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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.