Advance Access Publication 13 December 2006

eCAM 2007;4(4)419423
doi:10.1093/ecam/nel094

Original Article

Immunomodulatory Activity of Chlorophytum borivilianum Sant. F

Mayank Thakur, Shilpi Bhargava and V. K. Dixit
Department of Pharmaceutical Sciences, Dr H.S. Gour Vishwavidyalaya, Sagar, Madhya Pradesh, India
Chlorophytum borivilianum Santapau & Fernandes (Liliaceae) is a very popular herb in traditional
Indian medicine and constitute a group of herbs used as Rasayan or adaptogen. Ethanolic extract of the
roots and its sapogenin were evaluated for their immunomodulatory activity. Effect of azathioprineinduced myelosuppresion and administration of extracts on hematological and serological parameters
was determined. Administration of extracts greatly improved survival against Candida albicans
infection. An increase in delayed-type hypersensitivity response (DTH), % neutrophil adhesion and in
vivo phagocytosis by carbon clearance method was observed after treatment with extracts.
Immunostimulant activity of ethanolic extract was more pronounced as compared to sapogenins. The
results, thus justifies the traditional use of C. borivilianum as a rasayana drug.
Keywords: Chlorophytum borivilianum immunomodulator rasayana Safed Musli

Introduction
Scientific literature is continuously reporting plant drugs
having immunomodulatory activity. Most of the leads for
this activity are from traditional medicines from different parts
of the world (1,2). The Indian system of medicine Ayurveda,
conceptualizes a category of drug activity known as
Rasayana. The word Rasayana is composed of two words
Rasa meaning elixir and Ayana meaning house. The word
therefore signifies property of the plant that helps to rejuvenate the system, i.e. adaptogenic activity (3). Rasayan
therapy prevents diseases and counteracts the aging process
by means of optimization or homeostasis. Many plants
have been extensively used as Rasayana drugs in Ayurveda
for the management of neurodegenerative diseases, as
rejuvenators, immunomodulators, aphrodisiac and nutritional
supplements (47).
Safed Musli has been described in ancient Indian literature
such as Bhavaprakash nighantu, Rasendra Sarsangrah,
Raja Ballabh Nighantu as Vajikaran or aphrodisiac which is
a special type of immunomodulator (810). Safed Musli is a
controversial drug and various herbs are employed as
Safed Musli by practitioners of Indian system of medicine.
One of the popular and marketed herb under this nomenclature
For reprints and all correspondence: Prof. V. K. Dixit, Department of
Pharmaceutical Sciences, Dr H. S. Gour Vishwavidyalaya, Sagar, Madhya
Pradesh, India. Tel: 91-7582264582; E-mail: vkdixit111@rediffmail.com

is Chlorophytum borivilianum Santapau & Fernandes

(Liliaceae) (11,12).
Since the roots of C. borivilianum are employed as
Rasayana (13,14) and also because Safed Musli is a
constituent of Chyawanprash, an outstanding rejuvenator
(8). It was considered worthwhile to investigate this drug for
immunomodulatory activity.

Materials and Methods

Plant Material
C. borivilianum roots were obtained from Jeevan Agro
farms (Sagar, Madhya Pradesh, India). A herbarium of the
source was made and identified. A voucher specimen no.
CB-MT16 has been submitted at the departmental herbarium
of Department of Pharmaceutical Sciences Dr H.S. Gour
Vishwavidyalaya, Sagar (MP, India). The roots were dried in
sunlight and coarsely powdered for extraction.
Extraction and Fractionation
Powdered roots were defatted with petroleum ether (6080
C).
Ethanolic extract of the drug was prepared by extracting
defatted roots with 95% ethanol in Soxhlet extractor (yield
16% w/w). For isolation of sapogenin, ethanolic extract as
obtained above was suspended in water (100 ml). It was then
extracted with n-butanol (300 ml), the volume of n-butanol

2006 The Author(s).

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

420

Immunostimulant activity of Safed Musli

soluble portion was then reduced to half under reduced

pressure and finally saponins were precipitated by addition of
diethyl ether. The collected precipitate was hydrolyzed using
hydrochloric acid (2 N HCl) and the precipitated sapogenins
were collected (15).
Alternatively, 500 g defatted drug powder was hydrolyzed
by using 200 ml of 4 N H2SO4 in a round bottom flask which
was kept overnight at room temperature and filtered to remove
acidic solution, marc was washed three to four times with cold
distilled water. After washing and drying the marc was
refluxed for 30 min using fresh 200 ml of 4 N H2SO4 to
ensure complete hydrolysis of drug. The mixture was filtered
and filtrate was discarded. Hydrolyzed drug powder was
further washed with distilled water three times to ensure
complete removal of acid. The hydrolyzed drug powder was
dried in an air oven to ensure completely dried drug powder.
The hydrolyzed drug powder was fed in a Soxhlet extractor and subjected to extraction with ethanol (95%). Ethanol
was removed from the extract under reduced pressure and
sapogenins were collected (16). The sapogenins isolated by
this method were used for pharmacological studies.
Sapogenins isolated by either method exhibited identical
TLC profile suggesting suitability of both the process for
isolation of sapogenins from the drug. HPTLC analysis of the
ethanolic extract as well as isolated sapogenin fraction was
performed. In brief, the chromatographic analysis of ethanolic
extract was performed on silica gel-GF254 precoated plates
(E Merck, Germany) using chloroform:glacial acetic acid:
methanol:water (16:8:3:2, v/v) as mobile phase, 10 spots were
visualized upon derivatization with anisaldehyde sulfuric acid
reagent. Sapogenin fraction was characterized on chloroform:
diethylether (1:1, v/v) mobile phase against b-sitosterol as
standard marker. The pattern and standardization of the
sapogenins against standard marker (b-sitosterol) has been
reported by us previously (17).
Materials
Sheep red blood cells (SRBCs) were obtained from Haffkine
Biopharmaceuticals Ltd, Mumbai, India, and were washed
thrice with large volumes of pyrogen-free sterile saline and
adjusted to a concentration of 5 109 cells per ml for
immunization and challenge. Azathioprine was obtained as
gift sample from Troika Pharmaceuticals, Ahmedabad, India.
Candida albicans was purchased from IMTECH (Chandigarh,
India).
Rats
Wistar strain albino rats weighing between 140150 gm of
either sex were used. They were housed in departmental animal
room under standard condition of temperature (24 1
C),
12/12 h light/dark cycle and fed on standard pellet diet.
Ethanolic extract and sapogenin were administered orally as
suspension in 2% polyvinyl pyrolidine solution using metal
canula.

Toxicity studies were performed and dose of 1 gm kg1

body weight (bw) of ethanolic extract or sapogenins did not
cause any toxic effect.
Statistical Analysis
Data are expressed as mean SEM and analyzed for
significance by Dunnets test (comparing all versus control)
using Instat v.2.02 software (Graphpad software Inc.) residing
in Pentium IV processor run on Windows Xp.

Experimental
Treatment
Albino rats were divided into groups comprising of six animals
each. Group I served as control and was administered vehicle
only. Group II was administered 200 mg kg1 bw ethanolic
extract. Group III received 100 mg kg1 bw sapogenin extract.
Each experiment was performed on fresh group of animals
unless specified.
Non-Specific Immunity Determined by Survival
Rate Against Fungal Infection
Treatments of all the three groups began 14 days before
challenge. On the day of challenge all groups were injected
with 5 107 viable C. albicans cells and observed daily for
mortality for a period of 10 days.
In Vivo Phagocytosis Using Carbon Clearance Method
The method of Biozzi et al. (1953) (18,19) was used.
Treatments of all groups started 14 days before experimentation. On 15th day of treatment, mice were injected with 0.1 ml
of carbon suspension (Pellikan Tuschea Ink, Germany)
intravenously through tail vein. Blood samples (25 ml) were
collected from retro-orbital plexus just before and at 4, 8, 12
and 16 min after injection. Blood samples were lyzed with 2 ml
of 0.1% acetic acid and absorbance of samples recorded at
675 nm (20). The graph for absorbance versus time was plotted
for each animal in respective test group and phagocytic index
was calculated using the formula:
Phagocytic IndexPI

K sample
K standard

where Ksample represents the slope of absorbance versus time

curve for extract-treated samples and Kstandard represents the
slope of absorbance versus time curve for blood sample
collected before treatment.
SRBC-Induced Delayed-Type Hypersensitivity
Reaction (DTH Response)
The method of Lagrange et al. (1974) (21) was used.
Treatments with extracts began 14 days before challenge. All
the groups were immunized by injecting 20 ml of 5 109
SRBC per ml subcutaneously into the right foot pad. After

eCAM 2007;4(4)

421

bw azathioprine and 100 mg kg1 bw of sapogenin extract. All

the animals were sensitized by injecting 5 109 SRBCs
intraperitoneally before experimentation.
On 15th of treatment, all the mice were sacrificed and
blood was collected in heparinized vials. Blood samples for
animals of each group were subjected for hematological and
serological studies such as hemoglobin content, total RBC,
total and differential WBC count and platelet count (Fig. 1 and
Table 2).

14 days of treatment the thickness of left foot pad was

measured using calipers (Schnelltester, Germany) reading to
0.01 mm. The mice were then challenged by injecting 20 ml of
5 109 SRBC per ml intradermally on the left hind foot pad
(time 0). Foot thickness was measured after 24 and 48 h of
challenge. The difference between the thickness of left foot
just before and after challenge in mm was taken as a measure
of DTH (22).
Neutrophil Adhesion Test. The method described by
Wilkonson (1978) (23) was used for evaluating the effect of
extracts on neutrophil adhesion. After 14 days of treatment of
all the three groups, blood samples were collected by retroorbital puncture in heparinized vials and subjected to total as
well as differential leukocyte count. After initial counts the
blood samples were incubated with 80 mg ml1 of nylon fibers
at 37
C for 15 min. The incubated samples were again
analyzed for total and differential leukocyte count. The
product of total leukocyte count and % neutrophil known as
neutrophil index was determined for each of the respective
groups (24). The % neutrophil adhesion for each of the test
groups was determined as follows,

Results
Survival Rate Studies
The survival rate of the treated animals was considerably
enhanced after treatment with C. borivilianum extract.
Administration of ethanolic extract and sapogenin exhibited
94 5.56 and 88.86 5.6 percent survival after infection with
C. albicans. Sapogenin extract was 50% (P < 0.05) and
ethanolic extract 60 % (P < 0.05) more effective in reducing
mortality as compared to control group animals (Table 1). The
results suggest potentiation of non-specific immune response
on treatment with ethanolic extract and sapogenins.

% Neutrophil adhesion
Difference of neutrophil count in untreated and fiber-treated blood
100
Neutrophil count of untreated blood

Activity Against Drug-Induced Immunosuppression

DTH Using SRBC as an Antigen

The methods of Doherty (1981) (25) modified by Ziauddin

et al. (26) was used. Six groups of six albino mice each were
taken. Group I served as control. Group II was administered
ethanolic extract 200 mg kg1 bw. Group III was given
sapogenin extract 100 mg kg1 bw. Group IV received 100 mg
kg1 bw azathioprine. In Group V animals, 100 mg kg1 bw
azathioprine and 200 mg kg1 bw of ethanolic extract was
administered, whereas Group VI was treated with 100 mg kg1

In the control group animals, after 48 and 72 h of challenge

the DTH response was either equal or slightly more than the
0 h response; therefore, the peak edema after 24 h of
challenge was the evaluating parameter. Ethanolic extract
(200 mg kg1 per orally) was most effective (P < 0.05)
compared to sapogenin (100 mg kg1 per orally) treatment in
increasing the delayed-type hypersensitivity response
(Table 1).

Effect of C. borivilianum ethanolic extract/sapogenins on drug induced immunosuppression

Hematological parameters
Count

18
16
14
12
10
8
6
4
2
0
Control

Test gp II

Test gp III

Test gp IV

Test gp V

Treatment

Hemoglobin Concentration %g
RBC count in millions/cmm

Platelet Count in lakhs/cmm

WBC count in thousand/cmm

Figure 1. Effect of Chlorophytum borivillianum Sant. & F. on azathioprine induced suppression of hematological parameters.

Test gp VI

422

Immunostimulant activity of Safed Musli

Rate of Carbon Clearance

Rate of carbon clearance is the measure of competency of the
reticuloendothelial system and its granulopoetic activity (14),
the faster removal of carbon particles has been correlated with
the enhanced phagocytic activity. In the present study an
increased phagocytic activity was observed in treated groups
as compared to control. The rate of carbon clearance which
was determined as phagocytic index was 20 % (1.2 0.03)
and 25% (1.36 0.51) greater in sapogenins and ethanolic
extract-treated groups, respectively, clearly indicating an
enhanced in vivo phagocytic activity (Table 1).
Table 1. Effect of administration of C. borivilianum ethanolic extract/
sapogenins on non-specific and specific immune systems
Groups

Control
Test Group I
Test Group II

Survival
rate
percentage
33.3 9.642
94.43 5.56b
b

88.86 5.56

DTH response
(% increase in foot
pad thickness)

Phagocytic Index
(Ksample/
Kstandard)

0.89 0.04

1.51 0.02b (69.6%)

a

1.34 0.07 (50.6%)

1.36 0.51b
1.2 0.03b

P < 0.05 considered significant.

P < 0.005 considered extremely significant.
Control: no treatment. Test Group II: C. borivilianum ethanol extract treated
group (200 mg/kg b.w. p.o.). Test Group III: C. borivilianum sapogenin treated
group (100 mg/kg b.w. p.o.).
b

Table 2. Effect of administration of C. borivilianum ethanolic extract/

sapogenins on non-specific and specific immune systems
Groups

Haemoglobin
concentration
(g%)

Test Group I
Test Group II
Test Group III
Test Group IV

Platelet count
in thousand
per cmm

RBC count
in millions
per cmm

WBC count
in thousand
per cmm

14.5 0.31

468.5 0.51

4.7 0.31

10.1 0.38

15.3 0.25b

578.5 0.56c

5.9 0.57a

11.3 0.47a

496.6 0.56

5.2 0.12

10.8 0.28a

14.7 0.48

11.9 0.32

300.6 0.24

9.33 0.45a

3.13 0.43

Test Group V

14.3 0.11

560.45 0.42

5.99 0.35

10.8 0.38a

Test Group VI

14.0 0.09a

524.65 0.39b

5.86 0.31b

10.5 0.46a

Test Group I: control (no treatment); Test Group II: ethanolic extract 200 mg
per kg b.w; Test Group III: sapogenin extract 100 mg per kg b.w; Test
Group IV: azathioprine 100 mg per kg b.w; Test Group V: azathioprine 100 mg
per kg b.w; and ethanolic extract 200 mg per kg b.w; Test Group VI:
azathioprine 100 mg per kg b.w. and Sapogenin extract 100 mg per kg b.w.
a
P > 0.5 not significant.
b
P < 0.05 significant.
c
P < 0.005 extremely significant.

Effectiveness Against Drug-Induced

Immunosuppression
Administration of ethanolic extracts and sapogenins in general
had a beneficial effect on hematological profile wherein all the
parameters such as hemoglobin, platelets, RBC and WBC
counts were increased. Simultaneous administration of ethanolic extract and sapogenins along with azathioprine resulted
in restoration of suppressed values observed after azathioprine
treatment alone. Platelet, hemoglobin, RBC and WBC values
observed were better than untreated control groups. Among the
two test drugs ethanolic extract was pronouncedly more
effective (P < 0.005) followed by sapogenin treatment (P <
0.05) (Fig. 1 and Table 2).
Neutrophil Adhesion Test
This test is an indicative of the marginalization of phagocytic
cells in the blood vessels, i.e. an indication of immunostimulation. The % neutrophil adhesion in control group animals was
23.34 2.1, in ethanolic extract-treated group it was 27.21
1.6 whilst for sapogenin extract-treated group it was 25.62
1.4. As is evident from the results of neutrophil adhesion test,
nearly 10% (P < 0.5) increase in neutrophil adhesion is
observed after administration of sapogenin extract, whilst a
significant 17% (P < 0.05) increase in neutrophil adhesion is
observed in ethanolic extract (Table 3).

Discussion
The scientific evidences collected from the study supports the
traditional claims behind usage of the herb C. borivilianum,
which is being cultivated and marketed extensively in India
and abroad for medicinal purposes (12). The study affirms that
C. borivilianum root extract is an effective immunostimulatory
principle. The inference that can be drawn from the present
study is that the total ethanolic extract is superior over
sapogenin fraction of the plant as far as immunostimulatory
activity is concerned. The extract does not only potentiate nonspecific immune response, but is also effective in improving
humoral as well as cell-mediated immunity. The effectiveness
of extract-treated animals in overcoming the side effects of
drug-induced immunosuppression provides evidence for balancing and adaptogenic effectiveness of extracts.

Table 3. Effect of C. borivilianum ethanolic extract/sapogenins on neutrophil adhesion in rats (mean SE)
Animal group

TLC (103 mm3) (X)

% Neutrophil (Y)

Neutrophil Index (X Y)

% Neutrophil
adhesion

Un B

FTB

Un B

FTB

Un B

FTB

Group I

6.4 2.8

5.9 2.2

44 2.23

37 3.2

284.8 35.1

218.3 25.4

23.34 2.1

Group II

6.9 1.4

6.1 1.6

52 2.21

43 1.38

362.2 32.4

265.1 31.2

27.21 1.6b

Group III

6.8 1.2

6.1 1.4

51 2.23

44 1.47

356.6 31.4

268.8 32.1

25.62 1.4a

UnB: untreated blood; FTB: nylon fiber treated blood; Group I: control (no treatment); Group II: C. borivilianum ethanolic extract 200 mg per kg b.w; Group III:
C. borivilianum sapogenin extract 200 mg per kg b.w.
a
P < 0. 5 not significant.
b
P < 0.05 considered significant.

eCAM 2007;4(4)

Use of herbs for improving the overall resistance of body

against common infections and pathogens has been a guiding
principle of Ayurveda (27,28). Chlorophytum spp. have been
used and reported in many such formulations. The increase in
survival rate is a general marker exhibiting potency of the
ethanolic extract to overcome infectious condition. Increased
carbon clearance is an indicator of enhanced in vivo
phagocytic activity and competency of granulopoetic system
in removal of foreign particle, thereby an indicator of
enhanced immunological response against foreign particles
or antigens. Increase in percent neutrophil adhesion is
attributed due to marginalization of phagocytic cells, i.e.
improved defensive response under normal circumstances.
This study, apart from confirming the immunostimulant
activity of C. borivilianum also, presents evidence for the
presence of the substance other than sapogenins which induce
stimulation of immune response in treated animals. Therefore,
the plant holds promise for being used as an immunostimulating agent and an in-depth study on various fractions of the
extract effective as immunomodulating entities from the plant
is warranted to determine the most potent immunostimulating
fraction from C. borivilianum. Thus, the study validates the
traditional use of herb as a Rasayana in Ayurvedic system of
medicine (23).

Acknowledgments
The authors are grateful to Troika Pharmaceuticals (Ahmedabad, India) for providing gift sample of Azathioprine and to
Jeevan Agro farms Pvt. Ltd for providing C. borivilianum
roots. One of the authors Mayank Thakur is also thankful to
University Grants Commission for providing financial support.

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