INTRODUCTION:

Phytomedicines, complex chemical mixture prepared from plants, have been used in medicine since ancient times and continue to have widespread popular use. The use of phytosomes is a new advanced modern dosage formulation technology to deliver herbal products and drugs with improved better absorption and, as a result, produce better result than those obtained by conventional herbal extract^1,2. Most of the bioactive constituents of phytomedicines are water-soluble molecules (e.g. phenolics, glycosides and flavonoids). However, water soluble phytoconstituents are limited in their effectiveness because they are poorly absorbed when taken orally or when applied topically.

Many approaches have been developed to improve the oral bioavailability, such as inclusion by solubility and bioavailability enhancers, structural modification and entrapment with the lipophilic carriers^3,4. There are many factors which may contribute to the poor bioavailability. For example, many phytoconstituents have multiple rings and therefore, cannot be absorbed from the intestine into the blood by simple diffusion.

Also, some herbal phytomolecules are poorly miscible with oils and other lipids and often fail to pass through the small intestine because of its lipoidal nature. Plants are endowed with a multitude of medicinal and health giving substances, most of them are secondary metabolite, prominent among these being the flavonoids. First recognized for their antioxidant properties, flavonoid is widely distributed in plants. To date, more than 4,000 naturally occurring flavonoid have been identified from plant source having diverse biological activities⁵. The hypothesis of an interaction of flavonoid with phospholipids, which are ubiquitous in plant and animals, originated from the histo chemical finding indicating that anthocyanosides from Vaccinium myrtillus L. show a strong affinity for specific cellular structure rich in phospholipids⁶. The effectiveness of any herbal product is dependent upon delivering an effective level of the active compounds. Advanced biochemical and pre-clinical studies have proved the potential of plant flavonoids and other hydrophilic natural compounds for the treatment of skin disorders, different types of carcinoma, anti-aging and many other areas of therapeutics and preventive medicine. The hydrophilic nature and unique chemical structure of these compounds pose major challenge because of their poor bioavailability through the skin or gut. The use of phytosome is novel formulation technology which helps to overcome these problems.

PROPERTIES OF PHYTOSOME

Phytosomes are complex between a natural phytoconstituents and natural phospholipids, like soy phospholipids mostly phosphatidylcholine⁶. This complex results from the reaction of stoichiometric amount of phospholipids with the phytoconstituents in an aprotic solvent.

v Phytosomes can accommodate the active principle that is anchored to the polar head of the phospholipids, which finally becomes an integral part of the membrane.

v Phytosomes are advanced form of herbal drugs and which are better absorbed, utilized and which finally leads to better results than conventional dosage form. The increased bioavailability has been demonstrated by the

v Pharmacokinetic studies as well as by pharmacokinetic tests in experimental animals and human subjects.

v Phytosomes are lipophilic substances with definite melting point, freely soluble in non-polar solvents, and moderately soluble in fats.

v Phytosomes when treated with water assume a micellar shape, forming structurethat resemble liposomes exhibiting fundamental difference.

PREPARATION OF PHYTOSOMES⁷

Phytosomes are novel complexes which are prepared by reacting from 3-2 moles but preferably with one mole of natural or synthetic phospholipids, such as phosphotidylethanolamine or phosphotidylserine with one mole of component for example flavoliganans, either alone or by spray drying. 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. In the phytosome preparations, phospholipids are collected from the group consisting of soy lecithin, from bovine or swine brain or dermis, phosphotidylcholine, phosphotedylethanolamine, phosphotidylserine in which acyl group may be same or different and mostly derived from palmitic, stearic, oleic and lenoleic acid. Selection of flavonoids are done from the group consist of quercetin, kaempferol,quercretin-3, rhamnoglucosides, quercetine-3-rhamnoside, hyperoside, virtexine, diomine, 3-rhamnoside, (+)catechin, (-)epicatechin, apigenin-7-glucoside, luteoline, luteolineglucoside, ginkgonetine , isoginkgonetine and bilobetine 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 lyophilizaton or by spray drying. In the complex formation of phytosomes, the ratio demonstrated by pharmacokinetic studies or by pharmacodynamic tests in experimental animals and in human subjects. Phytosomes are formulated by patented processes in which the standardized extract (having a standardized content of active principles) and/or active ingredients of herbs (like flavoliganans and terpinoides) are bound to the phospholipids like Phosphatidylcholine (pc) through a polar end. The phytosome processes produces small cells which protect the valuable components of the herbal extract from destruction by digestive secretions and gut bacteria¹⁵. They improve trasition of constituents from the water phase to the enterocytes of gut wall ultimately they reach the circulation. The phytoactive components of these herbal extract are well suited to direct binding to phosphatidylcholine from soy. Pc is also the principle molecular building block of cell membrane and is miscible with water both oil/lipid mixtures , and is well absorbed orally.phospholipids are small lipid molecules in which the glycerol is bound to only two fattyacids , instead of three as in triglycerides, with the remaining site is occupied by a phosphate group. Specifically the choline head of the phosphatidylcholine molecule binds to phytoconstituents while the fat soluble phophotidyl portion, comprising the body and tail, then ratios of EFP-PVP precipitates by means of dissolution release. Silybin and phospholipids were resolved into the medium, after the organic solvent was removed under vaccum condition and a silybin-phospholipid complex was formed.

CHARACTERIZATION AND EVALUATION OF PHYTOSOMES⁸

The behavior of phytosomes in both physical and biological systems is governed by factors such as the physical size, membrane permeability, percentage of entrapped solutes, and chemical composition as well as the quantity and purity of the starting material. 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

Visualization

Visualization of phytosomes can be achieved using Transmission Electron Microscopy (TEM) and by Scanning Electron Microscopy (SEM) electron microscopic techniques used to assess liposome shape and size are mainly negative-stain transmission microscopy and scanning electron microscopy. The later technique requires dehydration of the sample prior to examination and is less preferred. Negative stain electron microscopy visualizes relatively electron transparent liposomes or phytosomes as bright area against a dark background (hence termed as negative stain). Liposome like structure is embedded in this method in a thin film of electron-dence heavy metal (salt) stain. The use of negative stain electron microscopy facilitates estimation of the liposome size range at the lower end of the frequency distribution. Irregular or ellipsoid shape can be treated mathematically to correct for perimeter irregularities thus estimations of original spherical diameter can be calculated.

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).

Entrapment efficiency

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

Transition temperature

The transition temperature of the vesicular lipid systems can be determined by differential scanning calorimeter.

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.

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 47.

Drug content

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

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.

1H-NMR: The NMR spectra of (+)-catechin and its stiochiometric complex with distearoyl phosphatidylcholine have been studied by Bombardelli et al. in polar solvents, there is a marked change of the 1H-NMR signal originating from the atoms 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 flavonoid are to be broadened that the proton cannot be relieved. In the phospholipids, there is broadening of all the signals while the singlet corresponding to the N-(CH3)3 of choline undergo an uplift shift. Heating the sample to 60°C and results in the appearance of some new broad bands, which correspond mainly to the resonance of the flavonoid moiety.

3C-NMR: In the 13C-NMR spectrum of (+)-catechin and its stoichiometric complex with distearoyl phosphatidylcholine, 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 60-80ppm) are broadened and some are shifted, while most of the resonance of the fatty acid chain retains their original sharp line shape. After heating to 60°C, all the signal belonging to the flavonoid moieties reappear, although they are still very broad and partially overlapping.

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 the solid form (phytosomes) with the spectrum of its micro-dispersion in water after lyophilization, 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.

In-vitro and in-vivo Evaluation

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 anti-hepatotoxic activity can be assessed by the antioxidant and free radical scavenging activity of the phytosomes.

ADVANTAGES OF PHYTOSOMES⁹

As compared to conventional herbal formulation, Phytosomes have following advantages.

ü Phosphatidylcholine used in preparation of phytosomes, besides acting as a carrier also acts as ahepatoprotective, hence giving the synergistic effect when hepatoprotective substances are employed.

ü Chemical bonds are formed between phosphatidylcholine molecule and phytoconstituent, so the phytosomes show better stability profile.

ü Added nutritional benefit of phospholipids.

ü Appreciable drug entrapment.

ü Application of phytoconstituents in form of phytosome improves their percutaneous absorption and act as functional cosmetics.

ü Phytosome process produces a little cell whereby the valuable components of the herbal extracts are protected from destruction by digestive secretions and gut bacteria.

ü Assured delivery to the tissues.

ü Phosphatidylcholine used in the phytosome process besides acting as a carrier also nourishes the skin, because it is essential part of cell membrane.

ü Phytosomes are also superior to liposomes in skin care products.

ü Significantly greater clinical benefit.

ü The particular structure of phytosome elicits peculiar properties and advantages in cosmetic application.

ü Enhanced ability of phytosomes to cross cell membranes and enter cells.

ü Their low solubility in aqueous media allows the formation of stable emulsions or creams.

CONCLUSION:

Phytosomes are novel compounds comprising of lipophilic complexes of components of various plants like Silybum Marianum, Ginkgo Biloba, ginseng etc with phospholipids. Preparation of phytosomes is usually carried out by non conventional method. Absorption of phytosome in gastro intestinal tract is appreciably greater resulting in increased plasma level than the individual component¹⁰. Complex formation ratio of component and phospholipids is 1:1 and 2:1. Phytosomes are used as a medicament and have wide scope in cosmeticology. Many areas of phytosome are to be revealed in future in the prospect of pharmaceutical application. Phytosomes forms a bridge between the conventional delivery system and novel delivery system. Phytosomes enables pharmaceutical manufacturers to provide new pharmaceutical products using water soluble drugs and provides new developments in medical industry. The technology can effectively deliver the product by topical and oral route.

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Received on 19.12.2012 Accepted on 26.12.2012

Res. J. Topical and Cosmetic Sci. 3(1): July-Dec. 2012 page 56-59