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Chapter 4
Toxicological and Histopathological investigations of the compound
from Andrographis paniculata.
4.1 Introduction
Pharmacological investigation is an important and decisive step towards drug
development of plant derived bioactive compound. For this, information on traditional
use, chemical content, toxicity, randomised selection or a combination of several
criteria are essential. Earlier studies indicated that inflammation caused by histamine
dimethyl benzene and adrenaline could be significantly reduced by administration of
dehydroandrographolide followed by neoandrographolide (Deng, 1978). Tripathi and
Kamat, (2007) examined aqueous extract for antioxidant activity using heat linear sub
cellular organelles as model systems and reported the extract had potent antiradical
activity against various pathological oxidants.
4.1.1 Toxicology
A greek physician by name (Dioscorides) in the court of the Roman Emperors
Nero, made the first attempt to classify plants according to their toxic and therapeutic
effect (Ernst Hodgson, 2010). Mathieu orfila is considered as the modern father of
toxicology having given the subject its first formal treatment in 1813. The relationship
between dose and its effects on the exposed organism is of high significance in
toxicology. The chief criterion regarding the toxicology of a compound is the dose
(i.e) the amount of exposure to the substance. A previous study demonstrated that
intraperitoneal (i.p) administration of methanol extract of Andrographis paniculata
for five consecutive days at 50mg/day inhibited 65% of production by peritoneal
144
macrophage and significantly inhibited carageenan indicated paw-odema formation is
mice (Sheeja et al., 2006).
Considering the potential for developing drug, the toxicology of the
compounds was studied using the African Earthworm Eudrilus euginae, as the human
genome and the earthworm genome resemble nearly about 80%. The approximate
dose of antibiotics given to human is 500mg for 50kg body weight. Suitable doses for
earthworms were derived and administrated to the worms in the studies.
4.1.2 LD50
LD50 is the median lethal dose that kills 50% of animal population in a
particular species. An approximate LD50 can be initially determined as a pilot study
by a so called staircase method using a small number of animals and increasing the
doses of the drug. Five doses can be chosen for determination of LD50 starting from
0% death to 100% mortality. The LD50 can be calculated by two methods
(Muhammad Akram Randhawa, 2009). LD50 estimations in animals are no longer
required for regulatory submissions as a part of preclinical development package
(Wenning Robert, 2009).
1 Graphical method (Miller and Tainter 1944)
2 Arithmetical methods (Karbers method)
Graphical method
The administered Eudrillus euginae were observed for 24 h. After 24 h, the
number of deceased animals was counted in each group and percentage of mortality
calculated. The percentage dead for 0 and 100 are corrected before the determination
of probits as given below.
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Corrected % formula for 0 and 100% mortality:
For 0% dead: 100 (0.25/n)
For 100% dead: 100 (n-0.25/n) where n=number of animals in each group.
The LD 50 was calculated from the following formula:
(Log LD84- Log LD16
LD50 = -----------------------------
√2N
Where N is the number of animals in each group
Arithmetical method
In the present study, the LD50 was calculated by the arithmetical method (ie) Karbers
method
LD50 = Maximum dose – (T/ Maximum dead)
Where: T is the product of difference in dosage of the drug and mean of the dead
worms.
146
4.2 Material and Methods
4.2.1 Collection and maintenance of earthworms
Eudrilus euginae is a species of earthworm native to tropical West Africa and
now widespread in warm regions. They are also called as African night crawlers.
(Johnson Ratnaraj Samuel et al., 2012) These worms were used for this study. The
worms Eudrilus euginae were collected from Pottalputhur village, Tirunelveli district
and maintained in the laboratory. The bed of the worm consisted of leaf litters, cow
dung and soil. They grew well at a temperature of 75-85 F (24-29
C). It reached its
maximum weight within 8-10 weeks. The night crawler has a uniform purple-grey
sheen and the posterior segments are evenly tapered to a point. The worms thus
maintained were utilised for the series of toxicological and histopathological studies
(Fig.83).
Figure 83: The normal apparently healthy worm of length 10 to 15 cm and weight 1
to 2 gm were taken for toxicological studies.
2 cm
147
4.2.2 Toxicology
Normal apparently healthy earthworms, Eudrilus euginae of approximately 1
to 2 g weight were taken. The individual weight and length of the worms were noted.
In general 500 mg of antibiotic was used for 50 kg body weight in humans. Based on
this calculation, 15 to 20 µg of the compounds was considered adequate for 1 to 1.5g
worms. A group of three worms for each concentration, control, water treated, ethanol
treated and worms without injection were taken. The compound 8 and 6 i.e.
andrographolide and neoandrographolide respectively were injected to the worms in
different concentrations such as 5, 10, 25, 40 and 50µg. The different dosage of the
compounds were prepared by using 5µl ethanol and injected to the worms in their first
three segments for three consecutive days. The worms were kept under constant
observation.
4.2.3 Excretion of the compounds
The excess compounds were excreted by the worms in two ways viz; (i) by
excreta and (ii) by autotomy (cutting the tail). The worms which were administered
with 50 µg compound were observed for changes. The administered worm removed
its tail by autotomy subsequently, the tail was regenerated and the worm was normal
and healthy. The weight of the worm was noted. The worm was kept under
observation for one month. The removed tail portion was carefully taken in a sterile
eppendorf tube and macerated with ethanol. The supernatant was taken for sample
analysis. The macerated samples were analysed using Thin Layer Chromatography.
For positive control, the compounds were loaded at 50µg concentration. For negative
control, only ethanol was loaded. The plates were then stained with iodine for
comparison. The weight and length of the worms were observed for one month.
148
4.2.4 Determination of the Maximum lethal dose of the compound
The worms were administered with the compound 8 and 6 in different ratios
i.e. 50, 100, 150 and 200µg and observed for 24 h to find out the maximum lethal
dose.
4.2.5 LD50
The LD50 was determined by Karbers method (Muhammad Akram Randhawa,
2009). For this, 5 groups of normal healthy worms were selected and in each group, 3
worms were maintained. The dose administered for the worms ranged between 40µg
and 200µg (40, 50, 100, 150 and 200) as the maximum lethal dose of the compounds
was found out to be 200µg. The worms were observed for 24h and the LD50 was
calculated by the formula:
LD50 = Maximum dose- (T/ maximum dead)
Where: T is the product of difference in dosage of the drug and mean of the dead
worms.
Histopathology
4.2.6 Bacterial sectioning
The histopathology of Escherichia coli and Salmonella typhi were studied and
the methodology followed is given below:
4.2.6.1 Collection of cells
Overnight broth cultures of Escherichia coli and Solmonella typhi were
prepared with heavy inoculam. The broth cultures were taken in 4 centrifuge tubes in
equal ratios. Two tubes for control; positive control – no treatment, negative control-
149
administered with ethanol. The sample tubes were treated with compound 1 & 2
(andrographolide and neoandrographolide). The steps which were involved in
histopathology are depicted in flow chart (P. 149).
4.2.6.2 Preparation for histology of bacterial cells
The cells were prepared for viewing under a microscope by chemical fixation.
The overnight broth cultures were centrifuged and the cells were collected. The
pellets were washed with water and mixed with formaldehyde for 15 minutes. This
was again centrifuged and the supernatant was discarded. The pellets were washed
with water and treated with serially increasing alcohol (treated with 70, 80, 90 and
100% isopropyl alcohol for 15 minutes each). Then the pellets were allowed to stand
in xylene for 15 minutes. After 15 minutes, the tube was centrifuged and the
supernatant was removed. The pellet was thoroughly mixed with vortex mixture, the
mixture was transferred to the molten wax and block was prepared.
Overnight broth culture of bacteria
Centrifuge
Supernatant (discarded) Pellet
Water wash
50% Formaldehyde
Centrifuge
Supernatant (discarded) Water wash
100% Alcohol treatment
150
Centrifuge
Supernatant (discarded) Pellet
100% Xylene
Centrifuge
Supernatant Pellet (Vortex mix)
Wax impregnation
Sectioning (2 micron thickness)
Staining
The ribbon was prepared by microtome sectioning about 2 micron thicknesses.
The albumenized slide was placed on the slide warming table at 50 C. One ml of
water was placed on the slide. To that slide, the ribbon was placed. The water was
removed with the tissue paper after the wax melted. The slide was kept on the slide
warming table for another 30 minutes to fix the sections. After heat fixing the slides
were placed in xylene 100% alcohol for 1 minute to remove the wax. Then the slides
were washed with water and placed in haematoxylin for 10 minutes. The slides were
then washed with water and acid alcohol to decolourise. After that, the slides were
immediately placed in running tap water for 15 minutes. After 15 minutes, the slides
were stained with eosin (counter stain). It was washed in water and alcohol. Then the
slides were allowed to air dry and mounted after a dip in xylene with DPX. The steps
involved are presented in the following flow chart:
151
De waxing in Xylene
10 minutes
100% Alcohol
Water wash
Haematoxylin
10 minutes
Water wash
Acid alcohol decolourization
Immediately running tap water
Eosin (30 sec)
Water wash
Alcohol wash
Air dry
Xylene dip
Mounted with DPX
4.2.7 Earthworm sectioning
Four groups of apparently healthy worms of uniform size were taken. Each
group contained two worms. Fifty µg concentrations of compound 8 and compound 6
152
were mixed in 5µl ethanol and injected in the second segment to the first and second
group of worms. Five µl of ethanol was administered to third group, which acted as
control. The fourth group of worms were without injection. The injection was given
for three consecutive days. The length and weight of the worms were noted before
and after injection. After three days, the worms were used for histopathological
studies. Thirty segments were taken for sectioning and the segments were divided into
6 samples:
Sample A (1-6)
Sample B (7-12)
Sample C (13-18) Clitellum
Sample D (19-24) Prostrate
Sample E (25-30) Intestine
4.2.7.1 Preparation for Histology (Tissue processing)
The samples were prepared by sacrificing the worms. The worms were cut
into segments and then placed in fixative (i.e.) 50% formaldehyde for 12 to 24 h.
After 24 h, the segments were washed with water and the moisture was removed by
using filter paper. The tissues were dehydrated by treating the samples in 70, 80, 90
and 100% isopropyl alcohol each for 1 h and
C. In this stage, the tissues looked transparent. The tissues were finally
transferred to hot liquid paraffin wax. C in oven. The
tissues were transferred to wax 1, 2 and 3 each for 2 h and the processed tissue was
set in mould. Thin sections of 2 to 7 µm were cut using a microtome Weswox optic
model MT-1090A13095.
153
4.2.7.2 Staining
The albumenized slide was placed in the slide warming table in 45 to C
and to expand the tissues one ml of water was allowed to stand in the slide. After the
water got heated, the ribbon with the sections was placed on the slide. When the wax
melted, the water on the slide was removed with the help of tissue paper. The slides
were placed in the slide warming table at 45 to C for ½ to1 h for heat fixing. The
slides were then placed in xylene and 100% alcohol for 10 minutes each for
dewaxing. The slides were then washed with water and dipped in hematoxylin for 10
minutes. Again it was washed with water and dipped in acid alcohol for
decolourization and immediately kept in running tap water for 15 minutes. The slides
were stained with Eosin. The slides were kept for air drying after washing with water
and alcohol. Then the slides were mounted with DPX.
154
4.3 Results
4.3.1 Toxicology
The weight of the worm Eudrillus euginae got reduced after the third day
injection. Though the weight was reduced, the worm appeared normal, healthy and
active. The decreasing weight of the worm corresponding to the dosage of the drug
was noted. The weights of the worms were noted for one month and it was presented
as a comparative graph (Fig. 84 A and B).
A
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Figure 84 A, B: Mortality and weight changes of andrographolide and neo-
andrographolide administered Eudrillus euginae
4.3.2 Excretion of the compounds
The 50µg compound injected worm had cut its tail after the third day of
injection (5 to 10 minutes) to eject the excess compound (Fig 85 and 86). The
compound was excreted by forming a knot in the tail region. The knot compressed
gradually and the tail was removed after 2 to 3 h. The worm behaved as normal
worm after the removal of the tail and the tail bud was regenerated after three days.
4.3.3. Thin Layer Chromatography of removed tail portion
The removed tail portion was macerated with methanol and TLC was carried
out. The 50µg of the compound loaded in the TLC plate served as control. The spots
were compared. The spots formed by the control (50µg of compound 6 and 8) were
B
156
more concentrated than the compound which was present in the tail region. From
this data, it was understood that the compounds were absorbed by the worm and the
excess compounds only excreted. Accordingly the compounds could be known to
exert mild toxic, as the removed tail regenerated on the consecutive days. The worm
was apparently normal and appeared healthy (Fig. 87 to 90).
4.3.4 Maximum Lethal Dose and LD50
The worm which was injected with 200µg andrographolide died in 10 h and
the worm which was injected with neoandrographolide died in 8h (Fig. 86). The
results of lethal dose could be indicated that the worm administered with 200µg died
within 24 h. LD 50 was calculated by arithmetical method. The calculated LD50 value
for andrographolide was 82µg/g and neoandrographolide was 55 µg/g (Table 25).
LD50 for andrographolide
The maximum dose given to the worm was 200µg. T was the product of the
dose difference and the mean of dead worms. The dose difference was calculated as
50, 50, 50, 10 (a) respectively for 150, 100, 50 and 40µg compound. The mean of
dead worms were 3, 2.5, 1.5 and 0.5 (b) and a*b was 150, 125, 75 and 5. The values
are tabulated in Table 25.
LD50 for neoandrographolide
The group of 3 worms administered with (200µg) neoandrographolide were
dead as the maximum dose was identified as 200µg. the mean of the dead worms were
calculated as 1, 2.5, 3 and 3(b) for 40, 50, 100 and 150µg (a) neoandrographolide the
product of the difference in dosage and the mean of the dead worms were 150, 150,
125 and 10 (Table 26).
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Figure 85: Andrographolide injected worms
Figure 86: Neoandrographolide injected worms
Figure 85, 86: A, B, C- Worms injected with 40, 50 and 25µg of andrographolide and
neoandrographolide. A knot was formed in the tail of the worm administered with
50µg compound (indicated by arrow). The worm administered with 40µg and 25µg
compound was normal.
A B
C
A B
C
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Figure 87: Maximum lethal dose
A and B administered with andrographolide and neoandrographolide. C and D
are dead worms (8, 10 h).
Bacterial Sectioning
The results indicated no morphological changes in the cells treated with
compounds (Fig. 91 to 94).
159
Figure 88: Excretion of andrographolide by autotomy
A, B, C, D are the steps noted in the autotomy. The excess compound was
removed from the body of the worm by removing its tail and the worm was normal
after the removal of the tail. E – worm without tail, F – removed tail portion, G –
worm with regenerated tail. (A - D) Photographs were taken at 1 h interval.
E F
D
B A
B
Regenerated tail
C
C D
F E
G
160
Figure 89: Figures depicting the excretion of neoandrographolide by autotomy
A, B, C, D are the steps noted in the autotomy. The excess compounds were
removed from the body of the worm by removing its tail and the worm was normal
after the removal of the tail. E – worm without tail, F – removed tail portion, G –
worm with regenerated tail. (A - D) Photographs were taken at 1 h interval.
F E
C
B A
G
Regenerated tail
C
D
161
Figure 90 A, B: Thin Layer Chromatography of compounds administered worms
(removed tail portion)
The Rf value of andrographolide (a) was 5/7 = 0.714 and for
neoandrographolide 3/7 = 0.42. 1 – loaded spot (compound), 2 – negative control
(methanol), 3- macerated tail sample, A- andrographolide, B - neoandrographolide
1 2 3 1 2 3
a a
A
a a
B
1 2 3 1 2 3
a
b b b
b
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Table 25: LD50 for andrographolide
Table 26: LD50 for Neoandrographolide
Group Dose (µg/g) Dose
Difference (a)
Dead Mean (b) Product
a*b
1 200 - 3 - -
2 150 50 3 3 150
3 100 50 2 3 150
4 50 50 2 2.5 125
5 40 10 0 1 10
T = 435
LD 50 = Maximum dose – (T / Maximum dead)
For andrographolide,
= 200 – (355/3) = 82 µg
LD50 for andrographolide was calculated as 82 µg / g.
For neoandrographolide,
= 200 – (435/3) = 55µg
LD50 for neoandrographolide was calculated as 55µg / g.
Group Dose (µg/g) Dose
Difference (a)
Dead Mean (b)
Product
a*b
1 200 - 3 - -
2 150 50 3 3 150
3 100 50 2 2.5 125
4 50 50 1 1.5 75
5 40 10 0 0.5 5
355
163
Figure 91: Sectioning of andrographolide treated Escherichia coli
Figure 92: Sectioning of andrographolide treated Salmonella typhi
Figure 91, 92: A – control organism without treatment, B – organism treated with
compound, C – organism treated with ethanol and observed under 100 X oil
immersion microscope.
2µm
2µm
2µm
C
B A
A
C
B
1.5µm 1.5µm
1.5µm
164
Figure 93: Sectioning of neoandrographolide treated Escherichia coli
Figure 94: Sectioning of neoandrographolide treated Salmonella typhi
Figure 93, 94: A – control organism without treatment, B – organism treated with
compound, C – organism treated with ethanol and observed under 100 X oil
immersion microscope.
B
C
2µm
A B
1.5µm 1.5µm
A
2µm
C
2µm
1.5µm
165
4.3.5 Earthworm sectioning
The segments 1 to 6, 7 to 12, 13 to 18, 19 to 24, 25 to 30 for the control (not
treated and ethanol treated worms) and compound treated worms are shown in Fig. 95
to 99. The histopathology of the injected worms was studied by microtome sectioning
(7µm thickness). One control worm without injection was kept as positive control.
The others injected with ethanol served as negative control and the compound treated
worms (Experimental group) were investigated in detail. Six samples from each worm
were taken which included: 1 to 5 segment, 6 to 10 segment, clitellum (13 to 18)
prostrate (19 to 24) and intestine (25 to 30). From the results, it could be seen that in
the ethanol treated worms: the intestinal layer, the dorsal and ventral blood vessel and
metanephridia were denatured and in the compound treated worm only the inner
intestinal layer cells was damaged. This could be noted in the 6 to 10th
segments as
given in Fig. 96
In clitellum and the prostrate region, the sections of both compound and
ethanol treated worms did not show any changes. In 25 to 30 segments, (intestine) the
typhlosole was damaged, the intestinal cells which are present in the inner lining of
the intestine were denatured in both ethanol and compound treated worm. These
effects might be due to the presence of ethanol in the compound. From this study, it
was confirmed again that the compounds purified from the plant Andrographis
paniculata could not be considered as toxic.
166
Figure 95: LS of Segment 3 (Head)
A – Body cavity; B – Circular muscle; C – Epidermis; D – Longitudinal
muscle; E – Ventral nerve cord; F – Intestine.
Ethanol treated Compound 6
Compound 8 Control
A
E
F
D C B
a b
c d
167
Figure 96: LS of Segment 7
A – Body cavity; B – Circular muscle; C – Epidermis; D – Longitudinal
muscle; E – Ventral nerve cord; F – Intestine; G – Dorsal blood vessel. In (b) – arrow
indicating denatures of metanephridia and nerve cord. In (c) and (d) – all the internal
organelles were denatured.
Control
G F
E
D C B
A
A
B
D
C
Compound 8
Compound 6 Ethanol treated
d c
b a
168
Figure 97: LS of segment 15
A – Body cavity; B – Circular muscle; C – Epidermis; D – Longitudinal
muscle; E – Ventral nerve cord. Red arrow indicating the denature of intestinal cells
in b, c, and d. In d another arrow indicating the denature of metanephrida
Ethanol treated Compound 6
Compound 8 Control
D B
E
C
A
D
A
C
B
d c
b a
169
Figure 98: LS of Segment 21
A – Body cavity; B – Circular muscle; C – Epidermis; D – Longitudinal
muscle.
Compound 8 Control
A
B D C
D B
A
C
c
b a
d
170
Figure 99: LS of Segment 25
A – Body cavity; B – Circular muscle; C – Epidermis; D – Longitudinal
muscle, F – Intestine. Red arrow indicating the denature of the inner intestinal layer
cells.
Ethanol treated Compound 6
Control Compound 8
D
C
B
A
A
D
B
C
F
F
c
b a
d
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4.4. Discussion
Earthworms are the first group of eucoelomate invertebrates who had
succeeded to inhabit terrestrial environment. They serve as bio indicators to
understand the physicochemical characteristics of their habitat. TLC of the removed
tail portion of 50µg (compounds) administered worm confirmed that the compounds
were absorbed by the worm. Plant extracts (7.2 mg/kg body weight) and partially
purified fractions (2.4 mg/kg body weight) when administered to mice experimentally
envenomed with rattle snake venom showed potent neutralising effect against the
venom. The isolated fractions effectively inhibited the toxic effect of snake venoms
in vitro than in vivo (Samy et al., 2008). In the present work 40 to 200µg of
compounds (andrographolide and neoandrographolide) were administered to
Eudrillus euginae.
The cytology of bacterial cellular division involves a number of structures and
events which are beyond the resolution limit of the light microscope. The complexity
of these structures and the possible variations which differences in species, age of
culture and growth conditions may introduce have made it impossible to make reliable
interpretations by indirect methods. This has led to considerable confusion as apparent
from any review of the literature. In the present thesis, bacterial sectioning was
carried out to check whether any changes were seen between the compounds treated,
ethanol treated and the organism without treatment. No visible changes were seen
among the organisms with and without treatment. This could be due to the resolution
limit of the light microscope.
There is no doubt that areas where wastes are usually dumped become toxic
with high concentration of metals and organochlorides. Intense coiling in earthworms
172
has been attributed to lack of sufficient soil moisture as coiling helped to conserve
body moisture (Callahan et al., 1991). Earthworms are known not to suffer significant
morbidity responses and direct mortality from exposure to organochlorides and heavy
metals but they may accumulate the residues in their tissues even above the amount in
the soil they inhabit (Ireland, 1979; Curry, 1994). The heavy metals have been
confirmed to affect sexual development and cocoon production (Cikutovic et al.,
1993). Heavy metals such as zinc, lead, cadmium, manganese and high concentration
of sodium chloride affect reproduction in earthworms (Fisher et al., 1997). The vital
organs of E. eugeniae are located in the first 13 segments and consist of a mouth,
simple brain, 2 pairs of heart, testis, seminal vesicle, rudimentary ovary, oviduct, and
accessory glands of the ovary. A thick cylindrical collar-like structure, called the
clitellum, which plays an important role in reproduction, is present in segments 13–18
(Gates, 1942). In the present study injection was given in the third segment and first
thirty segments were used for sectioning.
The wound of an amputated site was healed quickly at 24 h in the earthworm,
E. eugeniae. The injection that was given in the mouth may blocks the intake of
nutrients cause additional energy loss, and differentiation of the circular muscle cells
into the longitudinal cell layer could save the energy for the rest of the regeneration
process. Hence, there is a greater possibility for differentiation of the circular muscle
layer into the longitudinal cell layer (Johnson Retnaraj Samuel et al., 2012). Oboh et
al., (2007) reported that the dumpsite soil, petroleum effluent and lake sediments had
detrimental effects on the earthworm Eudrillus euginae to varying degrees. They also
reported that the weight and reproductive ability was reduced. The present thesis
revealed that the compounds andrographolide and neoandrographolide administered
173
worms showed reduction in weight. But no harmful effects were observed. This
indicated that the compounds could have exerted a mild toxic effect.
The principal systematic features of earthworms are that they are bilaterally
symmetrical, externally segmented with a corresponding internal segmentation. The
body wall consists of an outer cuticle, the epidermis, a layer of nervous tissue, circular
and longitudinal muscle layers and finally the peritoneum, which separates the body
wall from the coelom. The epithelial lining of the intestine is composed mainly of
glandular cells, and non glandular ciliated cells. Earthworms have a closed vascular
system consists of three principal blood vessels: one dorsal and two ventral. The
intestinal layer, dorsal and ventral blood vessel and metanephridia were denatured in
the ethanol treated worm and in the compound treated worm only the inner intestinal
layer was damaged. Comparing these results it was confirmed that the toxic effect was
due to the effect of ethanol as the parts of the ethanol treated worm was denatured.
The method of gaseous exchange depends upon a network of small blood
vessels buried in the body wall of terrestrial earthworms, so that oxygen dissolved in
the surface moisture film could be permeated through the cuticle and the epidermis to
the thin walls of these vessels, where it is taken up by the haemoglobin in the blood
and passed around the body. The nephiridia are the main organs of nitrogenous
excretion in earthworms, are paired in each segment except the first three and the last.
In the 25 to 30 segments, the typhlosole was damaged. Typhlosoles are the intestinal
cells which are present in the inner lining of the intestine were denatured in ethanol
and compound treated worm. The worm was observed for a month and found it was
normal and healthy. The removed tail portion was also regenerated normally. From
this data it was confirmed that the compounds andrographolide and
neoandrographolide could be exerted only mild toxic effect.