You are herePHYTOSOMES: AN EMERGING TECHNOLOGY FOR IMPROVING PHYTOCHEMICAL BIOAVAILABILITY- A REVIEW

PHYTOSOMES: AN EMERGING TECHNOLOGY FOR IMPROVING PHYTOCHEMICAL BIOAVAILABILITY- A REVIEW


IMPORTANCE OF PHOSPHATIDYLCHOLINE IN PHYTOSOME TECHNOLOGY
Properties of Phosphatidylcholine:
* Phosphatidylcholine is a natural component of lecithin.

* Phosphatidylcholine is found throughout the human body as an essential component of cell membranes.

*  The reaction of phosphatidylcholine with the herbal compounds creates new molecules known as phytosomes.

*  Phosphatidylcholine is a bifunctional compound, the phosphatidyl moiety being lipophilic and the choline moiety being hydrophilic in nature. Specifically the choline head of the phosphatidylcholine molecule binds to these compounds while the lipid soluble phosphatidyl portion comprising the body and tail which then envelopes the choline bound material. Hence, the phytoconstituents produce a lipid compatible molecular complex with phospholipids, also called as phyto-phospholipid complex. Molecules are anchored through chemical bonds to the polar choline head of the phospholipids, as can be demonstrated by specific spectroscopic techniques13-14.

*  The unit phytosome is usually a flavonoid molecule linked with at least one phosphatidylcholine molecule which results in a little micro sphere10.

*  Phosphatidylcholine is a very interesting molecule. It contains a water-soluble head (choline component) with two long, fat soluble tails (phosphatidyl component). Because of this dual solubility, phosphatidylcholine is an extremely effective emulsifier. Emulsifiers are substances which can mix together two seemingly incompatible liquids, such as oil and water. The emulsifying action of phosphatidylcholine is often used to greatly increase the absorption of fat-soluble vitamins and drugs.


Figure1: Chemical Structure of Phosphatidylcholine

*  Phosphatidylcholine functions in maintaining the "fluidity" of our cellular membranes.

*  Phosphatidylcholine plays a critical role in all membrane dependent metabolic processes. For example, membrane-bound enzyme systems, such as those involved in energy production within specialized cell compartments known as mitochondria, depend on phosphatidylcholine for stimulation. If phosphatidylcholine levels are inadequate, these enzymes will not become active. Since the mitochondria produce energy for the entire cell, all cellular processes are adversely affected when this occurs.

*  Phosphatidylcholine is a widely used pharmaceutical preparation in Europe for the treatment of liver disease and elevated cholesterol levels. In liver disease, phosphatidylcholine protects and enhances liver function; in high cholesterol, it improves the transport of cholesterol to the liver where it can be broken down.

*  In the United States, phosphatidylcholine is regarded as a food supplement because no therapeutic claims are made by manufacturers.

METHODS OF PREPARATION
Different methods of preparation as repoted in literature are given below:
1. Yanyu et al. (2006) reported the preparation of silybin phytosome which involved the formation of silybin-phospholipid complex using ethanol as a reaction medium15. The required amounts of drug and phospholipids were placed in a 100 ml round?bottom flask and dissolved in anhydrous ethanol. After ethanol was evaporated off under vacuum at 40oC, the dried residues were gathered and placed in desiccators overnight, then crushed in the mortar and sieved with a 100 mesh. The resultant silybin– phospholipid complex was transferred into a glass bottle flushed with nitrogen and stored in the room temperature16

2. Marena and Lampertico (1991), Jiang et al. (2001), Maiti et al. (2006) and Maiti et al. (2006) reported the methods of phytosome preparation17-20. According to literature procedures of phytosome preparations, phospholipids were selected from the group consisting of soy lecithin, from bovine or swine brain or dermis, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine in which acyl group was same or different and mostly derived from palmitic, stearic, oleic and linoleic acid. The flavonoids were selected from the group consisting of quercetin, kaempferol, quercretin-3, rhamnoglucoside, quercetin-3-rhamnoside, hyperoside, vitexine, diosmine, 3- rhamnoside, (+) catechin, (-) epicatechin, apigenin-7-glucoside, luteolin, luteolinglucoside, ginkgonetine, isoginkgonetine and bilobetine.

3. Another procedure for phytosome synthesis as given in literature involved the reaction of 3?2 moles but preferably, one mole of a natural or synthetic phospholipid, such as phosphatidylcholine, phosphatidylethanolamine or phosphatidyiserine with one mole of flavolignanans, either alone or in the natural mixture in aprotic solvent such as dioxane or acetone from which complex can be isolated by precipitation with non solvent such as aliphatic hydrocarbons or lyophilization or by spray drying. In the complex formation of phytosomes the ratio between these two moieties was in the range from 0.5?2.0 moles16. The most preferable ratio of phospholipid to flavonoids is 1:1

4. Naringenin–PC complex was prepared by taking naringenin with an equimolar concentration of phosphatidylcholine. The equimolar concentration of phosphatidylcholine and naringenin were placed in a 100 mL round bottom flask and refluxed in dichloromethane for 3 h. On concentrating the solution to 5– 10 mL, 30 mL of n?hexane was added to get the complex as a precipitate followed by filtration. The precipitate was collected and placed in vacuum desiccators21.

Common steps of preparation of phytosomes


Figure 2: Common stages for preparation of phytosomes22

1.      ADVANTAGES and disadvantages OF PHYTOSOMES23, 24, 25
Advantages

1.      It enhances the absorption of lipid insoluble hydrophilic polar phytoconstituents through oral as well as topical route and increases the bioavailability.
2.      It ensures appreciable drug entrapment.
3.       As the absorption of active constituent(s) is improved, it reduces the dose requirement.
4.      Phosphatidylcholine besides acting as a carrier, also acts as a hepatoprotective, hence it shows synergistic effect when hepatoprotective substances are employed.
5.      Phytosomes have better stability profile because chemical bonds are formed between phosphatidylcholine molecule and phytoconstituent.
6.      Application of phytoconstituents in form of phytosome improves their percutaneous absorption and act as functional cosmetics. Phytosome is widely used in cosmetics due to their more skin penetration and high lipid profile.
7.       Phytosomes also have nutritional benefit of phospholipids.
8.      The non?lipophilic phytoconstituent in phytosome can easily permeate the intestinal walls and is better absorbed in intestinal lumen.
9.      By improving the solubility of bile to herbal constituent, liver targeting can be facilitated by phytosomes.
10.  The phytosome process not only provides valuable phosphatidylcholine, it also intensifies the action of herbal compounds, by improving absorption, increasing biological activity, and enhancing delivery to the target tissue. Because of these effects, the phytosome is referred to as a delivery system.

Disadvantages
Phytoconstituent is rapidly eliminated from phytosomes. The duration of action is short.

PROPERTIES OF PHYTOSOMES
Chemical properties

Phytosomes is a complex between a natural product and natural phospholipids, like soy phospholipids which is obtained by reaction of stoichiometric amounts of phospholipid and the substrate in an appropriate solvent. It is confirmed by the spectroscopic analysis that the main phospholipid-substrate interaction is due to the formation of hydrogen bonds between the polar head of phospholipids (i.e. phosphate and ammonium groups) and the polar functionalities of the substrate. Phytosomes assumes a micellar shape forming liposomial-like structures in the presence of water.

Biological Properties
Phytosome, as an advanced form of herbal product is better absorbed, utilized and as a result produces better results than conventional herbal extract. The increased bioavailability of the phytosome over the non complexed botanical derivatives has been explained and verified by various pharmacokinetics and pharmacodynamic tests on experimental animal models and on human subjects26.

CHARACTERIZATION OF PHYTOSOMES26, 27
The Phytosomes are characterized by physical and biological tests and spectral analysis. The physical attributes include shape, size, its distribution, percentage drug capture, entrapped volume, percentage drug release, and chemical composition. Hence, the characterization of phytosomes is done by the following parameters:
1. Membrane permeability
2. Percent entrapped solutes
3. Chemical composition
4. Quantity and purity of the starting materials

I.  Physical tests
1. Visualization:
Visualization of phytosomes can be done using transmission electron microscopy (TEM) and by scanning electron microscopy (SEM) 28.

2. Particle 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) 29.

3. Drug entrapment efficiency: The entrapment efficiency of a drug by phytosomes can be measured by the ultracentrifugation technique 30.

4. Transition temperature: The transition temperature of the phytosomes can be determined by differential scanning calorimetry 31.

5. 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 32.

6. 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 33.

7. Drug content: The amount of drug can be quantified by a modified high performance liquid chromatographic method or by a suitable spectroscopic method 34.

II. Spectroscopic evaluations
To confirm the formation of a complex and interaction between the phytoconstituent and the phospholipids, the following spectroscopic methods are used 35.
1.     
1HNMR
The NMR spectra of (+) ?catechin and its stoichio?metric complex with distearoyl phosphatidylcholine have been studied by Bombardelli et al 36. In nonpolar solvents, there is a marked change of the 1H?NMR signal originating from the atoms that is involved in the formation of the complex, without any summation of the signal peculiar to the individual molecules. The signals from the protons belonging to the flavonoids are to be broadened that the proton cannot be relieved. In phospholipids, there is broadening of all the signals while the singlet corresponding to the N?(CH3)3 of choline undergoes an upfield shift. By heating the sample to 60?, the 1H?NMR shows the appearance of some new broad bands, which correspond mainly to the resonance of the flavonoid moiety.
2.     
13CNMR
In the 13C?NMR spectrum of (+) ?catechin and its stoichiometric complex with distearoyl phosphatidylcho?line, particularly when recorded in C6D6 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?, all the very broad and partially overlapping signals belonging to the flavonoid moieties reappear.
3.       FTIR
The formation of the complex can be also be analysed 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.

III. Biological tests (in vitro and in vivo evaluations)
The selection of models for in?vitroand in?vivoevaluations is based on the expected therapeutic activity of biologically active phytoconstituents present in the phytosomes 35. For example, in vitro antihepatotoxic activity is done by evaluating 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 examined37, 38. The in vivo safety evaluation of glycyrrhetinic acid?Phytosome® ointment, a commercial product, involves the skin sensitization and tolerability studies39.

DIFFERENCE BETWEEN PHYTOSOME AND LIPOSOME

Fig. 3: Shows difference between liposome and phytosome.

The molecular organization of the liposome (upper segment)
The molecular organization of phytosomes (lower segment)

Liposomes are prepared by same procedure as phytosomes.  A liposome is formed by mixing a water soluble substance with phosphatidylcholine in definite ratio under specific conditions. The main differences between liposomes and phytomes are:
1.      Main difference is that in liposomes no chemical bond is formed and the phosphatidylcholine molecules surround the water soluble substance. In a liposome, the material is simply emulsified.
2.      There may be hundreds or even thousands of phosphatidylcholine molecules surrounding the water-soluble compound. In contrast, with the phytosome process the phosphatidylcholine and the plant components actually form a 1:1 or a 2:1 molecular complex depending on the substance(s) complexes, involving chemical bonds. This difference results in better absorption of phytosomes than liposomes showing better bioavailability. Phytosomes have also been found superior to liposomes in topical and skin care products40 (Fig. 3).
3.      Another difference is size of liposomes. Liposomes, although composed of phosphatidylcholine, are much larger.
4.      Fundamental differences are that in liposomes, the active constituents are dissolved in the central part of the cavity, with no possibility of molecular interaction between the surrounding lipid and a hydrophilic substance41, 42. On the other hand the phytosome complex can somewhat be compared to an integral part of the lipid membrane, where the polar functionalities of the lipophilic molecule interact via hydrogen bonds with the polar head of a phospholipids (i.e. phosphate and ammonium groups), forming a unique pattern which can be characterized by spectroscopy43, 44, 45. This difference results in phytosome being much better absorbed than liposomes showing better bioavailability.
5.      Phytosomes are also superior to liposomes in skin care products while the liposome is an aggregate of many phospholipids molecules that can enclose other phytoactive molecules but without specifically bonding to them.
6.      Liposomes are touted delivery vehicles, but for dietary supplements, they are very less efficient. But for phytosome products numerous studies prove they are markedly better absorbed and have substantially greater clinical efficacy.
7.      Some liposomal drug complexes operate in the presence of the water or buffer solution whereas phytosomes operate with the solvent having a reduced dielectric constant. Starting material of component like flavonoids is insoluble in chloroform, ethyl ether or benzene. They become extremely soluble in these solvents after forming phytosomes. This chemical and physical property change is due to the formation of a true stable complex46.

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