INTRODUCTION:

Picrorhiza kurroa Royal Ex. Benth. has been used in several Indian herbal preparations to cure malfunctioning of liver (1). The ethanolic extract of Picrorhiza kurroa Benth. Exhibits hepatoprotective activity against CCl₄ and galactosamine induced liver damage by Plasmodium berghei infection in mastomys. This activity is located only in kutkin rich fraction of the extract. Kutkin is a stable mixed crystal of two glycosides, glucoside-A and kutkoside in the ratio of 1:2. Chemically glucoside-A is 6'O-cinnamoyl catalpol (V) designated as picroside-I¹.

Picroside-I normally exhibits considerable hepatoprotective effect when administered orally or experimented in vitro. But certain approaches must be made to increase the absorption and to render the compound more bioavailable.

In present study the hepatoprotective action of picroside-I was improved by complexing irridoid glycoside with natural or synthetic phospholipids (Phytosome term patented by Indena). One of the constituent of complex picroside-I is employed in the treatment of liver diseases of various origin, while the other i.e. phospholipids (particularly phosphatidylcholine) are essential for the normal structure and function of the liver cells, intervening in the regulation of the fluidity and permeability of cell walls and phospholipids as hepatoprotectants ³.

The phytosome are cell or vesicle like structures resulted from chemical interaction between the choline head of phosphatidylcholine molecules and terpenoid or flavonoid component of herbal extract ⁴.

These structures contain the active ingredient of herb surrounded and bound with phospholipids. These are lipophilic substances with a definite melting point different from that of the individual components, freely soluble in aprotic solvents, moderately soluble in fats and insoluble in water. On treatment with water they assume a miscellar shape forming structures, which might resemble liposomes, but is conceptually, practically and fundamentally different ⁴.

In liposome active principle is dissolved in the medium and contained in the cavity or intercalated in the layers of the membrane, while in phytosome active principle is an integral part of the membrane, where the molecule being anchored through chemical bonds to the polar head of the phospholipids⁴(Fig. 1).

Fig. 1: Main Difference between Liposome and Phytosome

Moreover, the bioavailability of the most of the botanical active principles isvery poor and it could be enhanced by complexing them with phosphatydylcholine. Therefore, phytosome are not only enhanced the bioavailability or GIT absorption of the herbal active principle but it also helps to target the active principle to desired site.

Hence the present project hepatoprotective action of active principle of Picrorhiza Kurroa was improve by making phytosomes.

EXPERIMENTAL

Plant Material :

Roots and rhizome of Picrorhiza kurroa Royale Ex. Benth. were collected from local market of Sagar (India) and identified.

Extraction and Isolation :

The Picroside-I was extracted and isolated from the Picrorhiza kurroa extract according to the procedure reported by Singh and Rastogi ⁵given in Indian Herbal Pharmacopoeia ⁶.

The isolated Picroside-I was characterized by qualitative tests (Legal test for lactone and godin's test, specific for picroside-I), UV, IR and ¹H-NMR spectroscopy.

Preparation of Drug Phosphatidylcholine Complex (Phytosomes)

The drug (Picroside-I) and phosphatidylcholine complex (Phytosome) was prepared according to the procedure described by Gabetta et al.,⁷. Briefly a mole of picroside-I (isolated from P. kurroa) was allowed to react with a mole of soya-phosphatidylcholine in an aprotic solvent and the reaction product was recovered by using non solvents.

The formation of complex was confirmed by comparing the UV, IR and ¹H-NMR spectra of picroside-I-phosphatidylcholine complex, picroside-I and phosphatidylcholine spectra.

Characterization of Phytosome

Size and size distribution :

After the dispersing the complex in the PBS pH 7.4 the size and size distribution of phytosomes were determine by laser diffraction based particle size analyzer (1064L Cilas, France).

Complexation efficiency :

Complexation efficiency was determined by taking different molar ratio of phosphatidylcholine (PC) and picroside-I (P-I) by passing through Sephadex G-50 mini column and then separated vesicles were disrupted using 0.2 ml of 2% Tritan X-100 and estimating drug content spectrophotometrically against blank at l_max 280 nm.

In vitro Absorption Studies:

In vitro absorption of picroside-I and phytosome was compared using everted small intestinal sac technique ⁸. Equimolar solutions of picroside-I (4 mg) and phytosome (10.1 mg) were prepared in 50 ml phosphate buffer saline solutions (pH 7.4). Freshly isolated goat intestine (5 inch) was isolated, washed properly with distilled water and buffer ringer solution. Two pieces of 2-2.5 inches in length of washed intestine were cut and everted with the help of blunt glass rod. The both ends of everted intestine were cannulated then intestine was filled with the buffer ringer solution and immersed in drug solution of known concentration. The solution was stirrered by continuous air flow and temperature was maintained at 37±1^oC using circulating water bath. After specified time, serosal fluid of each intestine was assayed spectrophotometrically at 280 nm for drug content and calculated the amount of drug absorbed through intestine.

In vivo Screening:

The phytosomes were assayed for their anti-hepatotoxic activity using Handa and Chakrabarti⁹models and the activity of phytosome was compared with plain picroside-I.

Animals :

Albino rat of either sex weighing between 75-100 gms. were selected.

Table 1: Various molar ratio of Picroside-I: PC complexes and their characteristics

S.No.	Formulation Code	Molar ratio of PC : P-I	% Drug complexed	Size (mm)
1.	V₁	1 : 5	17 %	2.80
2.	V₂	2 : 5	29%	2.84
3.	V₃	3 : 5	48%	2.93
4.	V₄	4 : 5	65%	3.12
5.	V₅	5 : 5	81%	3.45
6.	V₆	6 : 5	80%	3.41

PC = Phosphatidylcholine; P-I = Picroside-I

Table 2: Dose regimen of carbon tetrachloride model

Group	Treatment (Days)
Group	1	2	3	4	5
Normal	Veh.	Veh+liq. Para	Veh+ liq. Para	Veh.	Blood collected from retro-orbital plexus
Control CCl₄	Veh.	Veh + CCl₄	Veh + CCl₄	Veh.
Drug Solution (D.S.)	D.S.	D.S. + CCl₄	D.S. + CCl₄	D.S.
Phytosome Solution (P.S.)	P.S.	P.S. + CCl₄	P.S. + CCl₄	P.S.

Veh. = Vehicle (distilled water) = 1 ml/kg body weight

CCl₄=A 50% solution of CCl₄ in liquid paraffin administered S.C. at a dose of 2ml/kg body weight

D.S. = Drug solution (Solution of picroside in distilled water) 25 mg/kg body weight

P.S. = Phytosome solution = equivalent to 25 mg/kg body weight picroside I

liq. Para = Liquid Paraffin

Table 3 : Antihepatotoxic activity of picroside and 1:1 picroside-phosphatidylcholine complex in the albino rat, after poisoning with carbon tetrachloride

Group	SGOT (AST) Level ± S.D. (U/L)	% Protection	SGPT (ALT) Level ± S.D. (U/L)	% Protection
Normal	115.52±8.23	-	70.82±8.12	-
Control CCl₄	212.83±9.06	-	158.12±6.28	-
D.S.	178.12±5.62	35.66%	118.27±7.28	45.64%
P.S.	149.12±6.68	65.47%	92.14±5.13	75.55%

In vivo anti-hepatotoxic activity :

In vivo antihepatotoxic activity of picroside-I alone and phytosomes was compared using the method reported by Handa and Chakrabarti ⁹.

Twenty four albino rats were divided into four groups containing 6 animals in each group. Group-I animals were kept as normal and animals of
group-II (control) were given subcutaneous dose of toxin solution (CCl₄; 2 ml/kg). Picroside-I solutions and phytosome solutions (equivalent to 20 mg/Kg body weight of Picroside-I) were administered orally to the animals of group three and four, respectively. The drug solution (Picroside-I) and toxin(CCl₄) solution were administered according to schedule given in table 1.

On fifth day blood samples were collected from retro-orbital plexus of the rats and SGOT and SGPT level were estimated using auto blood analyzer (RA 50 Model) and reagent kit (Miles India Ltd., Mumbai, India)

RESULT AND DISCUSSION:

Compound-I isolated from Picrorhiza kurroa extract using chromatographic method and was confirmed to be picroside-I by performing qualitative tests and spectroscopical analysis. The isolated compound was a dark brown, amorphous powder having m.p. 130-132^oC. Positive legal's test suggested to have lactone ring in structure and further positive test with Godin's reagent (specific for picroside-I) suggested that compound could be picroside-I, which is in agreement with the findings of Singh and Rastogi⁵. UV spectroscopy of compound-I (Fig. 2) shows intense absorption near 238 nm indicating an heteroaromatic compound and low intensity absorption near 281 nm showing presence of heteroatom which could be oxygen having unshared pair of electron and indicative of the lactone ring presence. IR spectra (Fig. 3) indicated presence of hydroxyl, aromatic ester and enol ether groups, as band at 3420.9 cm^-1, which is due to hydroxyl group, 1057.1 cm^-1 and 1769.5 cm^-1 due to C-O and C=O, respectively suggesting aromatic ester and 1793.8 cm^-1 indicates lactone ring which is fused with another ring. Further the ¹H-NMR spectral data (Fig. 4) shows the presence of cyclohexane protons at chemical shift values 6.398 and 4.759, oxiran (epoxide) ring protons at 3.729, methine (CH protons of tetrahydropyran ring system (3.410, 3.726, 4.275, 5.033), ethylene protons of cinnamoyl moiety at 7.663 and benzene ring protons in cinnamoyl group at shift values near 7.147 (10). These findings are in agreement with spectral characteristics reported by Singh and Rastogi ⁵ and assigning compound-I as picroside-I.

This picroside-I present in kutkin rich ethanol fraction of Picrorhiza kurroa extract, possesses hepatoprotective activity against several intoxicants. The hepatoprotective activity of picroside-I is further investigated after complexing it with soya-phosphatidylcholine in stoichiometric amounts. The resulting phytosomes have marked lipophillic character and unexpectedly improve the oral absorption of complexed picroside-I and consequently shows improved specific in vivo activity.

Fig. 2 : UV Spectroscopy of Compound-I

Fig. 3 : IR Spectroscopy of Compound-I

Fig. 4 : ¹H-NMR Spectroscopy of Compound-I

Fig. 5 : IR Spectroscopy of Phosphatidylcholine

Fig. 6 : IR Spectroscopy of Phytosome

The profound interaction between irridoid glycoside and polar ends of soya- phosphatidylcholine is revealed in UV, IR and ¹H-NMR spectrum. IR spectrum of phosphatidylcholine (Fig. 5) shows characteristic bands near 2926 cm^-1 (due to C-H stretching of CH₃ group), 1651 cm^-1 (C=C stretching indicating unsaturation), 1738.6 cm^-1 (C=O stretch), 1460.2 cm^-1 (C-H bending of CH₂ adjacent to C=O), 3032 cm^-1 (C-H stretch showing CH₂=CH₂), 1070 cm^-1 (C-O stretch) and 1234 cm^-1 (P=O), while complex IR spectrum (Fig. 6) shows bands at 2925 cm^-1 (C-H stretch of -CH₃ group), 3046.9 cm^-1 (C-H stretch of CH₂), 1651.7 cm^-1 (C=C stretch showing unsaturation which was observed in phosphatidylcholine), 1093.9 cm^-1 (C-O stretch).

Fig. 7 : ¹H-NMR Spectroscopy of Phosphatidylcholine

¹H-NMR of phosphatidylcholine (Fig. 7) shows characteristic peaks at different chemical shift values as, peak at 6.298 shows presence of CH protons attached to -OC=O at 5.32 indicative of second CH₂ group protons near N atom, CH₂CH₂N), 4.513 (CH₂ protons attached to -OC (=O) C), peaks at 3.667-3.297 (Protons of CH₃ attached to N atom), 2.248 (CH₂ protons of aliphatic side chain of molecule) 0.968 (due to CH₂ protons of aliphatic side chain molecule), while 1:1 picroside-I and phosphatidylcholine complex (Fig. 8) shows peaks at shift value 0.968, 1.286 and 2.248 (CH₃ group protons of aliphatic side chain, CH₂ protons of aliphatic side chain and CH₂ porotns attached to -OC=C), 5.32 (second CH₂ protons near N atom CH₂CH₂N), 3.667-3.272 (protons of CH₃ joined to N-atom broadened significantly), also signals in range of 7.646 to 3.27 are also quite broadened.

Fig. 8 : ¹H-NMR Spectroscopy of Phytosome

The spectral characteristics of complexes are appreciably different from those of the individual constituents taken separately. The UV maxima of complex (Fig. 9) at 264 nm (intermediate between picroside-I 280-281 nm and soya-phosphatidylcholine (Fig. 10) 244 nm reveals significant changes in features of picroside-I as well as soya-phosphatidylcholine due to some chemical interaction. Similarly, IR spectra of soya-phosphatidylcholine at 1234.5 cm^-1 shows a band due to P=O which disappears in IR spectra of complex with picroside-I. Also IR spectra of picroside-I at 1769.5 cm^-1 due to C=O stretch also disappears in spectra of complex. Formation of complex is also shown in ¹H-NMR spectra. This is indicated by substantial widening of signals in ¹H-NMR of complex. Particularly the signals of the phosphatidylcholine which shows the presence of CH₃ group protons of aliphatic side chain, CH₂protons attached to -C(=O)-C and also the signals of picroside-I molecule due to hydroxyl protons and aromatic protons are broadened, even some of them disappears. This reveals strong interactions between choline portion of phosphatidylcholine and picroside-I moiety during complexation.

Fig. 9 : UV Spectroscopy of Phytosome

Fig. 10: UV Spectroscopy of Phosphatidylcholine

The size distribution analysis and complex efficiency are shown in table 2. Maximum complex efficiency (81%) is obtained when PC:P-I are taken in equimolar concentration ratio (5:5). On further increasing the molar concentration of PC in picroside-I (6:5) the complexation efficiency (80%) no longer increases.

Fig. 11 : Cumulative absorption between equimolar doses of Picroside-I and Phytosome solution

The amount of drug absorbed from mucosal fluid to serosal fluid at various time interval is shown in fig 11 and it was observed that 52 mg drug absorbed via intestinal membrane from picroside-I in 2 hrs, while equimolar doses of phytosome in the same time resulted in 156 mg of drug absorption. The value of rate constant K as estimated from slopes of plot for drug picroside-I and complex found to be 0.871 mg/min and 2.330 mg/min respectively. Thus, in vitro study shows that phytosome dispersion has significantly increased absorption as compared to drug solution when both are given in equimolar doses. This increased absorption may be contributed to increased liposolubility of phytosome as picroside-I forms complex with the polar ends of the phosphatidylcholine, while non-polar ends remain free to move, thus rendering phytosome to cross lipid soluble, biological barrier more easily.

Further, the hepatic protection action of phytosomes was studied by measuring SGOT and SGPT enzyme levels in CCl₄ induced hepatotoxicity models and compared with Picroside-I. Percent hepato protection offered by picroside-I alone and phytosomes determined in terms of decrease in SGOT and SGPT level, which were raised after CCl₄ intoxication and is shown in table 3 and fig. 12.

Fig. 12 : Graphical representation of in vivo serum parameter

It is observed that phytogen exhibited 65.47% hepato protection as compared to 35.66% hepatoprotection offered by picroside-I alone.

According to investigations, the picroside-I phytosomes could be utilized for effective treatment of acute or chronic liver diseases of toxic metabolic and for infective origin, degenerative liver diseases and preventive treatment against liver damage resulting from the use of drug and for luxury substances having injurious effect on the liver. It is hence concluded that the phytosomes can intensify the efficacy of herbal compounds, by simultaneous administration of phospholipids and picroside-1 both of which are hepatoprotectant individually –as phytosomes and can also improve the absorption, bioavailability and delivery of herbal compound to the tissues.

REFERENCES:

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Received on 21.03.2023 Modified on 19.04.2023

Research J. Pharm. and Tech 2023; 16(5):2353-2358.

DOI: 10.52711/0974-360X.2023.00387