World Journal of Pharmaceutical Sciences

ISSN (Print): 2321-3310; ISSN (Online): 2321-3086

Published by Atom and Cell Publishers All Rights Reserved
Available online at: http://www.wjpsonline.org/
Original Article

In Vitro Modulatory Effects of Certain Plant Extracts on T-lymphocytes Proliferation

Farid Abd-Elrehim Abd-Elaziz Badria1*, Fatma Mohamed Abdel Rahman Abdel Bar1, Diaaeldin Mohamed
Abdelkawi Elimam1, Mohammad Hosam El-Deen Zaghloul2
1
2

Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt.

Department of Clinical pathology, Faculty of Medicine, Mansoura University, Mansoura 35516, Egypt.
Received: 29-01-2016 / Revised: 11-02-2016 / Accepted: 23-02-2016 / Published: 28-02-2016

ABSTRACT
Herbal drugs are believed to enhance the natural immunity of the human body against viral and microbial
infection and improve response; their immunomodulatory activities have been reported in numerous plants. In
this study, fifty plant alcoholic extracts were screened for their potential immunomodulatory activity using invitro human lymphocyte transformation assay (LTA). Among the screened extracts, Aralia victoria, Boswellia
carteri, Cyperus rotundus, Ginkgo biloba, Glycyrrhiza glabra, Olea europaea and Rosmarinus officinalis were
capable to induce 96.67%, 32.22%, 11.11%, 5.56%, 25.56%, 13.33% and 2.22% respectively of lymphocyte
transformation in comparison to Concanavalin-A (20.5%). Subsequently, each extract was further subjected to
bio-guided fractionation using LTA. The bio-guided fractionation revealed that Dichloromethane and
Petroleum-ether fractions from Glycyrrhiza glabra and Boswellia carteri stimulate the transformation of human
lymphocytes by 86% and 28% respectively. This finding stimulated the interest to pursue a comprehensive
study on the possible contribution of the commonly known and structurally related triterpens of Glycyrrhiza
glabra and Boswellia carteri and their derivatives as potential immunomodulators and their possible use as
immune enhancer in many infectious and cancer diseases.
Keywords: Immunomodulators, LTA, Con-A, Glycyrrhiza glabra, Boswellia carteri.

INTRODUCTION
In recent years, there is an upsurge in the clinical
use of natural products or their derivatives due to
being free from serious toxic effects and better
efficacy. Additionally, steady increase in the
resistant microorganism strains to antibiotics and
serious averse manifestations brought about by the
synthetic drugs has incited researchers to search for
natural immunomodulators to fight different
infections [1]. Natural drugs are reckoned to
enhance the innate resistance of the human body
towards various infections and numerous plants
have been reported to have immunomodulatory
activities [2]. Many natural anti-infective
medications are believed to exert their effects not
just by affecting the pathogen directly, alternately,
at least a part of their impaction is underhanded, by
stimulating innate and adaptive defensive
mechanisms of the body [1]. Plant extracts have
been demonstrated to possess versatile biological
activities
including
anticancer
and
immunomodulation activity. In several studies, the
ability of medicinal plants to enhance or reduce the

immune response has been reported. The

immunomodulatory effect of garlic on cell
mediated immunity has been studied [2] Milk
thistle has been proven to increase humoral and
cellular activity [3] Ginseng enhances production
of macrophages, B and T cells [4] Echinacea is
being tested as an immune stimulant [5]. Therefore,
this study was designed to utilize the plethora of
data on the commonly available and traditionally
reputed immune enhancer herbs by using a simple
in vitro LTA assay. The outcomes of this study
may furnish a new class of immunomodulators
which may could be possibly use as immune
enhancer in many infectious and cancer diseases.
MATERIALS AND METHODS
Materials: Methanol (MeOH), Dichloromethane
(DCM), Petroleum-Ether (Pet-Eth), Ethyl-actate
(EthOAc), Dimethylsulfoxide (DMSO), Distilled
water (d.H2O), 100 glass containers (Jars), (AlGomheria Co., Mansoura, Egypt). RPMI-1640
medium, Fetal Bovine Serum (FBS), Lglutamine (2 mM, GIBCO, Grand Island, NY). An

*Corresponding Author Address: Prof. Farid A. Badria, Department of Pharmacognosy, Mansoura University Faculty of Pharmacy,
Mansoura 35516, Egypt; E-mail: Faridbadria@gmail.com

Badria et al., World J Pharm Sci 2016; 4(3): 299-308

antibiotic/anti-mycotic solution containing 1000

U/ml penicillin, 1000 g/ml streptomycin and 25
g/ml fungisone, Ficoll/Hypaque (Histopaque),
Concanavalin A (Con-A), Phosphate Buffer saline
(PBS) and 3-(4,5-dimethyl-thiazoyl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, St
Louis, MO, USA)), (Costar, 96-well plates, Tissue
Culture Treated Polystyrene # 3512, Corning Inc.,
NY, USA), ELISA BioTek Lx800 microplate
reader (BioTek, Bedfordshire, UK) were used.

assay was performed for the evaluation of LTA and

clonal expansion.
Calculation: Optical densities were measured
using an ELISA microplate reader at primary wave
length 570 nm (primary absorbance value) and a
reference wavelength of 630 nm (background
absorbance value).
The background absorbance was subtracted from
the primary values and cells viability was
expressed with Induction percentage (%) which
was calculated using the following formula:

Plant collection: The dried powder of 50 plants

(250 gm of each) represented in Table1 were either
collected from the farm of Faculty of Pharmacy,
Mansoura University or purchased from
professional local herbal market, after botanical
identification by department of Pharmacognosy,
voucher specimens were deposited at the
Department
of
Pharmacognosy,
Mansoura
University, Mansoura, Egypt.

% Induction =

ODcontrol - ODtest
ODcontrol

x 100

Analysis of data was done with GraphPad Prism

program (Version 6.01), Mean comparisons were
made with significant differences reported at P <
0.05, Replication N = 3.

Extracts Preparation: The plants were washed

with distilled water and shade dried, ground in a
tooth miller, 100 gm of each plant powder
extracted by 70 % MeOH until exhaustion, the
solvent was removed by rotary evaporation under
reduced pressure at temperatures below 45C.
Crude extracts was refrigerated at 0C until used in
the assay. RPMI-1640 medium was used to prepare
serial dilutions from DMSO stock solutions,
DMSO limit 0.1% v/v.

RESULTS
Immunomodulation activity of 50 plants was
assessed using simple in-vitro LTA assay. Out of
tested plants, 12 extracts were active, Table 2. The
percent induction of most active plants in
comparison with positive control ConA is
represented in Figure 1.

Liquid-liquid Fractionation: The different plant

extracts were suspended in d.H2O, partitioned
successively with solvents with increased polarity
including Pet-Eth, DCM, EthOAc and MeOH
followed by separation of the organic layer and
evaporation under reduced pressure.

Aralia victoria, Boswellia carteri, Cyperus

rotundus, Ginkgo biloba, Glycyrrhiza glabra, Olea
europaea and Rosmarinus officinalis were capable
to induce 96.67%, 32.22%, 11.11%, 5.56%,
25.56%, 13.33% and 2.22% of lymphocyte
transformation respectively in comparison with
Con-A ,20.5%.

Lymphocyte transformation assay (LTA): Adult

healthy volunteers venous blood was used to
obtain Peripheral Blood Monocytes (PBMC) by
centrifugation of heparinized blood over
Ficoll/Hypaque. Buffy layer at the interphase
corresponding to PBMC were collected after Ficoll
separation and washed three times in PBS buffer
before further processing, Trypan Blue staining
was performed to assess cellular viability after
isolation. [6] In flat bottomed 96-well plates,
1x106 cells/ml (1.5x105 cells per well) were
cultured in complete RPMI-1640 medium
supplemented with 10% (v/v), FBS and Lglutamine. Antibiotic/anti-mycotic was added to
control bacterial and fungal contamination.
Stimulation (Transformation) of the cultures were
done with 50 g/ml Con-A or by the plant extract
at 50, 100 g/ml and incubated for 72 h at 37C in
a humidified atmosphere containing 5% CO2. MTT

Fractionation of plant extracts yielded five

fractions of each. The induction activity at two
different concentrations, 50 and 100 g/ml for each
fraction is shown in Table 3 and represented in
Figures 2 - 8.
Fractions of Rosmarinus officinalis leaves and Olea
europaea leaves (EthOAc, MeOH & d.H2O)
showed higher activity than that of total extract,
Figures 2 and 3.
Moreover, DCM and EthOAc fractions of Cyperus
rotandus rhizomes, Figure 4, exhibited more
activity than its total extract. In contrast, the total
extracts of Ginkgo biloba and Aralia victoria
leaves, Figures 5 and 6, appeared to produce
superior activities rather than any fraction alone.
The Pet-Eth and DCM fractions of Boswellia
carteri and Glycyrrhiza glabra showed activities
300

Badria et al., World J Pharm Sci 2016; 4(3): 299-308

apparently equal to and higher than that of total

extract in both respectively, Figures 7 and 8.

Moreover, DCM and EthOAc fractions of Cyperus

rotandus rhizomes exhibited more activity than its
total extract, Figure 4. The intermediate polarity
components that extracts mainly in such solvents as
glycosides, flavonoids and some terpenoids are
suspected to be involved in its activity.

DISCUSSION
Lymphocyte transformation assay is simple, rapid
and uncomplicated technique that used to assess
immune responses. The assay investigates the
mitogenic effect of the crude drug on Tlymphocyte proliferation. Immunosuppression is
indicated by anti-proliferative activity on Tlymphocyte culture while immunostimulation is
represented when promotion of T-lymphocyte
proliferative response occurs [7].

In contrast, the total extracts of Ginkgo biloba and

Aralia victoria leaves, Figures 5 and 6, appeared to
produce superior activities over than any fraction
alone. The activity proposed to be due to
cumulative effects of different composition,
ranging from low polarity to highly polar
compounds. Notice that, d.H2O fraction of Ginkgo
biloba showed higher activity than any other
Ginkgo biloba fraction, outweighing the activity
toward polar compounds presented as phenolic
compounds, tannins and flavonoids

Upon evaluation of 50 plant extracts, Table 1, with

known reputation as immunomodulators, 12 plant
extracts were most active toward lymphocyte
promotion, Table 2. Based on the results of the
LTA and depending on the promising outcomes,
triterpenoid-containing plants were chosen for
further fractionation and evaluation. Seven
triterpenoid-containing plants were fractionated
using successive liquid-liquid partitioning into five
fractions; Pet-Eth, DCM, EthOAc, MeOH and
d.H2O.

The Pet-Eth and DCM fractions of Boswellia

carteri and Glycyrrhiza glabra, containing mainly
triterpenoid compounds[8][9], showed activity
apparently equal to and higher than total extracts of
both respectively, Figure 7 and 8. In Boswellia
carteri, its interesting to observe that, nearly, the
activity of Pet-Eth fractions in ranking equals that
of total extract, which toggle the activity of
Boswellia carteri as total extract to the Pet-Eth
soluble fraction, triterpenoid acids. While in
Glycyrrhiza glabra, d.H2O soluble fraction showed
the best activity in comparison with other
Glycyrrhiza glabra fractions, proposing water
soluble triterpen saponin glycosides are responsible
for the activity. The activity of Glycyrrhiza glabra
and Boswellia carteri is the most interesting due to
their nearly pure triterpenoid contents that have
structure similarities between boswellic acid and
glycyrrhizin, (Figure 9) that showed in previous
work [10], we suggest that these triterpen acid may
have a potential immunomodulatory activity
without involvement of any other components like
flavonoids and polyphenolics which present in
almost all other plants [11],[9].

The percent induction of the active plants in

comparison with positive control Con-A is shown
in Figure 1. Aralia victoria had shown the highest
activity with 96.67% induction, while the lowest
induction effect 2.22 % was produced by
Rosmarinus officinalis. Induction activity of each
plant fraction at two different concentrations, 50
and 100 g/ml 8 was assessed, that represented in
Figure 2-8.
Polar and semi-polar fractions (soluble in polar
solvents) of Rosmarinus officinalis and Olea
europaea leaves (d.H2O, MeOH & EthOAc)
showed higher activity than that of total extract,
Figures 2 and 3. These findings suggest that the
major activity is contributed by polar compounds
as polyphenolics, flavonoids and tannins rather
than being due to less polar components like
triterpens.

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Badria et al., World J Pharm Sci 2016; 4(3): 299-308

Table 1. Medicinal Plants Screened using In vitro T-lymphocytes Proliferation Assay

No.

Plant name

Part used

1. Ammi visnaga

Fruits

2. Anethum graveolens

Flowers & Fruits

3. Aralia victoria

Leaves

4. Atropa belladona

Fruits

5. Atropa belladona

Leaves

6. Boswellia carteri

Resins

7. Brassica alba

Seeds

8. Brassica nigra

Seeds

9. Capsicum annuum

Fruits

10. Carthamus tinctorius

Flowers

11. Carum carvi

Flowers

12. Centella asiatica

Leaves

13. Ceratonia siliqua

Bulbs

14. Cinchona officinalis

Barks

15. Citrullus colocynthis

Fruit

16. Chamaemelum nobile

Flowers

302

Picture

Badria et al., World J Pharm Sci 2016; 4(3): 299-308

17. Coriandrum sativum

Fruits

18. Curcuma longa

Rhizomes

19. Cyperus rotandus

Rhizomes

20. Date stone

Stones

21. Datura stramonium

Leaves

22. Digitalis purpurea

Leaves

23. Emblica officinalis

Fruits

24. Ferula assa-foetida

Resin

25. Foeniculum vulgare

Fruits

26. Fragaria ananassa

Leaves

27. Ginkgo biloba

Leaves

28. Glossostemon bruguieri

Roots

29. Glycyrrhiza glabra

Rhizomes

30. Hibiscus sabdariffa

Flower

31. Hyoscyamus muticus

Leaves

32.

Melaleuca leucadendra

Leaves

33. Mentha piperita

Leaves

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34. Morus alba

Leaves

35. Nerium oleander

Leaves and flowers

36.

Ocimum sanctum

Leaves

37. Olea europaea

Leaves

38. Pimpinella anisum

Fruits

39. Pistacia lentiscus

Resins

40. Psidium guajava

Leaves

41. Rheum rhabarbarum

Roots

42. Rosa damascena

Flowers

43. Rosmarinus officinalis

Leaves

44. Salvadora persica

Stems

45. Silybum marianum

Leaves

46. Solenostemma argel

Leaves

47. Syzygium aromaticum

Flowers

48. Tanacetum cinerariifolium

Flowers

49. Trigonella foenum-graecum

Leaves

50. Zingiber officinale

Rhizomes

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Table 2. Results of Tested Total Extracts In T- lymphocytes Assay

No.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.

Mitogen
Media/DMSO
Con-A
Aralia victoria
Boswellia carteri
Centella asiatica
Cinchona officinalis
Cyperus rotundus
Emblica officinalis
Ginkgo biloba
Glycyrrhiza glabra
Mentha piperita
Olea europaea
Rosmarinus officinalis
Strawberry leaves

% Induction
0.00
20.53
96.67
32.22
33.33
18.89
11.11
88.89
5.56
25.56
60.00
13.33
2.22
67.78

Table 3. Results of Tested Fractions in T-lymphocytes Assay ( % induction)

Plant

Aralia
victoria
Boswellia
carteri
Cyperus
rotundus
Glycyrrhiza
glabra
Ginkgo
biloba
Olea
europaea
Rosmarinus
officinalis

% Induction
Total alcoholic
extract
50
100
g/mL g/mL

Pet-Eth
fraction
50
100
g/mL g/mL

63.578

125.27

18.45

27.98

28

50
g/mL

100
g/mL

EthOAc
fraction
50
100
g/mL g/mL

66.24

20.73

20.333

20.70

21.18

21.95

23.16

26.26

16.63

16.66

12.48

13.33

42.08

19.24

20

46.26

100.68

65.27

69.24

70

20.29

21

110.18

111

109.3

252.87

3.21

3.5

23.62

44.80

68.78

3.22

64

39.64

36.79

12.20

13

DCM fraction

MeOH
fraction
50
100
g/mL g/mL

d.H2O fraction
50
g/mL

100
g/mL

21.667

30.40

30.833

20.99

18

16.74

18

147.14

40.04

72.72

29.45

35.35

48.13

49

24.74

26

42.29

134.11

31.99

41.63

64.98

71.33

142.77

76.50

195.62

33.72

38.01

73.05

136.46

44.16

71.68

41.09

76.48

16.17

17

19.08

20

35.51

82.16

46.82

63.32

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Badria et al., World J Pharm Sci 2016; 4(3): 299-308

Figure 1: The percent induction of most active

Figure 2: The percent induction of Rosmarinus

plant extracts as lymphocyte transforming agents

officinalis fractions at two concentrations, 50 and

at concentration of 100 g /ml in comparison with

100 g/ml each, in comparison with standard

standard mitogen Con-A 50 g/ml.

mitogen Con-A at concentration of 50 g/ml.

Figure 3: The percent induction of Olea

Figure 4: The percent induction of Cyperus

europaea fractions at two concentrations, 50 and

rotundus fractions at two concentrations, 50 and

100 g/ml each, in comparison with standard

100 g/ml each, in comparison with standard

mitogen Con-A at concentration of 50 g/ml.

mitogen Con-A at concentration of 50 g/ml.

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Figure 5: The percent induction of Aralia

Figure 6: The percent induction of Ginkgo

victoria fractions at two concentrations, 50 and

biloba fractions at two concentrations, 50 and

100 g/ml each, in comparison with standard

100 g/ml each, in comparison with standard

mitogen Con-A at concentration of 50 g/ml.

mitogen Con-A at concentration of 50 g/ml.

Figure 7: The percent induction of Boswellia

Figure 8: The percent induction of Glycyrrhiza

carteri fractions at two concentrations, 50 and

glabra fractions at two concentrations, 50 and

100 g/ml each, in comparison with standard

100 g/ml each, in comparison with standard

mitogen Con-A at concentration of 50 g/ml.

mitogen Con-A at concentration of 50 g/ml.

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H3C

CH 3

COOH

H3 C
R3

O
H3C

CH3

CH3

H3 C

CH3

CH 3

CH 3
CH 3

R1O

R2O
H3C

CH3

COOH

H3C

1 R2= H
2 R2= Ac
3 R2= H
4 R2= Ac

R1= D-glucuronic acid-D-glucuronic acid

R3= H2
R3= H2
R3= O
R3= O

-boswellic acid.
3-O-acetyl--boswellic acid.
11-keto--boswellic acid.
3-O-acetyl-11-keto--boswellic acid.

Boswellic acids

Glycyrrhizin

Figure 9: Similarity between Chemical structures of glycyrrhizin and

boswellic acids

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