injury [58]. Prompted by these observations, a phase II randomized open clinical trial to evaluate the dose response relationship of Silipide in patients with viral or alcoholic hepatitis was carried out, finding a dose-dependent improvement [59]. In a pilot study on eight patients with chronic active hepatitis B and/or C, treatment with Silipide, at 240 mg silybin, for two months, the liver enzymes alanine aminotransferase (ALT) and aminotransferase (AST) were significantly reduced, while glutamyltranspeptidase (GGT) and malondialdehyde (MDA) levels, a byproduct of lipid peroxidation were not statistically improved [60]. Healthy volunteers (total number not disclosed) receiving 360 mg Silipide three times daily for three weeks did not show any adverse effect [61], while treatment of 232 patients with liver disorder for up to four months with either 240 or 360 mg Silipide daily showed an excellent tolerability. Minor adverse effects (nausea, heartburn, dyspepsia, transient headache) were reported only in 12 patients (5.2% of the total studied), compared with 8.2% of patients who received uncomplexed silybin and 5.1% of patients treated with the placebo. In this study, Silipide produced no clinically relevant blood changes, and its efficacy was evaluated by measuring serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma-glutamyltranspeptidase (GGT). Besides antioxidant activity, Silipide has also the ability to scavenge ethanol-derived free radicals damage, and has therefore potential to prevent liver damage from alcohol abuse [62]. Silipide was also able to scavenge free radicals generated by CCl4 or INH [63]. Silipide was also investigated in rodents in various models of liver damage. After oral administration, Silipide exhibited a significant and dose related protective effect against hepatotoxicity induced by CCl4, acetaminophen, ethanol and galactosamine [64]. A controlled clinical study on the effect of Silipide in chronic persistent hepatitis was carried out [65]. The drug treatments available for this condition have limited efficacy, do not work at all for many patients, and have major adverse effects. In this study, patients were randomized to receive either 240 mg Silipide (n =31) or placebo (n=34) twice daily for three months. The Silipide group showed significant lowering of both serum ALT and AST, while in the placebo group these markers worsened. The Silipide treatment was well tolerated, with fewer adverse events than for the placebo group, and no patient discontinued the trial due to adverse effects. A quercetin–phospholipid complex was also developed, and investigated in a murine model of liver injury (carbon tetrachloride-induced damage) [23]. In this study, treatment with free quercetin (20 mg/kg) as well as quercetin– phospholipid complexes (10 mg/kg and 20 mg/kg) showed significant (p b 0.05) decrease in thiobarbituric acid reactive substance (TBARS) levels in liver homogenate when compared to CCl4-treated animals. It was also observed that lower doses of uncomplexed quercetin (10 mg/kg) failed to produce significant results. Quercetin at 20 mg/kg in phospholipid complex gave better results than free quercetin (20 mg/kg) restoring the normal enzyme levels (Fig. 6).
Fig. 6. Effect of quercetin–phospholipid complex on TBARS. Values are mean ± SEM (n = 6); *p b 0.05 [significant with respect to control (CCl4-treated group)], complex denotes quercetin–phospholipid complex.
The phospholipid complexes of naringenin [66] and curcumin [33] were also investigated. The phospholipid complex of naringenin produced better antioxidant activity than the free compound with a prolonged duration of action, while the curcumin–phospholipid complex was active on carbon tetrachloride-induced acute liver damage in rats, with significantly higher aqueous and n-octanol solubility and higher antioxidant activity at all doses investigated. 5.5. Anticancer properties Over the past few years, silymarin and silybin have received a growing attention for properties unrelated to liver protection, like anticancer, chemopreventive, hypocholesterolemic, cardioprotective, neuroactive and neuroprotective activities [67]. The anti-radical potential and the cytoprotective activity may be responsible for the use of silymarin as a chemoprotective and anticancer agent. Due to their chemopreventive activity, silybin and silymarin can inhibit the carcinogenic activity of many chemicals [68], and have the potential to be successfully used in the adjuvant therapy of cancer. In this context, silybin might act essentially as an antioxidant, protecting tissues against the oxidative stress generated by chemotherapeutics, preventing at the same time hepatotoxicity, a major side-effect of many chemotherapy treatment. A phase I and pharmacokinetic study of silybin-PHYTOSOME® in prostate cancer patients was carried out [69]. The study concluded that oral silybinPHYTOSOME® at a daily dose of 13 g seems well tolerated in patients with advanced prostate cancer, and this dosage was recommended for the phase II study [69]. Silybin and its phospholipid complex IdB1016 have been shown to have antitumor activity and to potentiate the effect of cisplatin [70], and repeated administration of IdB1016 to mice bearing human ovarian cancer xenografts significantly inhibited tumor growth [71]. Turmeric is probably the most thoroughly researched dietary supplement from the standpoint of potential value in cancer management [72,73]. Compared bioavailabilities of pure curcumin, turmeric active principle, and Meriva™ (curcumin-PHYTOSOME®) has been evaluated in rats, showing that Meriva™ is five times more bioavailable than the
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parent compound [24]. Recent studies have confirm that the interaction between curcumin and lipids layers plays a fundamental role in the wide bioactivity of this compound [74], and Meriva™ thus appears to be a favorite candidate to enhance biological action of curcumin through a higher bioavailability. 5.6. Weight management Results of a clinical trial investigating the role of GreenSelect® PHYTOSOME® in the treatment of obesity were recently published [75]. The study showed the efficacy of green tea PHYTOSOME® to potentiate weight loss in dieting obese subjects. 6. Conclusions A PHYTOSOME® is a complex between polar polyphenolics and dietary phospholipids that shows definite physicochemical and spectroscopic features. PHYTOSOME ® complexes were first investigated for cosmetic applications, but mounting evidence of potential for drug delivery has been cumulated over the past two decades, with beneficial activity in the realms of cardiovascular, anti-inflammatory, hepatoprotective and anticancer applications. While the exact nature of the supramolecular interactions between specific phospholipids and polyphenolics still awaits clarification, the burgeoning literature on the biomedical applications of the PHYTOSOME® bodes well for the elucidation of the structural bases of these remarkable interactions as well as for their translation in biomedical research. Acknowledgments A. Semalty, M. Semalty and M.S.M. Rawat acknowledge the grant provided by the Department of Science and Technology, Govt. of India for the research work and are also thankful to LIPOID GmbH Germany for providing the gift sample of phosphatidylcholine for the research work. They also thankfully acknowledge UGC-DAE Consortium for Scientific Research, Indore (M.P.) for the provided facilities. References [1] Manach C, Scalbert A, Morand C, Rémésy C, Jiménez L. Polyphenols: food sources and bioavailability. Am J Clin Nutr 2004;79:727–47. [2] Bombardelli E, Curri SB, Della Loggia R, Del Negro P, Tubaro A, Gariboldi P. Complex between phospholipids and vegetal derivatives of biological interest. Fitoterapia 1989;60:1–9 [Suppl. to issue N.1]. [3] Mauri PL, Simonetti P, Gardana C, Minoggio M, Morazzoni P, Bombardelli E, et al. Liquid chromatography/atmospheric pressure chemical ionization mass spectrometry of terpene lactones in plasma of volunteers dosed with Ginkgo biloba L. extracts. Rapid Commun Mass Spectrom 2001;15:929–34. [4] Kidd PM, Head K. A review of the bioavailability and clinical efficacy of milk thistle phytosome: a silybin–phosphatidylcholine complex (Siliphos®). Altern Med Rev 2005;10:193–203. [5] Rossi R, Basilico F, Rossoni G, Riva A, Morazzoni P, Mauri PL. Liquid chromatography/atmospheric pressure chemical ionization ion trap mass spectrometry of bilobalide in plasma and brain of rats after oral administration of its phospholipidic complex. J Pharm Biomed Anal 2009;50:224–7. [6] Citernesi U, Sciacchitano M. Phospholipid/active ingredient complexes. Cosmet Toilet 1995;110:57–68. [7] Kidd PM. Phosphatidylcholine: a superior protectant against liver damage. Altern Med Rev 1996;1:258–74.
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Contents lists available at ScienceDirect
Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f i t o t e
Review
Supramolecular phospholipids–polyphenolics interactions: The PHYTOSOME® strategy to improve the bioavailability of phytochemicals Ajay Semalty a,⁎, Mona Semalty a, Mohan Singh Maniyari Rawat b, Federico Franceschi c,⁎ a b c
Department of Pharmaceutical Sciences, H.N.B. Garhwal University Srinagar (Garhwal), India Department of Chemistry, H.N.B. Garhwal University Srinagar (Garhwal), India Research and Development Laboratories, Indena S.p.A. (Settala), Italy
a r t i c l e
i n f o
Article history: Received 20 October 2009 Accepted in revised form 3 November 2009 Available online 14 November 2009 Keywords: Flavonoid Bioavailability PHYTOSOME® Herbal drugs Phospholipid complex
a b s t r a c t The poor and/or erratic oral bioavailability of polyphenolics can be improved using the PHYTOSOME® 1 delivery system, a strategy that enhances the rate and the extent of solubilization into aqueous intestinal fluids and the capacity to cross biomembranes. Phospholipids show affinity for polyphenolics, and form supramolecular adducts having a definite stoichiometry. This article reviews the preparation and characterization of PHYTOSOME® complexes and their activity in various medicinal (cardiovascular, anti-inflammatory, hepatoprotective, anticancer) and cosmetic (skin aging) realms of application. © 2009 Elsevier B.V. All rights reserved.
Contents 1. 2. 3. 4.
Introduction . . . . . . . . . . . . . . . . . . . . Physical and chemical properties . . . . . . . . . . Methods of preparation . . . . . . . . . . . . . . . Characterization . . . . . . . . . . . . . . . . . . 4.1. Spectroscopy . . . . . . . . . . . . . . . . 4.2. Thermal gravimetric analysis (TGA)/differential 5. Biological profile of PHYTOSOME® adducts . . . . . . 5.1. Cardiovascular properties . . . . . . . . . . 5.2. Anti-inflammatory properties . . . . . . . . . 5.3. Anti-aging properties . . . . . . . . . . . . 5.4. Hepatoprotective properties . . . . . . . . . 5.5. Anticancer properties . . . . . . . . . . . . 5.6. Weight management . . . . . . . . . . . . . 6. Conclusions . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . .
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⁎ Corresponding authors. Semalty is to be contacted at Department of Pharmaceutical Sciences, PB No.-32, H.N.B. Garhwal University Srinagar (Garhwal)-246174, India. Franceschi, Indena S.p.A., via Don Minzoni 6, 20090 Settala (MI), Italy. E-mail addresses: [email protected] (A. Semalty), [email protected] (F. Franceschi). 1 PHYTOSOME® is a registered trademark of Indena S.p.A. Milan, Italy. 0367-326X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2009.11.001
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1. Introduction For good bioavailability, natural products must have a good balance between hydrophilicity (for dissolving into the gastrointestinal fluids) and lipophilicity (to cross lipidic biomembranes). Many phytoconstituents like polyphenolics have good water solubility, but are, nevertheless, poorly absorbed [1] because of their large size, incompatible with a process of passive diffusion and/or their poor miscibility with oils and other lipids. As a result, the ability of flavonoids to cross the lipid-rich outer membrane of small intestine enterocytes is severely limited. Water-soluble phytoconstituents (mainly polyphenolics) can be converted into a lipid-compatible molecular complex known as PHYTOSOME®. A PHYTOSOME® is generally more bioavailable than a simple herbal extract due to its enhanced capacity to cross the lipid-rich biomembranes and reach circulation [2–5]. Phospholipids are small lipid molecules where glycerol is bonded to two fatty acids, with the third hydroxyl, normally one of the two primary methylenes, bearing a phosphate group [6]. Phospholipids from soy, mainly phosphatidylcholine, are lipophilic substances and readily complex polyphenolics. In this context, phosphatidylcholine, the major molecular building block of cell membranes and a compound miscible in both water and in oil/lipid environments, is well absorbed orally, and has the potential to act as a chaperon for polyphenolics, accompanying them through biological membranes [7]. PHYTOSOME® complexes were developed at Indena (Milan, Italy) in the late Eighties. Many popular standardized herbal extracts [e. g. Ginkgo biloba L., grape (Vitis vinifera L.) seeds, milk thistle (Silybum marianum (L.) Gaertn), green tea (Camellia sinensis (L.) O. Kuntze), ginseng (Panax ginseng C.A. Meyer), licorice (Glycyrrhiza glabra L.), horse chestnut (Aesculus hippocastanum L.), Centella asiatica (L.) Urban, olive (Olea europea L.), Terminalia sericea Burch. Ex. DC, Amni visnaga (L.) Lam, turmeric (Curcuma longa L.) and hawthorn (Crataegus spp.)] are currently commercially available in the PHYTOSOME® form (see Table 1). Flavonoids and terpenoids from herbal extracts undergo different absorption pathways
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when bound to phospholipids, and this article reviews various aspects and the latest trends of PHYTOSOME® research, highlighting recent advances in their therapeutic potential. 2. Physical and chemical properties The first PHYTOSOME® generation was prepared by combining selected polyphenols or polyphenol extracts with phospholipids in non-polar solvents [2], but, more recently, a newer PHYTOSOME® generation was developed using hydro-ethanolic solvents. Products obtained in this way comply with current food specifications [8,9], and expand the PHYTOSOME®' potential from the pharma/cosmetic field to the health-food one. A PHYTOSOME® is an amphiphilic substance with a definite melting point, generally soluble in nonpolar solvents (where its hydrophilic moiety is not), and moderately soluble in fats. The low solubility in aqueous media makes the formation of stable emulsions and creams possible (Fig. 1), improving the biopharmaceutical properties of both highly lipid insoluble and poorly water-soluble phytoconstituents. The PHYTOSOME® formulation increases the absorption of active ingredients when topically applied on the skin [10–19], and improves systemic bioavailability when administered orally [20–24]. In water medium, a PHYTOSOME® will assume a micellar shape, forming a liposome-like structure. Fundamental differences exit, however, between a PHYTOSOME® and a liposome. In liposomes, the active principles are dissolved in the central part of the cavity, with no possibility of molecular interaction between the surrounding lipid and a hydrophilic substance. On the contrary, the PHYTOSOME® complex can somewhat be compared to an integral part of the lipid membrane (Fig. 2), where the polar functionalities of the lipophilic guest interact via hydrogen bonds with the polar head of a phospholipid (i.e. phosphate and ammonium groups), forming a unique arrangement that can be evidenced by spectroscopy [10,11,18,25–28]. Thus, IR and multi-nuclear spectroscopic studies show that a PHYTOSOME® is not a mechanical mixture of two constituents,
Table 1 Available PHYTOSOME® complexes on the market. PHYTOSOME® and all other trademarks are owned by Indena S.p.A. Milan, Italy. Trade name
Phytoconstituent complexed with phospholipid
Indication
Escin ß-sitosterol Phytosome® Siliphos® Silymarin Phytosome® Meriva™ Virtiva® Ginkgoselect® Phytosome® Ginselect® Phytosome® Leucoselect® Phytosome® Centella Phytosome® 18 ß-glycyrrhetinic acid Phytosome® Crataegus Phytosome® Ginkgo biloba Dimeric Flavonoids Phytosome® Ginkgo biloba Terpenes Phytosome® Sericoside Phytosome® Greenselect® Phytosome®
Escin ß-sitosterol from horse chestnut fruit Silybin from milk thistle seed Silymarin from milk thistle seed Curcuminoids from turmeric rhizome Ginkgoflavonglucosides, ginkgolides, bilobalide from Ginkgo biloba leaf Ginkgoflavonglucosides, ginkgolides, bilobalide from Ginkgo biloba leaf Ginsenosides from Panax ginseng rhizome Polyphenols from grape seed Triterpenes from Centella asiatica leaf 18 ß-glycyrrhetinic acid from licorice rhizome Vitexin-2″-O-rhamnoside from Hawthorn flower Dimeric flavonoids from Ginkgo biloba leaf
Anti-oedema Hepatocyte protection Antihepatotoxic
Visnadex® PA2 Phytosome®
Visnadin from Amni visnaga umbel Proanthocyanidin A2 from horse chestnut bark
Ginkgolides and bilobalide from Ginkgo biloba leaf Sericoside from Terminalia sericea bark root Polyphenols from green tea leaf
Vasokinetic Vasokinetic Skin elasticity improver, adaptogenic Antioxidant, capillarotropic Cicatrizing, trophodermic Soothing Antioxidant Lipolytic, vasokinetic Soothing Anti-wrinkles Prevention of free radical-mediated tissue damages and weight management Vasokinetic Anti-wrinkles, UV protectant
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depending on the protocol employed. Thus, the phospholipid complex of curcumin [34] prepared in an aprotic solvent [35] showed remarkable differences from the one prepared in a protic solvent [8], and three different silybin–phospholipid complexes have been reported. Two of them were prepared in aprotic solvent [Silipide (IdB 1016) [30], a pharma-grade PHYTOSOME® which has been extensively characterized [27], and Siliphos®], while a third silybin–phospholipid complex was obtained in protic solvents [32]. Different phospholipids afford different complexes, as found for G. biloba extracts, whose PHYTOSOME® extract with phosphatidylserine complex is known as Virtiva®, while the standard phosphatidylcholine complex is known as Ginkgoselect® PHYTOSOME®) [31]. Fig. 1. Electron-microscopic examination of lyophilized microdispersion of Ginselect® PHYTOSOME® 0.1% in water. Magnification 1 × 4000.
but a specific complex between a hydrophilic guest and a lipophilic host characterized by specific dipolar interactions, in accordance with the spectroscopic differences between a PHYTOSOME and a mechanical mixture of its two constituents. 3. Methods of preparation A PHYTOSOME® is prepared by complexing a polyphenolic phytoconstituent or mixture with a phospholipid. Depending on the product, mass ratios in the 1:1.5–1:4 are observed. Various preparation methods for a PHYTOSOME® [3,8,9,12– 16,19,29–33] and the resulting complex can be different
Fig. 2. Major difference between liposome and PHYTOSOME®.
4. Characterization 4.1. Spectroscopy Complexation and molecular interactions between phytoconstituents and phosphatidylcholine in solution have been studied by 1 H-NMR [2,8,10,25–27], 1 3 C-NMR [2,8,10,27], 31P-NMR, [8–10] as well as by IR spectroscopy [18]. Complex formation is associated with changes of chemical shift and line broadening of some characteristic signals in NMR spectra and with appearance of new bands in IR spectra. 4.2. Thermal gravimetric analysis (TGA)/differential scanning calorimetry (DSC) Detection and measurement of thermal effects such as fusion, solid–solid transitions, glass transitions, loss of solvent, and decomposition, can be used to characterize a solid PHYTOSOME®. The TG/DT analyses of two complexes and the mechanical mixtures of phytoconstituent and phospholipid components are reported in Fig. 3 [36]. The DT profile of Silymarin PHYTOSOME ® (green solid line in Fig. 3a) shows an endothermic peak with a maximum at about 173 °C, corresponding to a ca. 3% weight loss, and a less intense and broader endothermic signal with a maximum at about 148 °C. The TG profile (blue solid line in Fig. 3a) of some PHYTOSOME® adducts shows an initial loss on drying below 100 °C (about 4%), and a massive weight loss starting at about 175 °C due to degradation. The TG/DT profiles of the phytoconstituent/phospholipid mechanical mixture (dashed lines) show two broad endothermic peaks, the first one with a maximum at about 146 °C and corresponding to a weight loss of ca. 2%, and the second one at about 167 °C with a loss of weight of ca. 3% followed by the massive weight loss expected for degradation. Fig. 3b shows the TG/DT profile of Ginkgoselect® PHYTOSOME® and the one of a Ginkgoselect®/phospholipid mechanical mixture. The DT profile of the PHYTOSOME® (green solid line in Fig. 3b) shows only an endothermic peak with a maximum at about 179 °C, linked to a weight loss of about 2%. Conversely, the TG profile (blue solid line) shows an initial loss on drying below 100 °C (about 2%) and a massive weight loss starting at about 180 °C due to degradation.
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Fig. 3. a: TG curves (blue) and DT profiles (green) of Silymarin PHYTOSOME® (solid line) and silymarin/phospholipid mechanical mixture (dashed line). b: TG curves (blue) and DT profiles (green) of Ginkgoselect® PHYTOSOME® (solid line) and Ginkgoselect®/phospholipid mechanical mixture (dashed line).
5. Biological profile of PHYTOSOME® adducts
PHYTOSOME® to treat peripheral vascular disease (e.g., Raynaud's disease and intermittent claudication) was found to be 30–60% higher than that of Ginkgoselect® [37]. Ginkgoselect® PHYTOSOME® was also recently validated as a cardioprotective agent [38]. The two G. biloba L. PHYTOSOME® complexes showed different cognitive effects, with the phosphatidylserine complex (Virtiva® ) being active at lower doses than the standard phosphatidylcholine complex (Ginkgoselect® PHYTOSOME®) [31,39]. The activity of Leucoselect® PHYTOSOME® to decrease low-density lipoprotein susceptibility to oxidation and oxidative stress damage in a group of heavy smokers was also investigated, with positive results [40].
5.1. Cardiovascular properties
5.2. Anti-inflammatory properties
Preliminary studies have shown that the PHYTOSOME® of G. biloba L. and that of grape seed extracts are more potent than their non-complexed forms. The efficacy of Ginkgoselect®
PHYTOSOME® complexes show better anti-inflammatory activity than their uncomplexed herbal extracts. Croton oilinduced dermatitis was used, as an inflammation model
The TG/DT profiles of the mechanical mixture of Ginkgoselect® and a phospholipid (dashed lines) shows two broad endothermic peaks: the first with a maximum at about 135 °C simultaneous with a loss of weight of about 3%, and the second with a maximum at about 171 °C with a loss of weight of about 2% followed by a massive weight due to degradation. In both cases, phytoconstituent–phospholipid molecular interactions in the complex lead to a radically different thermal profile and behavior of the PHYTOSOME® complex compared to its corresponding mechanical mixture.
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involving both tissue and vascular damage, to evaluate the anti-inflammatory activity of PHYTOSOME® complexes in animals [18]. The results obtained with glycyrrhetinic acid, a powerful anti-inflammatory and anti-allergic triterpenoid, and with silymarin, a free radical scavenger flavanolignan endowed with anti-phlogistic activity, were discussed. The products were applied topically as microdispersion in water, and the reduction of edema was used as the end point. Phospholipids are per se devoid of anti-inflammatory activity, but it was found that their complexation with glycyrrhetinic acid prolonged, its activity. Thus, the anti-edematous activity of 18-β-glycyrrhetinic acid gradually waned with time and was almost completely terminated after 24 h, a time at which its phosphatidyl complex still induced an 80% reduction in edema. The silymarin complex showed greater reduction of edema (76% reduction in 6 h) as compared to that with the free form (33% even after 12 h) in the croton oil test in mice (Fig. 4). Therefore, the PHYTOSOME® was about two-fold more effective than the free phytoconstituent. In another study, the 18-β-glycyrrhetinic acid complex exhibited potent activity against the erythema secondary to UVA radiation [28]. 5.3. Anti-aging properties PHYTOSOME® as a delivery system offers interesting applications and opens new opportunities for the use of active ingredients in the cosmetic field. The G. biloba PHYTOSOME® was investigated for the treatment of skin aging connected to superficial capillary blood flow circulation. Extracts from G. biloba are used orally to improve peripheral circulation [41], and their phospholipid complexes were found to improve skin microcirculation after topical application. Activation of microcirculation meliorated skin aging associated to dystrophic alteration of the epidermis and dermis, and the regressive abiotrophic pannicular disease of lower limbs and breast associated to venous stasis and/or chronic venous insufficiency. The role of PHYTOSOME® in
Fig. 4. Reduction of edematous response to croton oil-induced by silymarin and its complex with distearoylphosphatidylcholine at different times (dose 0.5 µmol/ear).
functional cosmetics was reviewed [25], and the use of Silymarin PHYTOSOME® on aging skin was also reported [18,42]. 5.4. Hepatoprotective properties Most studies on PHYTOSOME® are focused on Silybum marianum Gaertner and its liver-protectant flavolignans. The fruit of milk thistle (Silybum marianum) contains flavonoids showing hepatoprotective properties [43,44]. Silymarin has been shown to be effective in the treatment of liver diseases of various kinds, including hepatitis, cirrhosis, fatty infiltration of the liver (chemical and alcohol induced fatty liver), and inflammation of the bile duct [45–48]. The antioxidant properties of silymarin substantially boost the liver resistance to toxic insults [49]. Silymarin has a complex composition, based on three patterns of covalent interaction between its flavonol and lignan constituents (Fig. 5). Silybin, in turn the mixture of two diastereomers, is the major and more potent constituent of silymarin [43]. Silybin protects the liver by conserving glutathione in the parenchymal cells [49], while phosphatidylcholine helps repair and replace cell membranes [7]. These constituents act synergistically, sparing liver cells from destruction. While silybin was found active in many models of human disease, clinical translation of these results was hampered by its extremely poor oral bioavailability. In a pharmacokinetic study [32], the oral bioavailability of silybin was increased remarkably when administered as silybin– phospholipid complex. The increased lipophilicity of the silybin–phospholipid complex, have been claimed to explain the improved bioavailability [7]. AFB1 (aflatoxin B1, a fungal compound produced by various strains of Aspergillus flavus and Aspergillus parasiticus) causes aflatoxicosis in chickens, and is characterized by mortality, listlessness, anorexia, decreased growth rates, negative feed conversions, fatty liver, decreased egg production, poor pigmentation, and increased susceptibility to other diseases. For these reasons, intensive research has been carried out to develop cost-effective and safe methods to reduce the effects of AFB 1 . Remarkably, Silymarin PHYTOSOME® showed better antihepatotoxic activity than silymarin alone, and could provide protection against the toxic effects of aflatoxin B1 on safety and quality performance of broiler chickens [50]. A series of studies on silymarin administered as Silymarin PHYTOSOME® found that the complex could protect the fetus from maternally ingested EtOH [51]. In similar studies, it was reported that Silymarin PHYTOSOME® shows a better fetoprotectant activity against ethanol-induced behavioral deficits than uncomplexed silymarin on fetuses of both sexes [52]. In the first study, gamma glutamyl transpeptidase (GGTP) activity was determined for liver and brain tissue from both the fetus and the dams on day 21 of pregnancy, while the offspring were tested at age 90 days on the social recognition task and at 75 days on the radial arm maze in the later study. Female EtOHexposed offsprings performed more poorly on the radial arm maze than did female EtOH/Silymarin PHYTOSOME® offsprings and that of controls. In a similar way, male EtOH-exposed offsprings were less able to form social memories than the male EtOH/Silymarin PHYTOSOME® offsprings and the male offspring control.
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Fig. 5. Flavolignans of Silybum marianum and hydrogen bond interactions in phosphatidylcholine complex of silybin.
Silymarin PHYTOSOME®, was also found to have a much higher and specific activity and a longer lasting activity than any single silymarin component in terms of oedema, inhibition of myeloperoxidase activity, antioxidant and free radical scavenging properties [42]. When silybin was administered as Silipide (a pharmagrade silybin–phospholipid complex) at a dose of 200 mg per kg body weight, plasma concentration of silybin remained high 70 h after the oral dosing, while, after 25 h, silybin barely rose above the detection threshold [21]. In another study on rats, silybin given as Silipide at 200 mg/kg was detectable in the plasma within minutes, peaked after 1 h, and its plasma levels remained elevated for over 6 h. Silipide was also shown to rapidly reach the liver, cross the liver cells, and appear in the bile within 2 h. The amount of silybin reaching the bile from PHYTOSOME® dosing was at least 6.5 times higher than that from non-complexed silybin (13% versus 2%, over 24 h) [22]. In general, Silipide was found to show higher pharmacological potency in animal models of hepatic injury than uncomplexed silybin, and the oral bioavailability and organ distribution profile of Silipide was much better than that of silybin from silymarin [53]. The pharmacokinetics of Silipide in healthy human subjects showed that complexation with phosphatidylcholine improved the oral bioavailability of silybin 4.6 fold compared with silymarin, probably because
of a facilitated passage across the gastrointestinal mucosa [20]. A pilot study on the liver protective effect of Silipide in chronic active hepatitis (CAH) found that Silipide improved liver function tests related to hepatocellular necrosis and/or increased membrane permeability in patients affected by CAH [54]. The iron chelating properties of Silipide were also investigated during a 12 week study on 37 patients with chronic hepatitis C and Batts–Ludwig fibrosis. Treatment with silybin–phosphatidylcholine complex was clearly associated with a reduction in iron stores in patients with advanced fibrosis [55]. Hepatitis C infection was also improved by a combination of Silipide and vitamin E (SPV) [56]. In another study, the role of SPV complex for the treatment of non alcoholic fatty liver disease (NAFLD) was investigated [57]. Significant correlation among indexes of fibrosis, body mass, insulinemia, plasma levels of transforming growth factor-β, tumor necrosis factor-α, degree of steatosis and γ-glutamyl transpeptidase was observed. Reduction in ultrasonographic scores for liver steatosis and improvement of liver enzyme levels, hyperinsulinemia and indexes of liver fibrosis suggest that SPV complex could be used as a complementary approach to the treatment of patients with chronic liver damage.
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The addition of increasing concentrations of Silipide to isolated rat hepatocytes caused a dose-dependent inhibition of lipid peroxidation induced by cumene hydroperoxide, concluding that Silipide acted as potentially useful protective agent against free radical mediated toxic liver injury [58]. Prompted by these observations, a phase II randomized open clinical trial to evaluate the dose response relationship of Silipide in patients with viral or alcoholic hepatitis was carried out, finding a dose-dependent improvement [59]. In a pilot study on eight patients with chronic active hepatitis B and/or C, treatment with Silipide, at 240 mg silybin, for two months, the liver enzymes alanine aminotransferase (ALT) and aminotransferase (AST) were significantly reduced, while glutamyltranspeptidase (GGT) and malondialdehyde (MDA) levels, a byproduct of lipid peroxidation were not statistically improved [60]. Healthy volunteers (total number not disclosed) receiving 360 mg Silipide three times daily for three weeks did not show any adverse effect [61], while treatment of 232 patients with liver disorder for up to four months with either 240 or 360 mg Silipide daily showed an excellent tolerability. Minor adverse effects (nausea, heartburn, dyspepsia, transient headache) were reported only in 12 patients (5.2% of the total studied), compared with 8.2% of patients who received uncomplexed silybin and 5.1% of patients treated with the placebo. In this study, Silipide produced no clinically relevant blood changes, and its efficacy was evaluated by measuring serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma-glutamyltranspeptidase (GGT). Besides antioxidant activity, Silipide has also the ability to scavenge ethanol-derived free radicals damage, and has therefore potential to prevent liver damage from alcohol abuse [62]. Silipide was also able to scavenge free radicals generated by CCl4 or INH [63]. Silipide was also investigated in rodents in various models of liver damage. After oral administration, Silipide exhibited a significant and dose related protective effect against hepatotoxicity induced by CCl4, acetaminophen, ethanol and galactosamine [64]. A controlled clinical study on the effect of Silipide in chronic persistent hepatitis was carried out [65]. The drug treatments available for this condition have limited efficacy, do not work at all for many patients, and have major adverse effects. In this study, patients were randomized to receive either 240 mg Silipide (n =31) or placebo (n=34) twice daily for three months. The Silipide group showed significant lowering of both serum ALT and AST, while in the placebo group these markers worsened. The Silipide treatment was well tolerated, with fewer adverse events than for the placebo group, and no patient discontinued the trial due to adverse effects. A quercetin–phospholipid complex was also developed, and investigated in a murine model of liver injury (carbon tetrachloride-induced damage) [23]. In this study, treatment with free quercetin (20 mg/kg) as well as quercetin– phospholipid complexes (10 mg/kg and 20 mg/kg) showed significant (p b 0.05) decrease in thiobarbituric acid reactive substance (TBARS) levels in liver homogenate when compared to CCl4-treated animals. It was also observed that lower doses of uncomplexed quercetin (10 mg/kg) failed to produce significant results. Quercetin at 20 mg/kg in phospholipid complex gave better results than free quercetin (20 mg/kg) restoring the normal enzyme levels (Fig. 6).
Fig. 6. Effect of quercetin–phospholipid complex on TBARS. Values are mean ± SEM (n = 6); *p b 0.05 [significant with respect to control (CCl4-treated group)], complex denotes quercetin–phospholipid complex.
The phospholipid complexes of naringenin [66] and curcumin [33] were also investigated. The phospholipid complex of naringenin produced better antioxidant activity than the free compound with a prolonged duration of action, while the curcumin–phospholipid complex was active on carbon tetrachloride-induced acute liver damage in rats, with significantly higher aqueous and n-octanol solubility and higher antioxidant activity at all doses investigated. 5.5. Anticancer properties Over the past few years, silymarin and silybin have received a growing attention for properties unrelated to liver protection, like anticancer, chemopreventive, hypocholesterolemic, cardioprotective, neuroactive and neuroprotective activities [67]. The anti-radical potential and the cytoprotective activity may be responsible for the use of silymarin as a chemoprotective and anticancer agent. Due to their chemopreventive activity, silybin and silymarin can inhibit the carcinogenic activity of many chemicals [68], and have the potential to be successfully used in the adjuvant therapy of cancer. In this context, silybin might act essentially as an antioxidant, protecting tissues against the oxidative stress generated by chemotherapeutics, preventing at the same time hepatotoxicity, a major side-effect of many chemotherapy treatment. A phase I and pharmacokinetic study of silybin-PHYTOSOME® in prostate cancer patients was carried out [69]. The study concluded that oral silybinPHYTOSOME® at a daily dose of 13 g seems well tolerated in patients with advanced prostate cancer, and this dosage was recommended for the phase II study [69]. Silybin and its phospholipid complex IdB1016 have been shown to have antitumor activity and to potentiate the effect of cisplatin [70], and repeated administration of IdB1016 to mice bearing human ovarian cancer xenografts significantly inhibited tumor growth [71]. Turmeric is probably the most thoroughly researched dietary supplement from the standpoint of potential value in cancer management [72,73]. Compared bioavailabilities of pure curcumin, turmeric active principle, and Meriva™ (curcumin-PHYTOSOME®) has been evaluated in rats, showing that Meriva™ is five times more bioavailable than the
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parent compound [24]. Recent studies have confirm that the interaction between curcumin and lipids layers plays a fundamental role in the wide bioactivity of this compound [74], and Meriva™ thus appears to be a favorite candidate to enhance biological action of curcumin through a higher bioavailability. 5.6. Weight management Results of a clinical trial investigating the role of GreenSelect® PHYTOSOME® in the treatment of obesity were recently published [75]. The study showed the efficacy of green tea PHYTOSOME® to potentiate weight loss in dieting obese subjects. 6. Conclusions A PHYTOSOME® is a complex between polar polyphenolics and dietary phospholipids that shows definite physicochemical and spectroscopic features. PHYTOSOME ® complexes were first investigated for cosmetic applications, but mounting evidence of potential for drug delivery has been cumulated over the past two decades, with beneficial activity in the realms of cardiovascular, anti-inflammatory, hepatoprotective and anticancer applications. While the exact nature of the supramolecular interactions between specific phospholipids and polyphenolics still awaits clarification, the burgeoning literature on the biomedical applications of the PHYTOSOME® bodes well for the elucidation of the structural bases of these remarkable interactions as well as for their translation in biomedical research. Acknowledgments A. Semalty, M. Semalty and M.S.M. Rawat acknowledge the grant provided by the Department of Science and Technology, Govt. of India for the research work and are also thankful to LIPOID GmbH Germany for providing the gift sample of phosphatidylcholine for the research work. They also thankfully acknowledge UGC-DAE Consortium for Scientific Research, Indore (M.P.) for the provided facilities. References [1] Manach C, Scalbert A, Morand C, Rémésy C, Jiménez L. Polyphenols: food sources and bioavailability. Am J Clin Nutr 2004;79:727–47. [2] Bombardelli E, Curri SB, Della Loggia R, Del Negro P, Tubaro A, Gariboldi P. Complex between phospholipids and vegetal derivatives of biological interest. Fitoterapia 1989;60:1–9 [Suppl. to issue N.1]. [3] Mauri PL, Simonetti P, Gardana C, Minoggio M, Morazzoni P, Bombardelli E, et al. Liquid chromatography/atmospheric pressure chemical ionization mass spectrometry of terpene lactones in plasma of volunteers dosed with Ginkgo biloba L. extracts. Rapid Commun Mass Spectrom 2001;15:929–34. [4] Kidd PM, Head K. A review of the bioavailability and clinical efficacy of milk thistle phytosome: a silybin–phosphatidylcholine complex (Siliphos®). Altern Med Rev 2005;10:193–203. [5] Rossi R, Basilico F, Rossoni G, Riva A, Morazzoni P, Mauri PL. Liquid chromatography/atmospheric pressure chemical ionization ion trap mass spectrometry of bilobalide in plasma and brain of rats after oral administration of its phospholipidic complex. J Pharm Biomed Anal 2009;50:224–7. [6] Citernesi U, Sciacchitano M. Phospholipid/active ingredient complexes. Cosmet Toilet 1995;110:57–68. [7] Kidd PM. Phosphatidylcholine: a superior protectant against liver damage. Altern Med Rev 1996;1:258–74.
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