INTRODUCTION:

Most of the phytoconstituents of the plant extract are water soluble or polar constituents. Water soluble phytoconstituents like flavanoids, terpenoids, tannins are poorly absorbed due to their multiple-ring larger molecular size which cannot absorbed by passive diffusion or due to their poor lipid solubility, severely limiting their ability to pass across the lipid rich biological membranes, resulting poor bioavailability^1,2.

Various constituents of an extract may contribute to synergistic effect and process like separation and purification can lead to a partial loss of specific activity due to the removal of chemically related substance contributing to the activity of main substance. Most of time chemical complexity of the extracts seems to be important for the bioavailability of active constituents³. Water soluble or polar phytoconstituents can be converted into lipid-compatible molecular complex called phytosomes.

Phytosome is patented technology developed by Indeda, a leading manufacturer of drugs and nutraceuticals, to incorporate standardized plant extracts or water soluble or polar constituents to produce lipid compatible molecular complexes and improve their absorption and bioavailability. Phospholipids are complex molecules that are used in all life forms to make a cell membrane. Phospholipids are small lipid molecules where glycerol is bonded to two fatty acids, while the third hydroxyl, normally one of the two primary methylenes, bears a phosphate group bound to a biogenic amino or to an amino acid. Phospholipids are employed as natural digestive aids and as carriers for both water miscible and fat miscible nutrients. The phospholipids mainly used in preparation of phytosomes, is phosphotidylcholine, derived from soybean. Phosphotidylcholine is not only a carrier for phytoconstituents, but itself a bioactive nutrient with documented clinical efficacy for liver disease, including hepatitis, alcoholic hepatic steatosis, and drug induced liver damage. Many popular standardized herbal extracts comprising of flavanoids, polyphenolics, terpenes, alkaloids, volatile oils are employed for the preparation of phytosomes^1-4.

PHYTOSOME PREPARATION

Generally Phytosomes are prepared by reacting from 3-2 moles but preferably with one mole of natural or synthetic phospholipids such as phosphotidylcholine, phosphotidylserin or phosphotidylethanolamine, with one mole herbal of component, either alone or in the natural mixture in aprotic solvent such as dioxane or acetone. The phytosome complex can be then isolated by precipitation with non solvent such as aliphatic hydrocarbons like n-hexane or lyophilization or by spray drying. The dried residues were gathered and placed in desiccators over night, then crushed in motor and sieved with a 100 mesh. In the complex formation of phytosomes the ratio between these two moieties is in the range from 0.5- 2.0 moles. The most preferable ratio of phospholipids to flavonoids is 1:1.Phytosomes are prepared by solvent evaporation and mechanical dispersion methods. Phospholipid complex is sometimes prepared under reflux and stirring condition for complete interaction^5-10.

MECHANISM OF ACTION

Phytosomes was prepared form standardized herbal extract or pure components of extract and phospholipid. Phosphatidylcholine contains Phosphatidyl moiety (lipophilic in nature) and choline moiety (hydrophilic in nature). The choline hade of the Phosphatidylcholine molecule binds to polar phytoconstituents of herbal extract while lipophilic phosphatidyl moiety comprising the body and tail which then envelope the choline bonded material. Hence polar phytoconstituents converted in to phytophospholipid complex. In phytosomes week vandar walls or hydrogen bond are formed¹¹.

PROPERTIES OF PHYTOSOMES

Chemical properties:

A phytosomes is a complex between an herbal extract and natural phospholipids, like soy phospholipids. This complex results from the reaction of stoichiometric amounts of phospholipids with the selected polyphenol (like simple flavonoids) in a nonpolar solvent like n-hexane. Phospholipid-phytoconstituent interaction is due to the formation of hydrogen bonds between the polar head of phospholipids (i.e. ammonium and phosphate groups) and the polar functional groups of the herbal extract. Phytosomes are lipophilic substances with a definite melting point, freely soluble in nonpolar solvents, insoluble in water and moderately soluble in fats. When treated with water, phytosomes assume a micellar shape forming liposomal-like structures. In liposomes the active principle is dissolved in an internal pocket or floats in the layer membrane, while in phytosomes the active principle is anchored to the polar head of phospholipids, becoming an integral part of the membrane. Molecules are anchored through chemical bonds to the polar head of the phospholipids, as can be demonstrated by specific spectroscopic techniques.

Biological properties:

Clinical studies in experimental animals and in human subjects have been used to demonstrate the biological behaviour of phytosomes. Phytosomes are better absorbed from skin and gastrointestinal membrane because of their lipophilic nature and increased solubility^{11, 12}.

ADVANTAGES OF PHYTOSOMES

1. Phytosomes enhance the absorption of polar phytoconstituents through oral as well as topical route showing better bioavailability with significantly better therapeutic benefit.

2. Absorption of phytoconstituents is improved so its dose required is reduced.

3. Phosphotidycholine used in the preparation of phytosomes act as a carrier as well as hepatoprotective, hence giving synergistic effect when hepatoprotective substances are employed.

4. No compromise of nutrient safety.

5. In phytosome herbal extracts are protected from destruction by gut bacteria and digestive secretion.

6. Phytosomes show better stability because chemical bond formed between Phosphotidycholine and polar phytoconstituent.

7. Entrapment efficiency is higher.

8. Because of high skin penetration and high lipid profile phytosomes are widely used in cosmetic preparation.

9. Significantly greater clinical efficiency

10. Unlike liposome, there is no need of following the tedious, time consuming step for removing the free, entrapped drug from the formulation.

11. Leakage of drug during storage does not occur in phytosome, because drug is bonded with lipid, however loss may occur due to some chemical degradation i.e. hydrolysis.

12. They can be given orally, topically, extra or intravascularly.

13. In phytosome, phospholipid transfer/exchange is reduced and solubilization by HDL (high density lipid) is low ^{1, 4, 13-15}.

EVALUATION OF PHYTOSOMES^16-19

The behaviour of phytosomes in both physical and biological systems is governed by factors such as the physical size, membrane permeability, percentage of entrapped drug, and chemical composition as well as the quantity and purity of the starting materials. Therefore, phytosomes can be characterized in terms of their physical attributes i.e. shape, size, distribution, percentage drug captured, entrapped volume, percentage drug released and chemical composition.

Different characterization techniques used for phytosomes:

1. Visualization

Visualization of phytosomes can be done by using scanning electron microscopy (SEM) and by transmission electron microscopy (TEM).

2. Vesicle size and Zeta potential

The particle size and zeta potential can be determined by dynamic light scattering (DLS) using a computerized inspection system and photon correlation spectroscopy (PCS).

3. Entrapment efficiency

The entrapment efficiency of a drug by phytosomes can be measured by the ultracentrifugation technique.

4. Solubility studies:

Determination of solubility characteristics of drug, drug-phospholipid complex and physical mixture of drug and phospholipid were obtained by adding excess of the samples to 5ml of water or n-octanol in sealed glass container at room temperature. The liquids were shaken for 24 h and centrifuged at 5000 rpm for 10 min. The supernatant was filtered, and diluted with appropriate solvent. Concentration of drug was measured by using HPLC or UV spectrophotometer.

5. Differential scanning calorimetry (DSC)/ Thermal gravimetric analysis of the complex:

The samples were sealed in the aluminum crimp cell and heated at the speed of 10˚C/min from 0 to 900˚C in nitrogen atmosphere (60 ml/min). The peak transition onset temperature of drug, phospholipid, drug–phospholipid complex and physical mixture of drug and phospholipid were determined.

6. Surface tension activity measurement

The surface tension activity of the drug in aqueous solution can be measured by the ring method in a Du Nouy ring tensiometer.

7. Vesicle stability

The stability of vesicles can be determined by assessing the size and structure of the vesicles over time. The mean size is measured by DLS and structural changes are monitored by TEM.

8. Drug content

The amount of drug can be quantified by a modified high performance liquid chromatographic method or by a suitable spectroscopic method.

9. Spectroscopic evaluations

To confirm the formation of a complex or to study the reciprocal interaction between the phytoconstituent and the phospholipids, the following spectroscopic methods are used:

9.1 FTIR

The formation of the complex can be also be confirmed by IR spectroscopy by comparing the spectrum of the complex with the spectrum of the individual components and their mechanical mixtures. FTIR spectroscopy is also a useful tool for the control of the stability of phytosomes when micro-dispersed in water or when incorporated in very simple cosmetic gels. From a practical point of view, the stability can be confirmed by comparing the spectrum of the complex in solid form (phytosomes) with the spectrum of its micro-dispersion in water after lyophilisation, at different times. In the case of simple formulations, it is necessary to subtract the spectrum of the excipients (blank) from the spectrum of the cosmetic form at different times, comparing the remaining spectrum of the complex itself.

9.2 ¹³C-NMR

In the 13C-NMR spectrum of phytoconstituents (mainly containing flavanoids) and its stoichiometric complex with phospholipid, particularly when recorded at room temperature, all the flavonoid carbons are clearly invisible. The signals corresponding to the glycerol and choline portion of the lipid (between 60–80 ppm) are broadened and some are shifted, while most of the resonances of the fatty acid chains retain their original sharp line shape. After heating to 60˚C, all the signals belonging to the flavonoid moieties reappear, although they are still very broad and partially overlapping.

10. In vitro and in vivo evaluations

Models of in-vitro and in-vivo evaluations are selected on the basis of the expected therapeutic activity of the biologically active phytoconstituents present in the phytosomes. For example, in-vitro antihepatotoxic activity can be assessed by the antioxidant and free radical scavenging activity of the phytosomes. For assessing antihepatotoxic activity in-vivo, the effect of prepared phytosomes on animals against thioacetamide, paracetamol or alcohol induced hepatoxicity can be examined.

APPLICATION OF PHYTOSOMES

1. Improve bioavailability:

Bioavailability of Ginko biloba extract (GBE) was enhanced by preparing Ginko biloba extract phospholipids complexes (GBP) and Ginko biloba extract solid dispersion (GBS). The result shows that bioavailability of quercetin, kaempferol and isorhamnetin in rats were increased remarkably after oral administration of GBP and GBS comparing with GBE. The bioavailability of GBP is increased more than that of GBS²⁰.

The bioavailabilities of oximatrin in rats were increased remarkably after oral administration of the oxymatrine-phospholipid complex compare with those of oxymatrine or physical mixture. This was mainly due to an improvement of the solubility of oxymatrine-phospholipid complex with soya lecithin²¹.

The bioavailability of silybin in rats was increased remarkably after oral administration of prepared silybin-phospholipid complex due to improvement of the lipophilic property of silybin-phospholipid complex and improvement of the biological effect of silybin¹⁷.

2. Reduction in dose size

The antioxidant activity of curcumin-phospholipid complex (equivalent of curcumin 100 and 200 mg/kg body weight) and free curcumin (100 and 200 mg/kg body weight) was evaluated by measuring various enzymes in oxidative stress condition. Curcumin–phospholipid complex significantly protected the liver by restoring the enzyme levels of liver glutathione system and that of superoxide dismutase, catalase and thiobarbituric acid reactive substances with respect to carbon tetrachloride treated group. The complex provided better protection to rat liver than free curcumin at same doses. So less dose of phospholipid complex was required then free curcumin¹⁸.

3. Better absorbed than conventional herbal extracts

Phytosome of curcumin was developed to overcome the limitation of absorption and also to investigate the protective effect of curcumin–phospholipid complex on carbon tetrachloride induced acute liver damage in rats. The curcumin-phospholipid complex deppicted enhance aqueous or n-octanol solubility. The antioxidant activity of the product (phytosomes) was significantly higher than pure curcumin in all doses which were tested¹⁸.

WORK DONE ON PHYTOSOMES

1. Chen et al. (2010) improved the bioavailability of Ginko biloba extract (GBE) through preparing Ginko biloba extract phospholipids complexes (GBP) and Ginko biloba extract solid dispersion (GBS). The result shows that bioavailability of quercetin, kaempferol and isorhamnetin in rats were increased remarkably after oral administration of GBP and GBS comparing with GBE. The bioavailability of GBP is increased more than that of GBS²⁰.

2. Liu et al. (2009) prepared Luteolin-phospholipid complex using tetrahydrofuran as a reaction medium. Luteolin and phospholipid were dissolved in the medium and after the organic solvent is removed, the Luteolin-phospholipid complex could be obtained. The obtained complex showed strong antioxidant activity and could be used in oils or lipophilic foods²².

3. Yue et al. (2009) developed the oxymatrine-phospholipid complex. The bioavailabilities of oximatrin in rats were increased remarkably after oral administration of the oxymatrine-phospholipid complex compare with those of oxymatrine or physical mixture. This was mainly due to an improvement of the solubility of oxymatrine-phospholipid complex with soya lecithin²¹.

4. Cui et al. (2006) developed a novel insulin-phospholipid complex. The complex compared with native insulin, the physicochemical properties and solubility of insulin changed significantly after it was complexed with phospholipid. These characteristic, improved lipophilicity, will contribute to improved oral absorption of insulin²³.

5. Yanyu et al. (2006) developed the silymarin phytosome and studied its pharmacokinetics in rats. In the study the bioavailability of silybin in rats was increased remarkably after oral administration of prepared silybin-phospholipid complex due to improvement of the lipophilic property of silybin-phospholipid complex and improvement of the biological effect of silybin¹⁷.

6. Maiti et al. (2006) reported the phytosomes of curcumin and naringenin. Phytosome of curcumin was developed to overcome the limitation of absorption and also to investigate the protective effect of curcumin–phospholipid complex on carbon tetrachloride induced acute liver damage (in rats). The curcumin-phospholipid complex deppicted enhanced aqueous or n-octanol solubility. The antioxidant activity of the product (phytosomes) was significantly higher than pure curcumin in all doses which were tested¹⁸.

7. Maiti et al. (2005) prepared the quercetin-phospholipids complex and showed that the formulation exerted better therapeutic efficacy than the molecule in rat liver injury induced by carbon tetrachloride¹⁹.

8. Busby et al. (2002) developed silymarin phytosome and reported a better fetoprotectant activity from ethanol-induced behavioural deficits than uncomplexed silymarin²⁴.

9. Grange et al. (1999) reported silymarin phytosomes and conducted a series of studies on phytosome containing a standardized extract S. marianum, administed orally and found that it can protect the fetus from maternally ingested ethanol²⁵.

10. Mascarella et al. (1993), based on study on 232 patients with chronic hepatitis and treated with silybin phytosomes for 120 days, reported that liver function returned to normal faster in patients taking silybin phytosome compared to a group of controls²⁶.

11. Bombardelli et al. (1991) prepareded phytosomes, in which Silymarin - a standardized mixture of flavanolignans extracted from the fruits of S. marianum was complexed with phospholipids which showed much higher specific activity and a longer lasting action than the single components, with respect to percent reduction of odema, inhibition of myeloperoxidase activity, antioxidant and free radical scavenging properties²⁷.

12. Barzaghi et al. (1990) performed a human study designed to assess the absorption of silybin when directly bounded to phosphotidylcholine. The result depicted that the absorption of silybin from silybin phytosomes is approximately seven times greater compared to the absorption of silybin from pure regular milk thistle extract²⁸.

Table 1: Commercially available phytosome preparations^{4, 10, 29-32}

Sr. No	Phytosomes	Phytoconstituents complexed with phosphatidylcholine	Dose	Indications
1.	Silybin phytosomes	Silybin from Milk thistle seed	120 mg	Food Product, Antioxidant, Hepato-protective
2.	Ginkgo phytosomes	Flavanoids from Ginkgo biloba	120 mg	Protect brain and vascular linings, Anti-skin ageing agent
3.	Grapes seed phytosomes	Procyanidins from Vitis vinifera	50-300 mg	Systemic antioxidant, Food product,
4.	Curcumin phytosomes	Polyphenols from Curcuma longa	200-300 mg	Cancer chemopreventive agent
5.	Green select phytosomes	Epigallocatechin from Thea sinesis	50-300 mg	Antioxidant, Anticancer
6.	Hawthorn phytosomes	Flavanoids from Crataegus species	100 mg	Cardio protective, Antihypertensive
7.	Ginseng phytosomes	Ginsenosides from Panex ginseng	150 mg	Immunomodulator
8.	Olea select phytosomes	Polyphenols from Olea europea	_	Anti-inflammatory, Anti-hyperlipidemic
9.	Sericoside phytosomes	Sericoside from Terminalia sericea	_	Anti-wrinkles, Skin improver
10.	Echinacea phytosomes	Echinacoside from Echinacea angustifolia	_	Nutraceuticals, Immunomodulatory
11.	Visnadin phytosomes	Visnadin from Ammi visnaga	_	Circulation improver
12.	Bilberry phytosomes	Anthocyanoside from Vaccinium myrtillus	_	Antioxidant
13.	Centella phytosomes	Terpens from Centell asitica	_	Vein and skin disorder, Brain tonic

Table 2: Patents of phytosomes

Title of patent	Innovation	Patent No.	Reference
Complexes of saponins with phospholipids and pharmaceutical and cosmetic compositions containing them	Complexes of saponins with natural or synthetic phospholipids have high lipophilia and improved bioavailability and are suitable for use as active principle in pharmaceutical, dermatologic and cosmetic compositions	EP0283713	33
An anti-oxidant preparation based on plant extracts for the treatment of circulation and adiposity problems	Preparation based on plant extracts which has an anti-oxidant effect and is particularly useful in treatment of circulation problems such as phlebitis, varicose veins, arteriosclerosis, haemorrhoids and high blood pressure	EP1214084	34
Soluble isoflavone compositions	Isoflavone compositions exhibiting improved solubility (e.g., light transmittance), taste, color, and texture characteristics, and methods for making the same.	WO/2004/ 045541	35
Cosmetic and dermatological composition for the treatment of aging or photodamaged skin	Composition for topical treatment of the skin comprises a substance that stimulates collagen synthesis and a substance that enhances the interaction between extracellular matrix and fibroblasts Cosmetic or dermatological composition for topical treatment	EP1640041	36
Fatty acid monoesters of sorbityl furfural and compositions for cosmetic and dermatological use	Fatty acid monoesters of sorbityl furfural selected from two diff series of compounds in which side chain is a linear or branched C3 -C19 alkyl radical optionally containing at least one ethylenic unsaturation.	EP1690862	37
Treatment of skin, and wound repair, with thymosin beta 4	Compositions and methods for treatment of skin utilizing thymosin β4.	US/2007/ 0015698	38
Phospholipid complexes of olive fruits or leaves extracts having improved bioavailability	Phospholipids complexes of olive fruits or leaves extracts or compositions containing it having improved bioavailability.	EP/1844785	39
Compositions comprising Ginko biloba derivatives for the treatment of asthmatic and allergic conditions	Compositions containing fractions deriving from Ginkgo biloba, useful for the treatment of asthmatic and allergic conditions.	EP1813280	40

COMMERCIALLY AVAILABLE PHYTOSOME PREPARATIONS

There are different phytosomes available in market. In table 1 different phytosomes preparations are given with dose and indications.

PATENTS OF PHYTOSOMES:

In table 2 different patents of phytosomes with titles, patent numbers and its innovations are given.

DISCUSSION:

When polar phytoconstituents complexed with phospholipids like phosphatidylcholine give rise to a new delivery system called Phytosome. Phytosomes are new drug delivery system of herbal extract that are better absorbed than conventional herbal extract and solid dispersion of herbal extract. Phytosomes have improved pharmacokinetic and pharmacological parameter. Phytosomes being much better absorbed than liposomes. Phytosome can play a vital role in efficient herbal drug delivery of a broad spectrum of hepatoprotective phytoconstituents like flavones, terpenes and xanthones. Phytosomes are used in treatment of various acute diseases as more amount of active constituent becomes present at the site of action (heart, liver, brain, kidney etc) at similar or less dose as compared to the conventional herbal extract. Phytosomes have been therapeutically used for hepatoprotective, liver diseases, cosmetic preparation, cancer disease and inflammatory disease. Thorough study of literature reveals that several herbal extract are reported to possess different pharmacological effects.

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Received on 12.11.2013 Modified on 12.12.2013

Res. J. Pharm. Dosage Form. & Tech. 6(1): Jan.-Mar. 2014; Page 44-49