Introduction

Andrographis paniculata which also known as ‘King of bitters’ because it is one of the most
bitter plants that are used in traditional medicine. It belongs to the family Acanthaceae native to
India and Sri Lanka. It is a type of annual herb. There are 4 main parts for this herb which are
leaves, stems, flowers and fruits. Its stem is around 50-80 cm. The lower part of this herb is more
branched. Leaves ovate to round to lanceolate, 4 to 8 cm long and 1 to 2.5 cm wide.
Inflorescences on the upper axis is smaller. The corolla is white and small. There are purple
striate at lower lip. The anthers have 2-loculed, pubescent on one part of base and filiform. It has
flat capsule with a groove in the middle. It is wrinkled and consists of 12 seeds. It tastes very
bitter, even if it is swallowed, it still has a bitter taste in thecc taste bud and throat.

A.paniculata contains the active phytochemicals from the aerial parts which are leaves and
stems. The phytochemicals including andrographolide, neoAndrographolide, andropanoside,
terpenoids and sarponins. It produces a group of diterpene lactones from secondary metabolites.
From which Andrographolide is the major content present in leaves of A.paniculata.

The chemical structure of andrographolides is significant to its bio activities. It is found that
lactone moiety and its conjugated double bond was fount to be the core structure of
andrographolide and its analogues. The pharmaceutical activities will be lost when lactone or its
conjugated double bond is open. Proper modification on functional groups at carbon-3, carbon-
14 and carbon-19 helps to enhance the pharmaceutical activity.
Andrographolides posses various important biological properties like anticancer, anti-
inflammatory antipyretic, anti-liver damage and anti-venom. Andrographolide enhances the
immune system and inhibits the proliferation of tumor cell lines including human cancers. It also
help to reduce the intensity of joint pain, total number and grade of swollen joints and number of
tender joints in a prospective.

Benefits and clinical application of Andrographis Paniculata

A.paniculata exhibits diverse pharmacological activities.

1. Anticancer
 Andrographolide induces cell cycle arrest and mitochondrial-mediated apoptosis
in human leukemic cells. Andrographolide is found to inhibit the rapid growth of
various cells lines including leukimea, breast cancer, lung cancer and melanoma
cells. This compound also has strong anticancer activity against human colorectal
carcinoma (colon or bowel cells) by inhibiting cell cycle progression. Induction of
cell cycle arrest at G0 or G1 phase. It inhibits the growth of prostate cancer cells
without affecting the primary prostate epithelial cells. A potent growth inhibitory
effect of andrographolide has been demonstrated in acute promyelocytic leukemic
cells.
 It also exerts its anti-cancer activity through stimulation of lymphocyte
proliferation and activation. The anti-cancer activity of andrographolide is also
mediated by suppressing tumor angiogenesis, and inhibiting cell proliferation and
migration, and tube formation of vascular endothelial cells in vivo.

2. Effects on cardiovascular disease

 Andrographis paniculata had proven that can increase the blood clotting time,
more over pre and post-treatments of after sugery using the extract of
andrographis paniculata significantly prevent the constriction of the blood vessels,
thus it will decrease the subsequent closing of blood vessel after angioplasty
procedures.
 Several studies have uses animal model to investigate the effect of andrographis
paniculata before and after myocardial infarction. The extract of the plant proven
that it relaxed the smooth muscle in wall of blood vessels and helps to prevent the
blood vessel from constricting and limit the flow of blood to the brain,heart and
other organs by producing hypertensive effects.
Extraction of Andrographis

There are few methods to extract Andrographis from A.paniculata such as Soxhlet extraction,
Reflux extraction, Cold extraction, Sonication-assisted extraction and Microwave-assisted
extraction. With the help of HPLC analysis of sample, it is found that Reflux extraction is the
best extraction method which can extract the most andrographolide among the other methods.
Under reflux extraction with methanol, the extraction with methanol for one hour has been found
the best in terms of andrographolide content (2,04%) with total extract 15.111%.

Cell Culture

Cell cultures have a higher rate of metabolism because their initiation leads to faster proliferation
of cells and a condensed biosynthetic cycle. It is not restricted by environment but can be
controlled by human. With elicitor treatment, the secondary metabolite content in cell cultures
can be further enhanced. There are 2 methods to undergo tissue culture which are callus culture
and root culture.

Callus culture

Callus is an unspecialized, unorganized, growing and dividing mass of cells. It produced when
explants are cultured on the appropriate solid medium, with both an auxin and cytokinin in a
correct condition. It may be compact or friable.

It is started with Induction. The cells will begin to divide. It will then enter Proliferative stage
which the cell will divide rapidly. Finally, it will enter Morphogenensis Stage. The organized
structures are differentiated and formed. It leads the somatic cells to have plant regeneration.

a) The most suitable explant in callus culture

There are various part of A.paniculata can used as explants in callus culture. But the results for
the quality of callus and content of andrographolide are different.

Explants from in vitro-germinated A.paniculata such as leaf, shoot and root parts are evaluated
for callus induction on MS media supplemented with various concentrations of phytohormones
such as 6-benzylaminopurine (BAP).
 Vitro-grown plantlets for callus induction

From response of all the plant parts, the stem sections gave rise to compact callus whereas the
root parts gave scant response. Leaf sections were preferred in terms of callus propagation rate
and friability. Sharma had reported the leaves of A.paniculata contain the highest amount of
andrographolide which is 2.36 %. It is the most medicinally active phytochemical in the plant
in 1992.

b) The most suitable growth hormone combination in callus culture

Leaves of A.paniculata will be used as explants since it has the major content of
andrographolide. Sterilization of leaves of A.paniculata will be conducted by using 70% ethanol.
After sterilization of explants were inoculated aseptically on nutrient media containing various
concentration of growth hormone like dichlorophenoxyacetic acid (2,4-D), naphtaleneacetic acid
(NAA) and benzyladeninepurine (BAP).

Callus induction standardize by using different combination and concentration of growth

regulator. Induction started after 15 days and get the observation around 30th days. The result
show that, the maximum callusing frequency is 92.02% in 2,4-D combined with NAA. It is
observed that leaf response towards callogenesis is with 2,4-D combined with NAA. The callus
is found green in colour and compact in texture.

Callus initiation started after 15 days in full Excellent quality of callus observed in
MS media 2,4- D+NAA (1.5+2.0 mg/L) after 30 days.
The result is obtained through HPLC. Standard for Andrographolide is used with 98% purity.
Percentage of content is calculated by using formula:

%Content of Andrographolide
Standard area Sample weight Standard weight
¿ × × ×%Purity of the standard
Sample area Sample dilution Standard dilution

It is not necessary the higher the hormone concentration, the higher the content of
Andrographolide. 2,4- D+NAA (1.5+2.0 mg/L) shows the excellent quality of callus while 2,4-
D+NAA (2.5+3.0 mg/L) only shows the good quality of callus been observed after 30 days. This
is because hormone NAA is toxic to plants at high concentration.

0.5+1.0(mg/L) 1.0+1.5(mg/L)2 e Callus

1.5+2.0(mg/L)3 2.0+2.5(mg/L) 2,4-D 1.0 Poor
2.5 1.5 Poor
2.36
2,4- 0.5+1.0 Excellent
Andrographolide Content(%)

2
D+BAP 1.0+1.5 Excellent
1.5
1.5+2.0 Poor
1 2.0+2.5 Good
2,4- 1.0+1.5 Poor
0.5
D+NAA 1.5+2.0 Excellent
0
2,4-D+BAP 2,4-D+NAA
2.0+2.5 Excellent
2.5+3.0 Good
Growth Hormone

Hormon Concentration(mg/L) Quality of Table of relationship between

Graph of andrographolide content versus andrographolide content and hormone
growth hormone

Best results of callus initiation are possible in MS media containing 2,4-D with NAA which is a
type of auxin. Auxins helps to induce content of Andrographolide.
Root culture

Root culture can be defined as the culture of excised radical tips of aseptically germinated
seeds in a liquid medium where they are induced to grow independently under controlled
conditions. It is generally maintained in moving liquid medium. Root tips are induced to row like
that of root system of an intact plant.

JA(Jasmonic acid) SA( Salicylic acid)

Maximum andrographolide content is obtain after the first week using adventitious root cultures
elicited with 25µM JA. Among the concentration of SA and its derivatives, elicitation with
MeSA for seven days promote 2.6 fold increase in andrographolide content in adventitious root
cultures of A.paniculata.

High temperature, low temperature and high salt concentration activates the stress related protein
kinases which sends the signal to nucleus for defense related activation of genes which are
involve in biosynthesis of secondary metabolites.

As temperature increased from 25ͦC to 50ͦC, the andrographolide content decrease initially then
sudden rise in content with increased temperature. It is also reported that low temperature 12 and
18ͦC increase eleutheroside accumulation in somatic embryos.

Reference

2) Seema Sharma, Yash Pal Sharma (July 2018) Comparison of different extraction methods
and HPLC method development for the quantification of andrographolide from
Andrographispaniculata (Burm.f.) Wall. ex Nees, Department of Forest Products,
University of Horticulture and Forestry, Solan-173230, Himachal Pradesh, India
3) Suryakala Gandi, Kiranmayee Rao, Bhuvaneswari Chodisetti, Archana Giri (December
2012) Elicitation of Andrographolide in the Suspension Cultures of Andrographis
paniculata
https://link.springer.com/article/10.1007/s12010-012-9892-4

4) Pawar SD, Yeole PT, Bhadane PV and Kadam SR (2018) Standardization of callus
induction protocol and effect of hormone concentration on synthesis of Andrographolide
from Andrographis paniculate
http://www.chemijournal.com/archives/2018/vol6issue2/PartT/6-2-98-692.pdf

5)