Research Journal of Recent Sciences ________________________________________________ ISSN 2277 - 2502
Vol. 1(ISC-2011), 398-404 (2012)
Res.J.Recent.Sci.

Review Paper

Initiation of Pharmaceutical Factories depending on more Application of
Biotechnology on some Medicinal Plants Review Article
(In Vitro Production of some Antioxidant, Analgesic, Antibacterial, Antidiabetic agents)
Eman, A. Alam
Botany Department, National Research Centre, Dokki, Giza, EGYPT

Available online at, www.isca.in
(Received 15th October 2011, revised 10th January 2012, accepted 25th January 2012)

Abstract
Higher plants are an important source of all type of substances, especially medicines (glycosides, ethereal oils, steroids,
flavonoids, anthraquinones, alkaloids, tannins and saponins, etc.,). Traditionally the medicinal plants have been grown and
then the active components extracted and this is likely to remain the normal procedure. However the production of medicinal
plants can present problems, which have load to the search for other ways to produce naturally accruing substances: i.
Production in the field is strongly dependent on season, weather, climate, diseases and pests. ii. Naturally occurring sources,
especially in the tropics and subtropical zones, are becoming limited and some medicinal plants are extremely scarce. iiiThere may be technical and economic problems in production. iv. Production is labor intensive and therefore costs are high.
v. There may be political instability in the country where the plants are available resulting in an interrupted supply. For the
above mentioned reasons, attempts have been made to obtain substances from cell suspension cultures of higher plants,
either through accumulation in the callus (biomass) or sometimes by the release into the nutrient medium. In this review we
will discuss in vitro production of some antioxidant, analgesic, antibacterial, antidiabetic agents.
Keywords: Glycosides, ethereal oils, steroids, flavonoids, weather, climate, production antibacterial, antidiabetic agents.

Introduction
In vitro production of secondary metabolites advantages,
the effective factors etc1,2 : Organ, tissue and cell culture
and other biotechnological techniques are useful ways to
obtain biologically active constituents those play an
important roles in our life (They have pharmaceutical,
medicinal and economical importance in our life). These
ways (in vitro) of obtaining secondary metabolites are better
than the classical methods since:
They are natural in origin, so they are of little toxic side
effects compared with synthetic drugs. They are safe sources
and do not cause any environmental pollution as like as those
occurred by applications of pesticides and insecticides to
farm lands. They are performed under controlled conditions
(since the yield can be increased, with increasing replicates
number and by using elicitors, fomenters and bioreactors in a
large application scale), they are performed under aseptic
conditions (This means that, they are system free of
contaminating microbes “ fungi and bacteria”, consequently
they are clean sources of drugs).
Using these techniques we can direct the culture for
producing the organ that contains the highest amounts of the
product we need (for example, we can produce root culture

International Science Congress Association

using Agrobacterium rizogenesis to obtain substances that
produced in root cells only).
Production cycle is smaller than that of normal culture in
land (it takes little time), since callus cultures with short life
cycle may be a good source for production of
phytochemicals needed. Using these methods we can
conserve our natural resources (wild plants) instead of overcollection by herbalists. Finally, the cost can be decreased if
done on a large scale (instead of Fedens language “large
areas” we use jars in small place = higher productivity of
secondary metabolites).
Two main approaches have been followed in
connection
with
the production
of
secondary
metabolites in vitro : The rapid growth of suspension
cultures in large volumes which are subsequently
manipulated to produce secondary metabolites. The growth
and subsequent immobilization of cells which are used for
the production of compounds over a prolonged period.
The production of secondary metabolites is strongly
dependent on the rate of cell division, so the yield of
metabolites is dependent on a large number of factors:
The starting material. The pre-treatment before the in vitro
culture. Physical growth factors: light, temperature, aeration.
The composition of the medium (carbohydrate sources,
398

Research Journal of Recent Sciences ____________________________________________________________ ISSN 2277 - 2502
Vol. 1(ISC-2011), 398-404 (2012)
Res.J.Recent.Sci
growth regulator etc.,). The two stage culture system is
necessary sometimes. The first stage involves growing the
cells on maintenance medium and the second stage involves
transferring the cells to a production (of secondary
metabolites) medium. Inducing morphological structure in
culture can have effects.
i- To enable the accumulation of a group of compounds such
as the appearance of leaf bearing shoots in the culture, these
leaves contain oil glands. ii -To alter the qualitative
composition of the products accumulated, such as the
furanocoumarin, psoralen, is present at much higher levels in
the shooty culture. Also, the different compounds are
elicited to various levels depending on the concentration of
elicitor applied. This different culture types can be used to
study the responses of different chemical components to
exogenous factors. Chrosomal stability of the culture.
The development of biosynthetic production in vitro has
been extremely rapid because: There has been a gradual
introduction of growth and production from plant cells in
fermentors and bioreactors. The growth of plant cells can be
optimized by changes in the nutrient medium and the
physical growth factors (aeration, stirring); also it has been
attempted to increase biosynthesis by the addition of
precursors.
Immobilized plant cells can be used (packed in a jelly-like
mass) to increase production of the products and also to
accumulate the metabolites in the medium. This
accumulation means that, substances excreted by the cells
can be obtained by simply exchanging the medium. The
interest in the in vitro production of other compounds (e.g.,
biodegradable nematicides and insecticides) increases. It is
hoped that, it will be possible to produce substances in vitro
which are impossible to normally biosynthesize with plants
in the field.
With this in mind it is hoped that, genetic manipulation of
cells can result in them gaining the characteristic required of
being able to produce a particular substance. Selection and
screening techniques have been developed for the growth of
plant cells which is hoped will result in a higher production
of secondary metabolites. Biotransformation becoming more
important. This is a technique which utilized enzymes
located in the plant cells to alter the functional group
chemistry of externally supplied chemical compounds. There
are two types of biotransformation.
Via whole cells (immobilized or non-immobilized). Via the
use of immobilized compound preparations. A good example
of biotransformation is the conversion of digitoxin to
diogioxin by cells of Digitalis lanata. Interest has grown in
the increase in accumulation of secondary metabolites by the
use of elicitors. Elicitors are strictly speaking compounds of
biological origin involved in plant-microbe interaction.
Elicitors such as phytoalexins (biotic elicitors) which are
International Science Congress Association

mediator compounds of microbial stress or stress agents such
as osmotic pressure or heavy metal ions, UV light, polymers
like chitosan, or dilution into fresh medium (abiotic elicitors)
are used to increase accumulation of products in plant cell
cultures. Multiple shoot cultures are becoming a viable
alternative in in vitro systems for the production of plant
constituents.
Some examples of secondary metabolites obtained in vitro
from some medicinal plants will be discussed in the review:
Antioxidant agents, Analgesic agents,Antibacterial agents,
Antidiabetic agents.

Material and Methods
In vitro production of some antioxidant agents: Many
tests are used to determine the antioxidant activity in
different in vitro culture systems obtained from different
plants (such as; flower cell, cell suspension, callus, shoot in
bioreactor, hairy roots, UV irradiated callus and regenerated
plantlets cultures etc.,) such as β-carotene bleaching, lipid
peroxidation, oxidative DNA damage in cell culture, DPPH,
cell membrane peroxidation, XOD/NBT, singlet oxygen
quenching, alleviation of oxidative stress in cultured
mammalian
cells,
TBARS,
lipid
peroxidation,
overexpressing CHI, H2O2 induced cell damage, carotenelinoleic acid oxidation, brain lipid peroxidation and
hydroxyl radical scavenging and FRAP activity3.
In vitro production of some analgesic agents: Different
cultures of Hyoscyamus niger and H. albus such as; callus,
cell suspension culture and root culture can be used as a
source of analgesic agents such as; tropan alkaloids2.
In vitro production of antibacterial and antidiabetic
agents: Callus cultures of different species of Fagonia are
studied as an antibacterial agents4-6, while callus cultures of
Zygophyllum coccinum are used as an antidiabetic agents7

Results and Discussions
Examples of in vitro production of some antioxidant
agents: Example of in vitro production of some analgesic
agents2: Production of tropan alkaloids from callus, cell
suspension culture and root culture of Hyoscyamus niger
and H. albus. Root culture was found to be the most
containing culture of this compound.
Examples of in vitro production of antibacterial and
antidiabetic agents " organ culture and Zygophyllaceae":
In our works4-7, we focused and will focus in the future on
Zygophyllaceae regarding using organ culture technique as a
tool for producing large amounts of active constituents.
In vitro production of antibacterial agents " organ
culture and Fagonia4-6 Callus of F. arabica: Callus of F.
arabica
leaf explants (photo-1) is a good source of
399

Research Journal of Recent Sciences ____________________________________________________________ ISSN 2277 - 2502
Vol. 1(ISC-2011), 398-404 (2012)
Res.J.Recent.Sci
antibacterial agents such as phenolics, saponins, alkaloids
and flavonoids. These substances gave the callus its
importance regarding antibacterial activity against many
infectious human pathogenic bacteria that cause many
dangerous diseases such as vomiting, diarrhrea, urinary
infections, gastroenteritis "Escherichia coli", infections
around nose and spreading over the face, piles, carbuncles
may be also caused by bacteria "Streptococci", bacteria also
considered to be the major cause of impetigo
"Staphylococcus", fever “Salmonella typhi”, urinary tract
infections may be also caused by bacteria "Klebsiella".

this callus regarding its chemical composition and medicinal
importance, especially as antidiabetic agent.

(a)

Photo-1
Callus of F. arabica leaf explants Ultrastructural
study of callus cells of F. arabica leaf explants
using Transmission Electron Microscope (TEM)
Ultrastructural study (Photo-2) of these callus cells using
Transmission Electron Microscope (TEM) shows the study
of the internal cellular structure of callus. Ultra structural
study on the callus showed large cells with normal structure.
Cell organelles such as vacuoles, dense cytoplasm, nuclei,
endoplasmic reticuli, mitochondrion, golgi apparatus
enveloped by cell wall appeared after using Transmission
Electron Microscope.

(b)

Our work regarding phytochemical screening on both calli of
F. indica and F. bruguieri (table-5) revealed the presence of
many important bioactive constituents such as saponins,
flavonoids, alkaloids, tannins, cardiac glycosides, coumarins,
fatty acids and other constituents. In the future, we will try to
investigate calli of F. indica and F. bruguieri (those calli are
rich sources of many bioactive constituents) regarding their
medicinal importance as antioxidant agents, especially that
our results revealed that, these plants are good antioxidant
agents.

(Photo-2 a,b,c)
Ultrastructural study of callus cells of
F. arabica leaf explants using Transmission
Electron Microscope (TEM)

Callus of
Zygophyllum coccineum7:
Callus of
Zygophyllum coccineum (photo-5) was obtained in our
work without any contamination, however it is difficult to
obtain callus from this succulent plant without any
contamination in the culture. This plant is an important plant
in antidiabetic worlds, in our next studies, we will investigate

(Where:1=Klebsiella pneumoniae, 2=Proteus mirabilis,
3=Salmonella
typhi,
4=Providencia
alcalifaciens,
5=Serratia
marcescens,
6=
Escherichia
coli,
7=Acetobacter aceti subsp. Liquefaciens, 8=Staphyl
ococcus aureus, 9=Streptococcus
salivarius
and
10=Streptococcus
faecalis).

International Science Congress Association

(c)

400

Research Journal of Recent Sciences ____________________________________________________________ ISSN 2277 - 2502
Vol. 1(ISC-2011), 398-404 (2012)
Res.J.Recent.Sci
Table -1 (A)
Examples of in vitro production of some antioxidant agents (3)
Species
Ajuga reptans
Anchusa officinalis
Anthoceros agrestis
Arachis arabica
Artemisia judaica
Carthamustinctorius
Cistanche eserticola
Crocus sativus
Cynara cardunculus
Daucus carota
Fagopyrum
esculentum
Glehnia littoralis
Hemidesmus indicus

Hyssopus officinalis
Ipomoea batatas

Compound

Culture system

Antioxidant testing

Anthocyanins

Flower cell culture

β-carotene bleaching and lipid peroxidation

Rosmarinic acid
Rosmarinic acid and its
glucosides
Piceatannol
(a stilbene)

Cell suspension

Many in vitro chemical assays

Cell suspension

Many in vitro chemical assays

Callus

Oxidative DNA damage in cell culture

Flavonoids

Shoot cultures in
bioreactor

DPPH

Kinobeon A

Cell suspension

Cell membrane peroxidation, XOD/NBT and
singlet oxygen quenching

Phenylethanoid glycosides

Cell suspension

DPPH

Crocin

Callus

Alleviation of oxidative stress
in cultured mammalian cells

Cynarin and
chlorogenic acid

Callus

TBARS

Anthocyanins

Callus and
cell suspension

Lipid peroxidation

Rutin

Hairy roots

Many in vitro chemical assays

Anthocyanins

Callus and cell
suspension

Many in vitro chemical assays

Rutin

Callus, shoot culture

Many in vitro chemical assays

Rosmarinic acid and
lithospermic acid B

Hairy roots

Anthocyanins

Callus and cell
suspension

DPPH

Rosmarinic acid

Callus, cell suspension
and bioreactor

Superoxide radical scavenging

Rosmarinic acid

Bioreactor culture of
nodal explants and cell
suspension

Many chemical in vitro assays

Flavone-C-glycosides

UV irradiated callus

Flavonols and flavones

Cell suspension

In vivo (rats)

Carnosic acid

Callus and shoot
culture

Oxidative stress reduction in living cells and
many chemical in vitro assays

Rosmarinic acid, abietane
and
diterpenoids
Lithospermic acid B and
rosmarinic acid

Callus, shoot culture,
hairy roots and cell
suspension
Callus, regenerated
plantlets and hairy roots

Lavandula officinalis

Ocimum basilicum

Passiflora
quadrangularis
Petroselinum sativum
Rosmarinus
officinalis
Salvia officinalis

Salvia miltiorrhiza

International Science Congress Association

Many in vitro chemical assays

DPPH

DPPH and P-Mo, lipid peroxidation
DPPH

401

Research Journal of Recent Sciences ____________________________________________________________ ISSN 2277 - 2502
Vol. 1(ISC-2011), 398-404 (2012)
Res.J.Recent.Sci
Table -1 (B)
Examples of in vitro production of some antioxidant agents 3
Apigenin
Baicalin,
wogonoside

Hairy roots
Hairy roots and
cell suspension

Overexpressing CHI , H2O2 induced cell damage

Flavonoids

Callus

FRAP and DPPH

Abietane and
diterpenoids

Cell suspension

LDL oxidation and nitric oxide inhibition

Anthocyanins

Cell and aggregate
suspension

Many in vitro chemical assays

Vitis vinifera

Stilbenes,
procyanidins

Cell suspension

DPPH and lipid peroxidation

Withania somnifera

Withanoloids

Hairy roots

DPPH, carotene-linoleic acid oxidation, brain lipid
peroxidation and hydroxyl radical scavenging

Saussurea arabica
Scutellaria baicalensis
Stevia rebaudiana
Torreya nucifera
Vaccinium pahalae

Table -2
Preliminary phytochemical screening on intact leaf and
callus of F. arabica leaf explants
Callus
Callus of F.
Experiment
Arabica leaf
explants
Carbohydrates and / or
+
+
Glycosides
Saponins
+++
++++
Tannins
++
Sterols and / or
+
+
Triterpenoids
Alkaloids
++
++++
Cardiac glycosides
+++
+
Cyanogenic glycosides
+
+
Flavonoids
+
+
Anthraquinones
+
Coumarins
+
++
Irodoids
+
+
A-Chlorides
+
++
-B-Sulphates
+
+
Table - 3
Determination of total phenol contents, total alkaloids,
total flavonoids and total saponins in callus of F. arabica
leaf explants
Concentration of different active ingredients
(mg/g fresh weight)
Total
phenols

Total
alkaloids

Total
flavonoids

Total
saponins

1.95

113.40

0.78

10.00

International Science Congress Association

Many in vitro chemical assays

Study of antibacterial activity of both callus of F. arabica
leaf explants and intact leaf:

Conclusion
To conclude, Plant biotechnology is an alternative and
effective source for production of valuable phytochemicals.
It is also useful for propagation and germplasm conservation,
and thus represent a way to protect plant biodiversity. As
well as being an alternative source of bioactive compounds 812
.
Table - 4
Study of antibacterial activity of both callus of F. arabica
leaf explants and intact leaf
Tested
Clear inhibition zones
Organisms
(mm), (volume of extract =
0.1 ml/disc)
Callus
Leaf extract
extract
Gram -ve
**
1.833
12.417**
1-Klebsiella pneumoniae
2-Proteus mirabilis
16.083**
21.000**
**
3-Salmonella typhi
10.417
22.667**
**
4-Providencia alcalifaciens
13.167
27.083**
**
5-Serratia marcescens
21.417
32.667**
*
6- Escherichia coli
6.083
33.917**
7-Acetobacter aceti subsp.
16.167**
34.833**
Liquefaciens
Callus
Gram + ve
Leaf extract
extract
1-Staphylococcus aureus
.000
2.000
2-Streptococcus salivarius
9.877**
26.420**
3 –Streptococcus faecalis
21.880**
26.920**
L.S.D. (0.05)
5.434
4.311
L.S.D.(0.01)
7.443
5.906
402

Research Journal of Recent Sciences ____________________________________________________________ ISSN 2277 - 2502
Vol. 1(ISC-2011), 398-404 (2012)
Res.J.Recent.Sci

35
30
25
20
15
10
5
0

callus
leaf

1

2

3

5

6

7

8

9

10

Figure -1
Antibacterial activity study on intact leaf and callus of F.
arabica leaf Callus of F. indica:

Photo-4
Callus growth of terminal bud explants of F.bruguieri

Photo - 3
Callus growth of stem segments explants of F. indica
Callus of F. bruguieri

Photo-5
Callus of stem segments explants of Zygophyllum
Coccineum

Table- 5
Preliminary phytochemical screening on both calli of F. indica
(stem segments explants) and Fagonia bruguieri (leaf and terminal bud explants) on different media
Experiment

Carbohydrates and / or Glycosides
Saponins
Tannins
Unsaturated sterols and / or Triterpenoids
Alkaloids
Cardiac glycosides
Cyanogenic glycosides
A- Chlorides
B- Sulphates
Irodoids
Flavonoids
Coumarins
Anthraquinones

International Science Congress Association

Calli of F. indica stem explants
1a
+
+
+
+
+
+
+
+
-

1d
+
+
+
+
+
+
+
+
-

1e
+
+
+
+
+
+
+
+
+
+
+
-

2b
+
+
+
+
+
+
+
+
-

Calli of Fagonia bruguieri
( leaf and terminal bud explants)
3a
3c
3d
3f
+
+
+
+
+
++
++
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-

403

Research Journal of Recent Sciences ____________________________________________________________ ISSN 2277 - 2502
Vol. 1(ISC-2011), 398-404 (2012)
Res.J.Recent.Sci

Reference
1.

Pierik R.LM., In vitro culture of higher plants, Martinus
Nijhoff Publishers, Dordrecht, Netherland, (1987)

2.

Dixon R.A. and Gonzales, Plant cell culture, Oxford
University Press, Walton Street, Oxford, New York,
USA. 2 nd Ed. (1994)

8.

Aguirre Y.A.G., Zamilpa A., Cortazar M.G. and Tapia
G.T., Adventitious root cultures of Castilleja tenuiflora
Benth. as a source of phenylethanoid glycosides,
Industrial Crops and Products, 36, 188– 195 (2012)

9.

Georgiev M., Müller J.L., Weber J., Stancheva N. and
Bley T., Bioactive metabolite production and stressrelated hormones in Devil’s claw cell sus-pension
cultures grown in bioreactors, Applied Microbiology
and Biotechnology, 89, 1683– 1691 (2011b)

3.

Matkowski A., Plant in vitro culture for the production
of antioxidants – A review, Biotechnology Advances,
26, 548- 560 (2008)

4.

Eman A. Alam, Gehan H. Amin, Yassin M. ElAyouty
and Mohamed S. Abdel-Hady, Chemical composition
and antibacterial activity studies on callus of Fagonia
arabica L., Academia Arena, 2(12), 91-106 (2010)

10. Stancheva N., Weber J., Schulze J., Alipieva K., Müller
J. L., Haas C., Georgiev V., Bley T. and Georgiev M.,
Phytochemical and flow cytometric analyses of Devil’s
claw cell cultures, Plant Cell Tissue and Organ
Culture, 105, 79–84 (2011)

5.

Eman A. Alam, Cytological and Ultrastructural studies
on callus of Fagonia arabica, New York Science
Journal, 3(12), 154-157 (2010 a)

11. Lubbe A. and Verpoorte R., Cultivation of medicinal
and aromatic plants for specialty industrial materials,
Industrial Crops and Products, 34, 785–801 (2011)

6.

Eman A. Alam, Phytochemical screening on calli of
Fagonia indica and Fagonia bruguieri Dc., New York
Science Journal, 3(12), 158-164 (2010 b)

7.

Eman A. Alam, In vitro production of callus from
Zygophyllum coccineum L., Academia Arena, 3(1), 69 (2011)

12. Sarasan V., Kite G.C., Sileshi G.W. and Stevenson
P.C., Applications of phytochemical and in vitro
techniques for reducing over-harvesting of medicinal
and pesticidal plants and generating income for the
rural poor, Plant Cell Reports, 30, 1163–1172 (2011)

International Science Congress Association

404

