36 

 

Acute Toxicity Study of the Combination of 
Azadirachta indica A. Juss. and Gynura procumbens 
(Lour.) Merr. Leave Extracts    
 

Nofran Putra Pratama*, Kurnia Rahayu Purnomo Sari; Ririn Irma Marliana 
Faculty of Health, Universitas Jenderal Achmad Yani Yogyakarta, Jl. Ringroad Barat, Gamping Kidul, Ambarketawang, Gamping, Sleman, 
Yogyakarta 55294. 
 

Abstract 
Azadirachta indica A.Juss. and Gynura procumbens (Merr.) are 
medicinal herbs widely used in traditional medicine. Recent 
research on the combination of these two plants showed 
aggressive antioxidant activity. The combination results can 
improve insulin and beta-cell morphology and can increase insulin 
expression. The variety of activities performed is the basis for 
conducting acute toxicity tests on the ethanol extract of 
Azadirachta Indica A. Juss. and Gynura procumbens (Lour.) Merr. to 
increase public confidence in its efficacy and ensure the safety of 
its use. The acute toxicity test on the ethanol extract of 
Azadirachta Indica A. Juss. and Gynura procumbens (Lour.) Merr. 
was carried out on female Wistar rats by following the 423 
procedures of OECD (Organization for Economic Cooperation and 
Development) Guideline with five groups of experimental animals, 
namely normal treatment, aquadest solvent treatment and 0.5 Na-
CMC 0.5%, a separate treatment of the ethanol extract of 
Azadirachta Indica A. Juss., a separate treatment of the ethanol 
extract of Gynura procumbens (Lour.) Merr., and combination 
treatment of the ethanol extract of Azadirachta Indica A. Juss. and 
leaves of Gynura procumbens (Lour.) Merr. Furthermore, it was 
proceeded by observing the liver of the experimental animals 
histopathologically. The acute toxicity test results utilizing the 423 
procedures of the OECD did not confirm any death or signs of 
toxicity in the experimental animals, and histopathological 
observations did not show any major histopathological damage. 
Based on these results, according to Globally Harmonized 
Classification System (GHS), the combination of the ethanol 
extract of Azadirachta Indica A. Juss. and Gynura procumbens 
(Lour.) Merr. is included in the unclassified category (> 2,000 
mg/kg BW. 
 

Keywords: Acute toxicity; Azadirachta Indica A. Juss. and Gynura 
procumbens (Lour.) Merr; Histopathology; OECD 

 
 
 
 
 
 
 
 
 

 

 
Data of article 

Received 
Reviewed 
Accepted 

: 
: 
: 

21 Nov 2020 
12 Dec 2020 
22 Feb 2021 

 
DOI 
10.18196/jfaps.v1i2.9868 
 
Type of article: 
Research 
 

  

 
* Corresponding author, e-mail: nofranputrapratama@gmail.com 



Nofran Putra Pratama, Kurnia Rahayu Purnomo Sari, Ririn Irma Marliana | Acute Toxicity Study of the Combination of 
Azadirachta indica A. Juss. and 

Gynura procumbens (Lour.) Merr. Leave Extracts  
 

57 

INTRODUCTION 
 
The use of natural materials, especially 
those derived from plant materials, has 
been carried out by the Indonesian people 
from generation to generation to prevent 
and treat diseases. This natural ingredient 
is known as traditional medicine/herbs as 
its manufacture, and use principles are still 
traditional, and most of its properties to 
date have merely been based on empirical 
experience. The use of traditional 
medicine is considered an alternative 
treatment with few side effects. 
Therefore, further research is required on 
the safety testing of these traditional 
medicines/herbs. Research related to 
traditional medicines/herbs derived from 
plants is still mainly in the form of 
separated extracts, while the combined 
extracts are still rarely conducted. The 
combined extracts can be an alternative in 
the development of medicinal plants to 
obtain better results. 
 
One example of a combination of extracts 
that has been widely researched is the 
combination of the ethanol extract of 
Azadirachta Indica A. Juss. and Gynura 
procumbens (Lour.) Merr. having been 
known to have many properties, including 
antioxidant activity1, decreased blood 
glucose levels2, decreased glycemia and 
improving insulin morphology and beta 
cells and increasing insulin expression3, 
significantly decreased glycemia in 
streptozotocin-induced rats4, and 
hypoglycemic effect in mice5. 
 
An acute toxicity test is needed to 
determine the safety of the combination 
of the ethanol extract of Azadirachta 
Indica A. Juss. and Gynura procumbens 
(Lour.) Merr. This test is intended to obtain 
information regarding the symptoms of 
poisoning, the cause of death, the 
sequence of the death process, and the 

lethal dose range for the experimental 
animals (LD50)6. Based on the data above, 
an acute toxicity test was carried out 
utilizing a method adopted from the 
Organization for Economic Co-operation 
and Development (OECD) and 
histopathological observation on the lab 
rats' liver. 
 
METHODS 
 
Materials 
The materials for the extract of 
Azadirachta Indica A. Juss. and Gynura 
procumbens (Lour.) Merr. were obtained 
from Moyudan Sleman Yogyakarta, such 
as 70% ethanol (Onemed). CMC Na 0.5%, 
buffer formalin 10%, NaCl physiology 
(Otsuka), Hematoxillin Eosin (Merck)1, 
female Wistar rats. 
 
Research Process 
 
1. Animal grouping 

An acute toxicity test of a combination 

of the ethanol extract of Azadirachta 

Indica A. Juss. and Gynura procumbens 

(Lour.) Merr. utilized the 423 

procedures of OECD guidelines7. The 

experimental animals handled were 15 

female Wistar rats aged 8-12 weeks, 

not pregnant, with a bodyweight of 

150-250 grams. Female experimental 

animals were more sensitive than 

males, allowing toxic effects to appear 

in female animals. 

 

The experimental animals were divided 

into five groups, consisting of 3 animals 

in each group. The control groups 

(without treatment) were the solvent 

control group (aquades and 0.5% Na-

CMC), the group of the ethanol extract 

of Azadirachta Indica A. Juss., the group 



Journal of Fundamental and Applied Pharmaceutical Science, 1(2), Febuary 2021 58 

of the ethanol extract of Gynura 

procumbens (Lour.) Merr, and the group 

of the combined extract of both. 

 

2. Provision of test preparation 

In the first stage of the test, the 

experimental animals were fasted 

overnight before administrating the 

test preparation, and its weighed. After 

that, the pressure was carried out on 

three experimental animals with an 

initial 300 mg/kg BW dose. 

Furthermore, the toxic symptoms were 

observed, especially during the first 4 

hours, and were continued during the 

first 24 hours after the treatment. 

Observation of toxic symptoms was 

continued every day until the 14th day. 

When 2-3 experimental animals died, 

the dose was reduced to 50 mg/Kg BB. 

 

Meanwhile, when 0-1 experimental 

animals died, administration of test 

preparations of 300 mg/Kg BW was 

continued. When 0-1 experimental 

animals died in the second round, the 

test was continued by administering a 

test dosage of 2000 mg/Kg BB to 3 test 

animals, as shown in Figure 1. The 

weight of the experimental animals was 

weighed on day 1, 2, 5, 8, 11, 14. On the 

14th day, the tested animals were 

dynecropsied, and a histopathological 

examination was performed. 

 

3. Observation of toxic symptoms 

The physical observation was carried 

out on toxic symptoms in all rats from 

the five test groups. This examination 

was carried out continuously during the 

first four hours after giving the test 

preparation. Observation of toxic 

symptoms qualitatively followed the 

criteria in the Balazs table8. 

 

However, not all criteria in the Balazs 

table were observed in this study. The 

criteria observed were those that could 

be seen with the naked eye, such as 

changes in behavior, body weight, 

seizures, tremors, paralysis, sedation, 

coma, and salivation. 

 

4. Histopathological observations  

Histopathological observations were 

carried out in several stages, namely 

organ harvesting, making 

histopathological preparations carried 

out at Balai Besar Veteriner 

Yogyakarta, and histopathological 

examinations by comparing them with 

the organ's histological preparations 

and histology of the treated groups of 

experimental animals qualitatively. 

RESULTS AND DISCUSSION 
 
Data on experimental animals' body 
weight were used to determine whether 
the extract treatment affected the 
experimental animals' weight. In addition, 
one of the symptoms of toxicity can be 
weight loss8. Based on the results of body 
weight observations, a graph of the 
experimental animal weight's average was 
created to illustrate the experimental 
animals' changes in body weight for 14 
days (Figures 1 and 2).



Nofran Putra Pratama, Kurnia Rahayu Purnomo Sari, Ririn Irma Marliana | Acute Toxicity Study of the Combination of 
Azadirachta indica A. Juss. and 

Gynura procumbens (Lour.) Merr. Leave Extracts  
 

59 

Figure 1. A graph of the average weight of the experimental animal after the 
administration of test preparation of 300 mg/kg BW for 14 days of observation.

  

   
Figure 2. A graph of the average weight of the experimental animal after the 

administration of test preparation of 2,000 mg/kg BW for 14 days of observation 
 
Based on the experiment's weight average 
figure after administrating the test 
preparation, there was no decrease in the 
experimental animals' weight. It indicates 
one of the toxic symptoms, namely weight 
loss, was not found. The difference in 
weight between each treatment might be 
due to each experimental animal's amount 
of food consumed. The amount of food 
consumed was different as the amount of 
feed given was not weighted. 

Dosage for the Azadirachta Indica A. Juss 
test was 50 mg/kg BW and 112.5 mg/kg 
BW was Gynura procumbens (Lour.) Merr3. 
For the essential dosage of acute toxicity, 
the selection of the dosage was carried out 
based on the 423 procedures of OECD 
Guideline consisting of dosages of 5, 50, 
300, and 2,000 mg/kg BW, as well as 
additional tests with a dose of 5.000mg/kg 
BW when it was needed and possible7. The 
initial dose for the test preparation used in 

0

50

100

150

200

250

1 2 5 8 11 14

W
e

ig
h

t 
(g

ra
m

)

Control

Solvent control

Gynura procumbens
extract

Azadirachta Indica
extract

combination extract of
both



Journal of Fundamental and Applied Pharmaceutical Science, 1(2), Febuary 2021 60 

this study was 300 mg/kg BW. According 
to the 423 procedures of the OECD 
Guideline, the initial dose for the test 
preparation was unknown, or there was no 
information related to its toxicity that was 
300 mg/kg BW. The test preparation was 
administrated at an initial dose of 300 
mg/kg BW to 3 tested animals per group. 
Upon the treatment with this dosage, no 

toxic symptoms and death signs were 
found in the experimental animals. The 
experiment was then continued at a dose 
of 2.000 mg/kg BW, and the condition of 
the experimental animals was re-
observed. Up to 14 days after the 
treatment, there were no toxic symptoms 
and death signs in the experimental 
animals.  

 
Table 1. The number of dead animals in the observation for 14 days after the 

administration of the test preparations at a dose of 300 mg/kg BW and 2,000 mg/kg BW 

Treatment 
Group 

Number of 
Early Rats 

Number of Final 
Rats, the dosage 
of 300 mg/kg BW 

Number of Final 
Rats, the dosage of 

2,000 mg/kg BW 

LD50 cut 
off 

(mg/kg 
BW) 

Dead  Live Dead Live  

Control 3 0 3 0 3  

Solvent 
Control  

3 0 3 0 3  

Gynura 
Procumbens 
extract  

3 0 3 0 3 >2,000 

Azadirachta 
indica extract 

3 0 3 0 3  

Combination 
extract of 
both 

3 0 3 0 3  

Total 15 0 3 0 3  

Table 1 shows that administering the test 
preparation at a 300 mg/kg BW dose did 
not cause death in the experimental 
animals until the 14th day. Based on these 
results, the potential for acute toxicity or 
LD50 cut-off of the test preparation 
administered to female Wistar rats falls 
into category 5 or unclassified category7. 
The 423 procedures of the OECD Guideline 
were used to calculate the LD50 cut-off 
because this method was relatively simple; 
the number of experimental animals used 
was small, and the test results' validity was 
not inferior compared to other methods. 

 
Observation of toxic symptoms is one of 
the qualitative measures of toxicity. Based 
on the observed toxic symptoms, it did not 
display any toxic symptoms appearing 
after treatment. Hence, it can be 
perceived that the test preparations 
administered either single or in 
combination between the extracts of 
Azadirachta Indica A. Juss. and Gynura 
procumbens (Lour.) Merr. were declared 
safe in terms of acute toxicity.  
In the final stage, the experimental 
animals were sacrificed by means of neck 



Nofran Putra Pratama, Kurnia Rahayu Purnomo Sari, Ririn Irma Marliana | Acute Toxicity Study of the Combination of 
Azadirachta indica A. Juss. and 

Gynura procumbens (Lour.) Merr. Leave Extracts  
 

61 

dislocation. Prior to the surgery, rats' heart 
rate and breath were re-observed to 
ensure the experimental animal had 
actually died. Furthermore, the liver organ 
was taken. The reason for examining the 
liver is because the liver has various vital 
functions in the body, like neutralizing 
toxic substances, synthesizing serum 
protein, regulating nutrition and secreting 
bile salts9. The taken liver organs were 
then weighed and preserved in a 10% 
formalin solution. Observation of the 
experimental animal organs was carried 
out macroscopically and microscopically. 
Macroscopic observations were employed 

to determine any visible damage to the 
organs due to the test's provision that did 
not appear visually when the experimental 
animal was still alive. Based on the results 
of macroscopic observations, no damage 
and abnormalities were found after 
treatment. Based on the vision, there was 
no difference between the organs of each 
group. Furthermore, organ weighing was 
carried out to determine the effect of 
treatment on the experimental animal 
organs seen from their weight. The 
average results of each treatment showed 
no significant difference from the 
experimental animals' organ weights. 

 
Table 2. The weight of the experimental animal's liver 

No Treatment Rat Weight 
(gram) 

Average weight 

1 Control 1 5.6277  

2 7.5499 7.1289 

3 8.2091  

2 Solvent Control 1 7.6987  

2 6.2049 7.3045 

3 8.0099  

3 Gynura Procumbens extract 1 10.3372  

2 7.6726 8.8553 

3 8.5562  

4 Azadirachta indica extract 1 7.2171  

2 7.2091 7.4488 

3 7.9202  

5 Combination extract of both 1 5.6412  

2 7.1454 7.2778 

3 9.0470  

The formalin solution's liver organs were 
then transferred into histopathological 
preparations at the Pathology Laboratory 
of Balai Besar Veteriner, Wates, 
Yogyakarta. The dye used was 
Hematoxylin-Eosin (HE). HE is a common 
coloring compound used for cells and 

tissues. Hematoxylin would color the cell 
nucleus blue, while Eosin would color the 
cytoplasm and extracellular matrix red. 
The results of observations on the liver are 
as follows:  
 



Journal of Fundamental and Applied Pharmaceutical Science, 1(2), Febuary 2021 62 

 
Figure 3. Histopathological microscopic profile of the rats’ liver of the dosage of 2.000 

mg/kg BW 
 

The histological profile of normal treated 
rats, namely the group of rats without 
administering the test preparation, 
includes A) The cell nucleus structure is 
located in the middle and shows a radial 
arrangement pattern. (40 x 10) (Figure 
3.1). The histological profile of the solvent-
treated rats was a group of rats that were 
given distilled water and 0.5% Na-CMC. A) 
The structure of the cell nucleus is located 
in the middle, B) Sinusoid (40 x 10) (Figure 
3.2). The rats' histological profile was 
treated with the ethanol extract of leaves 
of longevity spinach (Gynura procumbens 
(Lour.) Merr.). A) The nucleus is located in 
the center, B) Dilation of the central vein, 

C) Sinusoid. (40 x 10) (Figure 3.3). 
Histological profile of rats treated with 
ethanol extract of neem leaves 
(Azadirachta Indica A. Juss.). A) The 
nucleus is located in the middle, B) Dilation 
of the central vein, C) Fat hepatocytes, D) 
Sinusoids (40 x 10) (Figure 3.4). 
Histological profile of rats treated with a 
combination of ethanol extracts of neem 
leaves (Azadirachta Indica A. Juss.) and 
leaves of longevity spinach (Gynura 
procumbens (Lour.) Merr.), A) Dilation of 
the central vein, B) Sinusoids (40 x 10) 
(Figure 3.5). 
 

 
Table 2. The score of Hepatocyte Damage 

Test 
Hepatocyte Damage (%) 

Total 
Normal Inflammation Degradation Necrosis 

Control 60 0 0 0 60 

Solvent Control 52 0 0 0 52 

Gynura Procumbens 
extract 58 22 0 4 84 

Azadirachta indica extract 58 20 0 4 82 

Combination extract of 
both 58 14 0 8 80 

 
Determination of liver damage score is a 
modification of Manja Roenigk’s scoring 
histopathology. The interpretation of the 

histological preparations on the liver 
organs of the experimental animals did 
not show any damage to the cells in the 



Nofran Putra Pratama, Kurnia Rahayu Purnomo Sari, Ririn Irma Marliana | Acute Toxicity Study of the Combination of 
Azadirachta indica A. Juss. and 

Gynura procumbens (Lour.) Merr. Leave Extracts  
 

63 

normal and solvent group. The control 
group showed the highest percentage of 
the normal cell than the other groups. In 
the group treated with the ethanol extract 
of Gynura procumbens (Lour.) Merr., there 
was no liver cell damage, indicating that 
there were only a few widenings of the 
central veins. It was probably due to the 
high content of flavonoids in the extract. In 
the group treated with the ethanol extract 
Azadirachta Indica A. Juss., it did not show 
any significant damage and only a few 
widenings of the central veins leading to 
inflammation, and a little fatty hepatocyte 
was shown as empty vacuoles in the 
cytoplasm of the liver cells. This fatty 
process was reversible and caused by an 
inhibited release of lipid transfer from 
inside cells and an imbalance in synthesis 
and the release of triglycerides carried by 
parenchyma cells into the circulation10. In 
the combination treatment group, 
Azadirachta Indica A. Juss's extract and 
leaves of Gynura procumbens (Lour.) Merr. 
was seen to widen the central vein. In all 
treatments except the normal treatment 
group, several sinusoids were indicated by 
the presence of a flat and dark cell nucleus 
with little cytoplasm. 
 
In the experimental animals' liver cells, the 
possibility of necrosis was indicated by the 
destruction or loss of the nucleus (cell 
nucleus). However, the amount of necrosis 
found was not too much, and no tested 
animals had died during the treatment. 
Moreover, in general, there was no 
significant damage to the liver of the 
experimental animal. It was likely due to 
the presence of flavonoid and phenolic 
compounds in both extracts. Hence, these 
compounds can act as antioxidants that 
can minimize toxin compounds' harmful 
effects1. 
 
 
 

CONCLUSION 
 
The test compound of a combination of 
the ethanol extract of Azadirachta Indica 
A. Juss. and Gynura procumbens (Lour.) 
Merr. after acute toxicity test with the 423 
procedures of OECD up to a dose of 2,000 
mg/kg BW did not show any death or signs 
of toxicity in the experimental animals; 
hence, the combined extract was 
categorized as unclassified category 
according to GHS. 
 
ACKNOWLEDGMENT 
 
We would like to thank the Faculty of 
Health, Jenderal Achmad Yani University 
Yogyakarta, for funding this research 
 
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