4.1.

Material
Plant material: Leaf part of Cordia Dichotoma & Carissa Carandas were collected from
Jalandhar Cant., Jalandhar in the month January 2010. The plant material was identified
by Dr. H. B. Singh of Raw Material Herbarium & Museum, NISCAIR, New Delhi where
a

voucher

specimen

(No.NISCAIR/RHMD/Consult/-2009-10/326/128)

has

been

deposited at the herbarium unit.

Chemicals: Lecithin soya, 30%, HiMedia Laboratories Pvt. Ltd., Cholesterol, KEM
LIGHT Laboratories Pvt. Ltd. And all other chemicals are of analytical grade.

4.2. Method
Part A: preparation of aqueous extract of cordio dichotoma/Carissa Carandas
The leaves were dried under the shade and ground into coarse powder. The powder
(500 g) was macerated in 2.05 L of distilled water at room temperature for 24 h. it was
then filtered using a filter paper (Whatman size no. 1 ) and the filtrate evaporated to
dryness in the water bath at 600 C. A brownish residue weighing 20.6 g/ 20.16 g (yield of
4.12 & 4.02 % w/w) was obtained. This was kept in air tight bottles in a refrigerator until
used.
Part B: method of preparation of cordio dichotoma Phytosomes/Carissa carandas
Accurately weighed quantity of lecithin 45 mg and cholesterol 15 mg dissolved in
10 ml of chloroform in round bottom flask (RBF) and sonicated for 10 minutes using bath
sonicator. Organic solvent removal is done by Rotary evaporator (45-50C). After
complete removal of solvent thin layer of phospholipids mixture was formed. This film
was hydrated with aqueous extract of cordio dichotoma/ Carissa Carandas in Rotary
evaporator (37-40C for 1 hour). After hydration of mixture of lipid and plant extract was
sonicated for 20 minutes by using Ultrasonic Probe Sonicator (Pci Analytics) in presence
of ice bath for heat dissipation. Then prepared phytosomes were stored in freezer (2-80 C)
until used.

(1)

78

Table 4.1: Formulation composition of phytosome formulation of cordia dichotoma.

S. No.

cholesterol

Lecithin

chloroform

Type of
formulation
CDL1

10 ml

Aqueous extract of
cordio dichotoma
10 ml

Cholesterol:
lipid ratio
1.5:4

15 mg

40 mg

CDL2

15 mg

45 mg

10 ml

10 ml

1.5:4.5

CDL3

15 mg

50 mg

10 ml

10 ml

1.5:5

CDL4

15 mg

55 mg

10 ml

10 ml

1.5:5.5

CDL5

15 mg

60 mg

10 ml

10 ml

1.5:6

CDL6

15 mg

65 mg

10 ml

10 ml

1.5:6.5

CDL7

15 mg

70 mg

10 ml

10 ml

1.5:7

Table 4.2: Formulation composition of phytosome formulation of Carissa carandas.

S. No.

cholesterol

Lecithin

chloroform

Type of
formulation
CCL1

10 ml

Aqueous extract of
carissa carandas
10 ml

Cholesterol:
lipid ratio
1.5:4

15 mg

40 mg

CCL2

15 mg

45 mg

10 ml

10 ml

1.5:4.5

CCL3

15 mg

50 mg

10 ml

10 ml

1.5:5

CCL4

15 mg

55 mg

10 ml

10 ml

1.5:5.5

CCL5

15 mg

60 mg

10 ml

10 ml

1.5:6

CCL6

15 mg

65 mg

10 ml

10 ml

1.5:6.5

CCL7

15 mg

70 mg

10 ml

10 ml

1.5:7

4.3. Characterization
4.3.1. Quantification of CDL & CCL
The both formulation (CDL & CCL) contains the aqueous extracts of Cordia Dichotoma
& Carissa carandas. Standardization of extract done by HPTLC method. In which
79

quercetin taken as a marker to quantify the plant extracts. In HPTLC method used the
silica gel 60F254 precoated TLC aluminum plates [E-Merck], Toluene: Ethyl acetate:
Acetone: Formic acid (10:5:15:1) as solvent system and the plate was scanned at UV 366
nm and 254 nm using CAMAG TLC Scanner-3 and LINOMAT-V. (1)

4. 3.2. Size analysis of phytosomes

The Size distribution of phytosomes was analyzed using Malvern mastersizer S laser
diffraction size analyzer (Malvern Instruments Ltd., UK). A magnetically stirred cell
dispersion unit (Malvern Instruments Ltd., UK) was employed with medium speed
stirring in order to keep the phytosome dispersed during size measurement. The
measurement position was 1.05 mm and polydisperse mode of analysis was chosen.
These set up conditions permitted accurate measurement of particles having a size range
between 0.1 and 10000 d.nm. The size and size distribution were measured by the
instrument as ZAve.

(2)

4.3.3. Morphology of phytosomes

The morphology of phytosome was observed by Transmission electron microscopy
(TEM). The phytosome dispersions were deposited on carbon coated copper grids (400
mesh) and negatively stained with 1% w/v phosphotungstic acid. Phytosome morphology
was viewed using a Philps CM 120 BioTwin transmission electron microscope (Philips
Electron Optics BV, The Netherlands.

(1, 2)

4.3.4. Separation of entrapped and unentrapped plant extract

Phytosomes (10 mg/ml) were centrifuged for 50 min at 41,000 g and 40 C using R-8C,
REMI centrifuge machine (Lab Centrifuge, Ambala).The supernatant was collected and
phytosomes pellets in the centrifuge tubes were redispersed in 10 ml distilled water to
remove phytoconstituents (as for quercetin) adsorbed onto phytosomes. Centrifugation
was repeated for 50 min and the supernatant was again collected and added to the first
supernatant to

comprise the unentrapped

fraction of the phytoconstituents. The

phytosomes pellets were solubilized using Triton X-100 (1%, w/v) and the released
phytoconstituents was the phytosome-entrapped fraction. The entrapment efficiency (EE)
80

of phytoconstituents (as for quercetin) in phytosomes was calculated after quantification

of entrapped and unentrapped drug using HPTLC. EE (%) = (amount of quercetin
entrapped in phytosomes/overall amount of the quercetin in formulation) 100 %. (2)

4.3.5. Zeta potential

The measurement were made with a zetasizer 2000 DTS52013 (Malvern Instruments,
UK) at 250 C. The phytosomes were dilute with distilled water, loaded into capillary cell
mounted on the apparatus and all experiment was performed at least in triplicate.

(3, 4)

4.3.6. In Vitro Release Studies

The in vitro releases of cordio dichotoma were studied by using simple diffusion cell
apparatus. The diffusion cell apparatus consist of glass tube with an inner diameter of 2.5
cm open at both ends, on end of the tube is tied with sigma dialysis membrane, which
serves as a donor compartment. Phytosomes equivalent to 100 mg of cordio dichotoma
phytosomes was taken in test tube and placed in 100 ml of phosphate buffer. The medium
was stirrer and the temperature at 35-37C. Periodically 10 ml of sample were withdrawn
and after each withdrawn same volume of medium was replaced. Then the samples were
assayed spectrophotometrically/HPTLC . The releases of all formulations were compared
with pure cordio dichotoma extract.

(5, 6)

4.3.7. Drug Release Kinetics

In the present study,

the formulations (CDL2 & CCL2) were evaluated for

phytoconstituents release kinetics. The drug release data were plotted using various
kinetic equations (Zero order, first order, Higuchis kinetics, Korsmeyers equation, and
Hixson-Crowell Cube root law) to evaluate the drug release mechanism and kinetics.
The data obtained from in vitro drug release studies were plotted as cumulative amount of
drug released vs time, Zero order equation 1, log cumulative percentage of drug
remaining vs time, first order equation 2 , cumulative percentage of drug released vs
square root of time, Higuchis model equation 3
C = K0t

(1)

81

Where K 0 is the zero order rate constant expressed in units of concentration/time and t is
the time in hours. A graph of concentration vs. time would yield a straight line with a
slope equal to K 0 and intercept the origin of the axes.
Log C = LogC0 -kt/2.303

(2)

Where C0 is the initial concentration of drug, k is the first order constant, and t is the
time.
Q = Kt1/2

(3)

Where K is the constant reflecting the design variables of the system and t is the time in
hours. Hence, drug release rate is proportional to the reciprocal of the square root time.
To evaluate the drug release with change in the surface area and the diameter of the
particles, the data were also plotted using the Hixson-Crowell cube root law:
3

Q 0 - 3 Qt = kHC X t

(4)

Where Qt is the amount of drug released in time t, Q 0 is the initial amount of the drug in
the microsphere, and K HC is the rate constant for the Hixson-Crowell rate equation, as the
cube root of the percentage of drug remaining in the matrix vs. time.
Mechanism of Drug Release

(7, 8)

Koresmeyer et als equation (Equation 5). Log

cumulative percentage of drug released vs log time was plotted to evaluate the
mechanism of drug release from microspheres and the exponent n was calculated through
the slop of the straight line
Mt / M = Ktn

(5)

Where Mt/M is the fractional solute release, t is the release time, K is a kinetic constant
characteristic of the drug/polymer system, and n is an exponent that characterizes the
mechanism of release of tracers.
If the exponent n= 0.45, then the drug release mechanism is Fickian diffusion, and if
0.45 < n < 0.89, then it is non-Fickian or anomalous diffusion.

82

4.4. Statistical analysis

The invitro release results of CDL2 & CCL2 are expressed as the mean standard
deviation. Multiple comparisons of means (Tukey Test) are used to substantiate statistical
differences between groups, while students t-test was used to comparison between CDL2
& CCL2 batches. Significance was tested at the 0.001 level of probability (p) (9, 10).

83

4.5. Results and discussion:

4.5.1. Selection of method of Preparation:
Solvent evaporation method was selected for the preparation of phytosomes containing
plant extracts.
4.5.2. Optimization of lipid and Lecithin Ratio:
Seven formulation batches of aqueous extracts of Cordia Dichotoma & Carissa
Carandas leaves with the different ratio of lipid & lecithin and selected on the basis of
formation of phytosomes, encapsulation efficiency and desired particle size. On the basis
of morphology (digital photographs & TEM), particle size and encapsulation efficiency,
CDL2 & CCL2 batches were selected as the optimized formulation.
4.5.3. Visualization
4.5.3.1. Digital Microscopy

CDL2

CCL2

Figure 4.1: Digital Microscopy Results of Phytosomes (CDL2 & CCL2).

84

Figure 4.2: TEM of Cordia Dichotoma phytosomes(CDL2)

Fig 4.3: TEM of Carissa Carandas phytosome (CCL2).

85

4.5.4. Quantification of CDL & CCL

For quantification of CDL2 & CCL2 phyto constituents (flavonoids), HPTLC method
was used and quercetin taken as the marker compound. The standard curve of quercetin
was prepared by HPTLC method & UV spectrophotometer method. In HPTLC method
obtained the Y = 532.67x + 539.83 and R2 = 0.9929 and in UV spectroscopy method
Y=0.0907x + 0.0655 and R2 = 0.9989. These above equations used in quantification of
quercetin, which represent the quantity of phyto constituents present in the phytosomes.

4.5.5. Size analysis of phytosomes

The Size distribution of phytosomes was analyzed using Malvern mastersizer S laser
diffraction size analyzer (Malvern Instruments Ltd., UK). The average size of the cordio
dichotoma & Carissa Carandas phytosomes were 153 & 257.3 nm (Table no: 4.3).

Table 4.3: physiochemical characteristics of the Cordia Dichotoma and Carissa

Carandas phytosome formulation

Type of phytosomes

n-Average diameter (nm)

Zeta potential (mV)

Cordia Dichotoma Phytosomes

153

-21.0

Carissa Carandas phytosomes

257.4

-23.5

86

Figure 4.6: Showing Size Distribution by intensity of cordio dichotoma Phytosomes

(CDL2)

87

Figure 4.7: Showing Size Distribution by intensity of Carissa Carandas Phytosomes

(CCL2)

88

4.5.6. Transmission electron microscopy (TEM) analysis

TEM provided the evidence of vesicle formation and their morphology evaluation
analysis showed small, spherical, and unilamellar vesicles (Figure: 4.2, 4.3).

4.5.7. Separation of entrapped and unentrapped plant extract

The entrapment efficiency of Cordia Dichotoma & Carissa Carandas phytosomes were 91
& 90 % respectively (Figure 4.8). (1)

Figure 4.8: Encapsulation efficiency of cordio dichotoma and Carissa Carandas

phytosome
4.5.8. Zeta potential
The membrane surface potential plays an important role in the rate of aggregation and
fusion of vesicles and hence in the physical stability of phytosomes. As far as zeta
potentials are concerned, all types of liposomes had negative electrophoretic mobilities
(Table: 4.3).
The presence of a net negative charge in Phosphatidylcholine conferred higher values of
zeta potentials, as function of percentage of phosphatidylcholine, was asymptotic, and the
values observed elsewhere.

(11)

Kinetic stability in majority of the preparations is related to the presence of charge on

membrane, and consequently to the existence of electrostatic forces of repulsion that
balance the London dispersion forces. Vesicles repel each other when their double layer
overlaps. The origin of repulsive force is entropic.
89

Thus surface charge produces an energy barrier and when this barrier far exceeds the
thermal energy (expressed as kT), the primary minimum becomes inaccessible and the
system is kinetically stable.

(6)

Figure 4.9 .Showing Zeta Potential distribution of cordio dichotoma Phytosomes

(CDL2)

90

Figure 4.10: Showing Zeta Potential distribution of Carissa Carandas Phytosomes

(CCL2)

91

4.5.9. In Vitro Release Studies

The in vitro releases of cordio dichotoma & Carissa Carandas were studied by using
simple diffusion cell apparatus. Phytosomes equivalent to 100 mg of cordio dichotoma
phytosomes was taken in test tube and placed in 100 ml of phosphate buffer. The medium
was stirrer and the temperature at 35-37C. The CDL2 & CCL2 batches showed the
89.73 & 87.09 % cumulative release. When compared the release profile of CDL2 &
CCL2, both were significant different (figure 4.9, 4.10 & 4.11)

Figure 4.11: In vitro release of cordio dichotoma phytosomes (CDL1-CDL7)

Figure 4.12: In vitro release of Carissa Carandas phytosomes (CCL1-CCL7)

92

Figure 4.13: comparison of In vitro release of cordio dichotoma (CDL2) and Carissa
Carandas phytosomes (CCL2).
.
4.5.10. Drug Release Kinetics

(A)

93

(B)

(C)

(D)

Figure 4.14: release kinetics of CDL2,(A)first order plot, (B) korsemeyer peppas
plot, (C) zero order plot, (D) Higuchi matrix plot.

94

(A)

(B)

95

(C)

(D)

Figure 4.15: release kinetics of CCL2,(A)first order plot, (B) korsemeyer peppas
plot, (C) zero order plot, (D) Higuchi matrix plot.
The kinetic treatment reflected that release data of CDL2 & CCL2 showed R2 value of
0.9938 & 0.9904 for first order or 0.9446 & 0.9601 for zero order equation respectively,
indicating that release of plant phytoconstituents follows first order kinetic further in
kosrsemeyer peppas & higuchi equations n value were 0.259 & 0.266 showed the Fickian
diffusion.
4.6. Statistical analysis
When compared the release profile of Cordia Dichotoma phytosomes (CDL1 CDL2)
and Carissa Carandas (CCL1 CCL2) at the p=0.001 probability, it was found that all
groups of Cordia Dichotoma & Carissa Carandas phytosomes were significant different.
Furthermore it also found that CDL2 & CCL2 were significant different, when compare
by students t-test.

96

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