Drug Delivery

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Bacopasaponin C: Critical Evaluation of Anti-

Leishmanial Properties in Various Delivery Modes

J. Sinha, B. Raay, N. Das, S. Medda, S. Garai, S. B. Mahato & M. K. Basu

To cite this article: J. Sinha, B. Raay, N. Das, S. Medda, S. Garai, S. B. Mahato & M. K. Basu
(2002) Bacopasaponin C: Critical Evaluation of Anti-Leishmanial Properties in Various Delivery
Modes, Drug Delivery, 9:1, 55-62, DOI: 10.1080/107175402753413181

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Drug Delivery, 9:55– 62, 2002
Copyright °c 2002 Taylor & Francis
1071-7544 /02 $12.00 + .00
Bacopasaponin C: Critical Evaluation of
Anti-Leishmanial Properties in Various Delivery Modes
J. Sinha, B. Raay, N. Das, S. Medda, S. Garai, S. B. Mahato, and M. K. Basu
Biomembrane Division, Indian Institute of Chemical Biology, Calcutta, India
Bacopasaponin C, an indigenous glycoside, was isolated from
Indian medicinal plant Bacopa monniera (b. brahmi) and was tested
for antileishmanial properties both in free and in various delivery
modes, e.g., niosomes, microspheres, and nanoparticles that are
used now as alternatives to more commonly used liposomes. The
different vesicles were prepared by published protocols. The per-
cent intercalation of Bacopasaponin C in liposomes, niosomes, and
micropspheres determined at its absorption maximal (¸max =
238 nm, 2 = 8:6 £ 103 M¡ 1 cm¡ 1 ) was found to be 30; for nanopar-
ticles it was 50. At equivalent dose of 1.75 mg/kg body weight, every
third day for a total of 6 doses in 15 days, Bacopasaponin C in all
the vesicular forms was found to be very active. An inverse lin-
ear relationship between the efŽ cacy and the size of the vesicles
was established. As analyzed from tissue histology, blood pathol-
ogy, and speciŽ c tests related to normal liver and kidney functions,
Bacopasaponi n C in each of the four vesicular forms was found to
be without any side effects. Thus, because of its indigenous origin
and non-toxic nature, Bacopasaponin C could very well be consid-
ered for application in the clinic through these alternative delivery
modes.
Keywords Bacapasaponin C, EfŽ cacy versus Size, Liposomes,
Microspheres, Nanocapsules, Niosomes
Biomedical science has illustrated the need to control, regu-
late, and target the release of drug in the body to the particular
tissue or cells of interest. In general, a primary requirement has
been to provide less frequent drug administration with a constant
Received 15 May 2001; accepted 6 August 2001.
Financial support from University Grants Commission, New Delhi,
India, and Dept. of Biotechnology, New Delhi, India, are gratefully
acknowledged. Thanks are also due to CSIR, New Delhi, India, for
providing Ž nancial assistance to M. K. Basu in the form of ES Scheme.
We are extremely grateful for helpful suggestions of Prof. S. P. Moulik,
Jadavpur University, for dynamic light scaterring measurements and
Dr. S. Chakraborty, Calcutta University, for hisopathological studies.
Address correspondence to M. K. Basu, Indian Institute of Chemical
Biology, 4, Raja S.C. Mullick Road, Jadavpur, Calcutta-700032, India.
E-mail: mukulkbasu@yahoo.com
55
therapeutic level of drug in the systemic circulation or at the spe-
ciŽ c target organ site furthering a reduction in any undesirable
side effects of the drug.
Among the different strategies for site-speciŽ c drug delivery,
colloidal/vesicular carriers were found to be effective because of
their structure function diversity in in vivo and in vitro milieus.
Colloidal carriers are of various types, such as liposomes, nio-
somes, nanoparticles, and microspheres. The most popular and
well-studied colloidal drug delivery systems are the liposomes,
though they have some fundamental problems, such as sensi-
tivity lamellae and difŽ culty in scaled up production (Guerrero
et al. 1998). Thus, to avoid such limitations of liposomes, al-
ternative drug delivery systems as niosomes, nanocapsules, and
micropheres are researched. Niosomes (Baillie et al. 1985) are
made from nonionic surfactants, that are quite stable, biodegrad-
able, and cheap. Polymeric delivery vesicles include the mi-
crospheres and nanoparticles and are made up of cost-effective
biodegradable polymers such as poly(esers), poly (orthoesters),
polyanhydrides, and different natural polymers. These vesicles
are found to be highly stable and biocompatible (Anderson and
Shive 1997; Guterres et al. 1995). These vesicles are also ca-
pable of encapsulating a wide variety of drugs and molecules
(Hiremath and Hota 1999; Kreuter 1996).
Our investigation aims to compare the structure and function
of four drug delivery vesicles—liposomes, niosomes, micro-
spheres, and nanocapsules—on the basis of their size, efŽ cacy,
and toxicity with liposomes as control. The efŽ cacy of these
vesicles is studied by incorporating an antileishmanial com-
pound, Bacopasaponin-C, extracted from an Indian medicinal
plant Bacopa monniera (b. brahmi) against experimental leish-
maniasis in a hamster model. Bacopasaponin-C is a glycosidic
pseudojujobogenin (Garai et al. 1996) having two carbohydrate
residues, glucose and rhamnose, attached to the triterpenoid
aglycone part. The unique presence of a glucose residue equipped
the compound to be a self-targeting molecule that can be directed
toward the glucose receptor present on the macrophage surface.
This facilitates a receptor-mediated drug delivery to the reticu-
loendothelial system.
56 J. SINHA ET AL.
MATERIALS AND METHODS
Polylactide-co-glycolide (PLGA) (Avr. Mol. wt. 113, 200);
polylactide(PLA) (Avr. Mol. wt. 106000); gelatin; Span-40
phosphatidylethanolamine (PE); cholesterol; dicetyl phosphate
(DCP); Tween-80; Span-40; isopropylmyristate; fetal calf serum
(FCS); and RPMI-1640 were purchased from Sigma Chemicals
(St. Louis, MO, USA). Bacopasaponin-C was supplied by our
Medicinal Chemistry Department (IICB) with optimum purity
and quality. All other chemicals were of analytical grade.
Preparation of Different Vesicles Systems
Bacopasaponin-C – loaded micropheres were prepared by the
solvent evaporation technique. In short, PLGA and Bacopasa-
ponin-C at ratio of 15:1 w/w was added to 2 ml of dichlorome-
thane. This was then added drop-wise into 100 ml of PBS
(pH 7.2) containing 1% gelatin as a dispersing agent. The solu-
tion was emulsiŽ ed by stirring for 2 hr resulting in the evapora-
tion of dichloromethane. The gelatin solution was centrifuged
at 8K – 10K rpm. The pellet was washed twice with PBS, then
collected and re-suspended in PBS (Wang et al. 1996).
MLV liposomes were prepared using PE, cholesterol, and
DCP in a molar ratio of 7:4:1. Bacopasaponin-C was added to
chloroform-methanol (2:1 v/v) solution of the lipids in a ratio of
1.16::10 w/w (Gregoriadis and Ryman 1972). A thin dry Ž lm of
the lipids and the drug was made on the inner surface of a round
bottom  ask by evaporating the organic solvents in a rotating
 ask evaporator. The suspension was sonicated for 30 sec at
4± C and unencapsulated Bacopasaponin-C was separated from
the vesicle-trapped components by centrifugation at 105,000 g
for 1 hr; the pellet was suspended in PBS.
Niosomes were prepared from the nonionic surfactant, sorbi-
tan monopalmitate (Span-40), cholesterol, and DCP in the molar
ratio 1:0.5:1. Chloroform was added to Span 40 and cholesterol.
The mixture was heated to 60± C in a water bath to form a so-
lution. After cooling, 1 mg of Bacopasaponin-C (per 10 mg of
Span 40) dissolved in 100 ¹l of DMSO was added and a thin
Ž lm prepared. The Ž lm was dried in an atmosphere of N2 and
kept overnight in desiccators. The Ž lm was swelled with 1 ml of
PBS (pH 7.2) for 1 hr and then sonicated for 30 sec. The entire
suspension was centrifuged at 100,000 £ g for 40 min; the pellet
formed was suspended in PBS to form the niosomal suspension
(Yoshioka et al. 1994).
Nanocapsules are prepared according to the method of Seyler
et al. (1995) with some modiŽ cations. In brief, polylactide
(70 mg) was dissolved in 10 ml of dichloromethane. Phospha-
tidylethanolamine (100 mg) was dissolved in 250 ¹l of isopropy-
lmyristate along with drug (9 mg) and added to dichloromethane
phase. The organic solution was added or allowed to run slowly
into 50 ml of PBS (pH 7.2) containing 0.4% of nonionic surfac-
tant (Tween-80), under moderate magnetic stirring. The organic
phase was evaporated out by moderate stirring for 3 hr. This solu-
tion was then ultracentrifuged at 35 K rpm, and the nanocapsules
were then collected (Seyler et al. 1995).
The percent intercalation of Bacopasaponin-C in various ves-
icular forms was calculated from a small aliquot taken and sol-
ubilized in the required solvent, knowing ¸max (238 nm) and
2M (8.6 £ 103 M¡1 cm¡1 ). For liposomes, niosomes, and
nanocapsules, the percent intercalation was found to be »30;
for microspheres, the value increased to 50.
Animal Experiments
A colony of Golden hamsters (Mesocristatus auratus) orig-
inally from Haffkine Research Institute, Bombay, was used to
maintain Leishmania donovani, strain AG83, from an Indian
Kala-azar patient by intracardial passage every 6 weeks. Each
animal was infected through intracardial passage with 2 £ 106
amastigotes. A group of 18 hamsters of average body weight
100 gm was infected and ready for drug testing after 1 month.
The hamsters were distributed into free drug, liposomal drug,
niosomal drug, microspehuralated drug, nanocapsulated drug,
and untreated control groups.
For MTD determination, a single dose treatment was fol-
lowed using varying amounts such as 1 mg to 10 mg/kg-body
weight of free drug; 4 such doses were given through subcuta-
neous route at an interval of 3 days. The MTD of the free drug
was found to be 1.75 mg/kg body weight (Figure 1).
The chemotherapy of multiple dose treatment was followed,
with average body weight of hamster at »100 gm. In practice,
0.175 mg of Bacopasaponin-C encapsulated in 0.5 ml of liposos-
mal, niosomal, microsphere, and nanocapsule suspension was
injected subcutaneously in each hamster every 3 days for a total
of 6 days in 15 days. Free Bacopasaponin-C at same dosage
(0.175 mg/0.5 ml PBS) was administered to one group. Para-
site burden of spleen was assessed from stained smears using
Stauber’s formula (Stauber et al. 1958).
Total number of amastigotes
D (Number of amastigotes=hostcell nucleus)
£(weight of spleen in mg) £ 2 £ 105
Investigation of Drug Toxicity
SpeciŽ c serum enzyme levels and splenic tissue histopathol-
ogy were assessed to Ž nd out drug toxicity. Serum gluta-
mate, pyruvate transaminase, and serum alkaline phosphatase
(Reitman and Frenkel 1957; Kind and King 1954) were assayed
to analyze the hepatotoxicity. Similarly serum urea and creati-
nine levels were measured to Ž nd nephrotoxicity of the drug. The
splenic histopathology was examined through haematoxylin-
eosin stained splenic section (Gurr 1962).
Determination of Vesicular Size
The average vesicle diameters of the niosomes and nanocap-
sules were determined through a dynamic light scattering
instrument (Otsuka Electronics, Japan) using Neon laser of
 BACOPASAPONIN C
FIG. 1. MTD determination curve of Bacopasaponin-C , assessed through single dose treatment using varying amounts of 1 – 10 mg/kg body weight. Four such
doses were given through subcutaneou s route at intervals of 3 days.
¸ D 632:2 nm and taking measurements at 90± angle (Majhi
and Moulik 1999; Moulik et al. 1999). The same for micro-
spheres and liposomes was carried out with light microscopy to
get optimum and accurate results. Size distributions of each of
these vesicles are exhibited through bar diagrams in Figure 3.
Trypan Blue Exclusion Test to Study Toxicity of
Bacopasaponin-C on Macrophages
Peritoneal macrophages were isolated from thioglycollate
injected Balb/c mice in RPMI-1640 medium containing 10%
FCS. In each eppendorf containing 500 ¹g of macrophage sus-
pension (5 £ 106 cells), Bacopasaponin-C solution was added
to prepare three effective concentrations (50 ¹g/ml, 250 ¹g,
and 500 ¹g/ml of macrophage suspension). The separate un-
treated controls were also prepared. The macrophages were in-
cubated with drug for 1 hr at 37± C. Negative control was used
with heat-killed macrophages; 500 ¹l of 0.4% Trypan blue so-
lutions was added to the untreated, heat-killed, drug-treated
macrophages and incubated for 10 min. Percent viability for
three sets of treatment and controls were calculated according to
relationship.
Cell viability D Total viable cells (unstained)
£100 =total cells (stained C unstained)
57
RESULTS
Size Determination and Distribution
The size determination of the nanocapsules and niosomes
was done by dynamic light scattering; the average diameter of
the vesicle was found to be 266.9 nm and 556 nm, respectively.
The distribution patterns are shown in Figure 2.
From size determination, the average diameter of micro-
spheres and liposomes of these vesicles was found to be
16.28 ¹m and 2.27 ¹m respectively. The size distribution of
each is demonstrated in Figure 3.
Toxicity of Drug to Macrophages
The drug was found to be nontoxic to the host peritoneal
macrophages when tested with Trypan blue exclusion test. Re-
sults show negligible macrophage mortality when treated with
different drug concentrations, as compared with untreated
controls (Figure 4).
Assessment of Drug-Induced Leishmania Killing
While testing the drug-induced antileishmanial activity (in
vivo) of the drugs in various forms, free Bacopasaponin-C low-
ered parasitic load of the spleen by 40% whereas liposomal,
niosomal, microencapsulated, and nanocapsulated drug lowered
58 J. SINHA ET AL.

FIG. 3. Size distribution of microspheres (B) and liposomes (A) determined

through light micrometry.

microencapsulated, or nanocapsulated drug. A similar result was

obtained with SGPT level.
Measuring serum urea and creatinine level as shown in
Table 3 assessed nephrotoxicity of the drug. The level of both
urea and creatinine increased in the free drug treatment. But
with the drug in different vesicular forms, values for both urea
and creatinine reduced to normal level, showing apparently no
nephrotoxic effect.

FIG. 2. Size distribution of nanocapsule s (B) and niosomes (A) determined

through dynamic light scattering, using neon laser of ¸ D 632.2 nm, measured
at 90± angle.

the spleen parasite burden by 81%, 86%, 79%, and 91%,

respectively (Table 1).

Assessment of Drug Toxicity

The hepatotoxicity of the drug was explored by assaying
the speciŽ c activities of two liver function enzymes: alkaline
phosphatase (AP) and serum glutamate pyruvate transminase
(SGPT). The results are shown in Table 2. The level of AP
increased on treatment with free drug, but decreased to nor- FIG. 4. Trypan blue exclusion test to study the toxicity of Bacopasaponin- C
mal level when treatment was done with liposomal, niosomal, on macrophages .
 BACOPASAPONIN C 59

TABLE 1 TABLE 3
Effect of Bacopasanonin-C loaded liposomes, niosomes, Effect of Bacopasanonin-C loaded liposomes, niosomes,
microspheres, and nanocapsules for treatment of experimental microspheres, and nanocapsules on urea and creatine levels
leishmaniasis on 30-day hamster model related to normal kidney functions

(Parasitic Percent supression Group Ureaa Creatinineb

load in of spleen parasite
Group spleen) £ 10¡9 load Infected untreated control 41.87 § 3.24 0.44 § 0.10
Free drug 50.45 § 2.83 0.58 § 0.15
Infected untreated control 2.65 § 0.24 – Liposomal drug 32.32 § 1.59 0.32 § 0.07
Free drug 1.60 § 0.75 40 Niosomal drug 36.89 § 7.05 0.47 § 0.07
Liposomal drug 0.50 § 0.10 81 Microencapsulated drug 31.97 § 2.61 0.33 § 0.04
Niosomal drug 0.38 § 0.21 86 Nanocapsulated drug 30.68 § 2.88 0.40 § 0.02
Microencapsulated drug 0.54 § 0.07 79
Values are expressed as mean § s.d. (n D 3).
Nanocapsulated drug 0.23 § 0.05 91 a
mg/ml. Normal level: 33.9 § 2.31.
b
Values are expressed as mean § s.d. (n D 3). mg/ml. Normal level: 0.37 § 0.06.

Histopathological Examination
numbers as compared with untreated infected controls.
Histopathological examination (Figure 5) was made through
But in microsphere encapsulated drug treatment (Figure 5D),
light microscopic examination of eosin-haematoxylin stained
both white pulp and red pulp region are of normal disposition. In
sections. Some positive changes were observed for the infected
venous sinuses, number of inŽ ltrating monocytes had reduced.
untreated control (Figure 5A). Comparing with normal ones the
In niosomal drug treatment, both white pulp and red pulp
white pulp region was enlarged and found to be invasive into
regions are of normal structure and the number of inŽ ltrating
red pulp region. Many small bulbs were observed in the venous
monocytes have reduced (Figure 5E).
sinuses. As a whole, the tissue becomes areolar.
In nanocapsulated drug treatment, white pulp region became
In the free drug treatments (Figure 5B), there was regression
much condensed and covered less area. Red pulp is of normal
of the white pulp region, though small white pulp follicles were
disposition. InŽ ltrating manocytes are found in much reduced
still observed. Sinusoidal distribution became nearly normal.
numbers in the venous sinuses (Figure 5F).
InŽ ltration of reticuloendothelial cells or monocytes was still
found. Red pulp region is more or less normal.
In liposomal drug treatment (Figure 5C) the white pulp region
Vesicle Size versus EfŽcacy
became condensed. The follicular artery was distinct, scattered
lymphatic cells were observed, and the red pulp region had nor- The log diameter of each type of vesicle (liposomes, nio-
mal disposition. InŽ ltrating monocytes were found but in less somes, microsphere, and nanocapsules) were plotted against the
parasite-killing efŽ cacy of each corresponding drug-loaded vesi-
cle. We observed that the efŽ cacy was inversely correlated with
TABLE 2
vesicle size (Figure 6). Best efŽ cacy was exhibited with the
Effect of Bacopasanonin-C loaded liposomes, niosomes, smallest vesicle, i.e., the nanocapsules, whereas least efŽ cacy
microspheres, and nanocapsules on speciŽ c enzyme levels was observed with the microspheres. The efŽ cacy gradient was
related to normal liver functions found as:
Serum glutamate
Alkaline pyruvate nanocapsule > niosomes > liposomes > microspheres
Group phosphatase a transaminase b
Infected untreated control 8.71 § 1.84 99.331 § 11.65
Free drug 13.66 § 1.31 121.85 § 13.47
Liposomal drug 8.72 § 0.68 57.02 § 3.25 DISCUSSION
Niosomal drug 9.10 § 0.22 46.3 § 11.60 Saponins have a long history of acting as antimicrobial, an-
Microencapsulated drug 8.89 § 0.45 60.65 § 5.18 tifungal (Otshudi et al. 2000), and antiprotozoal (Traore et al.
Nanocapsulated drug 8.66 § 0.45 61.83 § 5.18 2000; Delmas et al. 2000) agents. Although this is the Ž rst report
Values are expressed as mean § s.d. (n D 3). of Bacopasaponin-C as an antileishmanial agent, the mechanism
a
¹mol p-nitrophenol released/min/dl sera. Normal level: 8.8 § 1.50. of action of Bacopasaponin-C is not known. Besides being an-
b
¹mol of sodium pyruvate released/min L sera. Normal level: 49:6§ tileishmanial in property, it has a terminal glucose moiety and
11:63. as such it is self-targeting in nature to cells having the respective
60 J. SINHA ET AL.

(A) (B)

(C) (D)

(E) (F )

FIG. 5. Histological examination of thin sections of spleen under different experimental conditions (A) infected untreated control, (B) Free drug treated,
(C) liposomal drug treated, (D) microencapsulate d drug treated, (E) niosomal drug treated, and (F) nanocapsulate d drug treated (magniŽ cation £ 400).
 BACOPASAPONIN C
FIG. 6. A size versus efŽ cacy relationship of different delivery vesicles: li-
posomes, niosomes, microspheres, and nanocapsules .
receptors (Figure 7). On examination, it appears that the leish-
manicidal property is not due to the inhibitory effect of
Bacopasaponin-C on the two vital enzymes of leishmania dono-
vani viz topoisomerase I and adenosine kinase. The speciŽ c
activity of both the enzymes remains unaltered in the pres-
ence of varying concentrations of Bacopasaponin-C (Ray et al.
1998; Dutta et al. 1987). Moreover, its leishmanidal property
is parasite-speciŽ c as judged from Trypan blue exclusion test
involving both the host cells and the parasites.
A wide range of bioactive molecules are commercially avail-
able as drugs. Many of these are stable only under physiological
conditions and their therapeutic application is severely limited
by their short half-lives in vivo. Most of the administered amount
does not reach the target site. Thus, delivery of these molecules
FIG. 7. Structure of Bacopasaponin-C .
61
by encapsulation in appropriate delivery vehicles protects them
from degradation. Encapsulation not only increases their half-
lives, but also increases their efŽ cacy and as such minimizes the
required drug dose.
Besides conventional liposomes and niosomes, polymeric
vesicles have received much attention (Davis and Illum 1988;
Davis et al. 1993; Allerman et al. 1993; Stolnik et al. 1995;
Torchillin and Trubetskoy 1995; Gref et al. 1995). A signiŽ cant
challenge for polymer based drug delivery formulations is the
further development of drug carriers leading to higher therapeu-
tic efŽ cacy. A drug carrier can be efŽ ciently targeted if its size is
below 100 nm diameter (comparable to the size of a virus) and
is not scavenged by the body’s primary defense system such as
the reticuloendothelial system. Obviously, these particles should
have long circulation times in blood. In addition, the surface of
these nanometer-sized particles should be sufŽ ciently reactive
to chemically bound ligand molecules for receptor-mediated tar-
geting. The chemical properties of these nanocapsules should be
modulated so that they remain unaffected by circulating lipases,
they should be pH or temperature sensitive, or they should be-
have differently in hydrophilic and hydrophobi c environments.
In our research, drug-loaded nanocapsules turned out to be the
most efŽ cacious in killing parasites; the least effective was the
drug-loaded microspheres, the largest vesicular delivery system
tried so far. An inverse linear relationship between the size of
the vesicles and percent efŽ cacy in killing the parasites was
established suggesting that composition and the size are the two
important parameters that must be considered for a strategy for
efŽ cient delivery.
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