TOXICITY PROFILE OF CELASTRUS PANICULATUS SEEDS: A PRECLINICAL STUDY

Abstract

Objective: The objective of the study was to evaluate the toxicity profile of Celastrus paniculatus (CP) by performing a preclinical study on Swiss albino mice and demonstrate a safety description through monitoring their autonomic, neurological, behavioral, physical, and biochemistry profiles. Methods: The toxicity profiles (acute and subacute) of CP were evaluated using Swiss albino mice in which they were divided into four groups: Group I received 1% Tween 20 and dimethyl sulfoxide. Group II, III, and IV received CP seed oil orally, at doses of 300, 2000, and 5000 mg/kg body weight for both acute and subacute toxicity studies in accordance with Organization for Economic Cooperation and Development guidelines No. 423. Special attention was given during the first 4 h and daily thereafter for a total of 14 days. Behavioral profile, physical state changes, and other parameters such as tremors, convulsion, lethargy were noted. Clinical signs were observed daily during the 28 days of the treatment period. Body weights were measured once a week. On the 29th day, the animals were kept to overnight and blood samples were collected through retro-orbital puncture for biochemical analysis. Results: In both acute and subacute toxicity studies, the treatment with CP did not affect the normal health status of animals. It is suggestive that CP is considered practically non-toxic. Conclusion: The toxicity profile of CP seed oil was evaluated and found to be safe until 2000 mg/kg dose.

Full text

Vol 13, Issue 7, 2020
Online - 2455-3891
Print - 0974-2441
TOXICITY PROFILE OF CELASTRUS PANICULATUS SEEDS: A PRECLINICAL STUDY
BHARAT MISHRA*, ELEZABETH JOHN, KRUPAMOL JOY, BADMANABAN R, ALEESHA R
Department of Pharmacology, Nirmala College of Pharmacy, Ernakulam, Kerala, India. Email: bharatekansh@gmail.com
Received: 28 March 2020, Revised and Accepted: 08 May 2020
ABSTRACT
Objective: The objective of the study was to evaluate the toxicity profile of Celastrus paniculatus (CP) by performing a preclinical study on Swiss
albino mice and demonstrate a safety description through monitoring their autonomic, neurological, behavioral, physical, and biochemistry profiles.
Methods: The toxicity profiles (acute and subacute) of CP were evaluated using Swiss albino mice in which they were divided into four groups: Group
I received 1% Tween 20 and dimethyl sulfoxide. Group II, III, and IV received CP seed oil orally, at doses of 300, 2000, and 5000 mg/kg body weight
for both acute and subacute toxicity studies in accordance with Organization for Economic Cooperation and Development guidelines No. 423. Special
attention was given during the first 4 h and daily thereafter for a total of 14 days. Behavioral profile, physical state changes, and other parameters
such as tremors, convulsion, lethargy were noted. Clinical signs were observed daily during the 28 days of the treatment period. Body weights were
measured once a week. On the 29th day, the animals were kept to overnight and blood samples were collected through retro-orbital puncture for
biochemical analysis.
Results: In both acute and subacute toxicity studies, the treatment with CP did not affect the normal health status of animals. It is suggestive that CP
is considered practically non-toxic.
Conclusion: The toxicity profile of CP seed oil was evaluated and found to be safe until 2000 mg/kg dose.
Keywords: Celastrus paniculatus, Acute toxicity, Subacute toxicity, Treatment, Safety.
INTRODUCTION
Herbal plants have been used from ancient times for the treatment of
several diseases in the indigenous system of medicine [1]. Celastrus
paniculatus (CP) Wild. (Family: Celastraceae) [2], commonly known as
Malkangni (in Hindi) or Jyotishmati (in Sanskrit) and commonly known
as black oil plant, climbing staff tree (in English) [3] is a long established
medicinal plant which has been used extensively in the Ayurvedic
system for its recognized analgesic, anti-inflammatory, and marked
central effects such as memory boosting and antiepileptic effects [4].
The use of the plant parts in the treatment of several ailments can be
attributed to the potential effects of its various phytoconstituents. The
seeds of the plant when extracted with petroleum ether yield dark
brown oil known as Celastrus oil or Malkangni oil are known to have
an effect on the central nervous system, mainly the memory-enhancing
activity and stimulating effects [3].
The neuropharmacological effects of this herb are striking and the
seeds have been reported to possess antidepressant [5], anxiolytic [6],
antioxidant [7], hypolipidemic, anti-atherosclerotic, anti-stress, anti-
spermatogenic, nootropic activities, and relaxant effects on smooth
muscles [8]. Celastrus oil therapy in mentally retarded children results
in an improvement in their intelligence quotient as it contains a number
of fatty acids such as oleic, linoleic, linolenic, palmitic, stearic, benzoic,
and acetic acid as volatile acids and their glycerol esters mainly α,
α’ dipalmitoyl glycerol. They also contain sesquiterpene alkaloids –
celapanin, celapanigin celapagin, and malkangunine [9].
Toxicology is an important aspect of pharmacology that deals with the
adverse effects of bioactive substances on living organisms before the
use as drug or chemical in clinical use. Plants or drugs must be ensured
to be safe before they are employed as medicines because the potential
toxicity of herbal plants has been recorded [10]. A key stage in ensuring
the safety of drugs is to conduct toxicity tests in appropriate animal
models.
Although animal toxicity studies of the CP seeds have not yet been
established before, it is widely used because of its many beneficial
effects and this research is focused on establishing the safety profile of
the plant so that they may be credibly employed for various therapeutic
applications in medicine.
METHODS
Plant collection and extraction
CP (greenwood essential) seed oil was purchased from a registered,
authentic source. The seed oil is administered orally as o/w emulsion,
prepared by the wet gum method.
The vehicle employed was phosphate buffer saline, prepared using 4
g of sodium chloride, 100 mg of potassium chloride, 0.72 g of sodium
hydrogen phosphate, 120 mg of potassium hydrogen phosphate, and
500 ml distilled water. All the components were initially dissolved in
400 ml distilled water, then the pH was adjusted to 7.4 and was diluted
to 500 ml with distilled water [11].
Phytochemical screening
Phytochemical screening for carbohydrate, protein, amino acid,
alkaloid, tannins, steroid, terpenoid, volatile oil, glycoside, and fixed oil
had been carried out.
Preparation of 100 ml CP o/w emulsion by wet gum method
The proportion of oil:water:gum for preparing primary emulsion is
4:2:1. The required quantity of acacia was weighed and powdered in
a dry and clean glass mortar. To this, 28.56 ml water was added to the
powdered acacia slowly with trituration to form a smooth mucilage
followed by the addition of 0.28 ml CP seed oil and triturated to obtain
an even mixture. Then, 56.84 ml coconut oil was introduced in small
portions with rapid trituration until a clicking sound was produced
and the emulsion becomes white or nearly white to form the primary
emulsion after which more water in small portions to the primary
© 2020 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.
org/licenses/by/4. 0/) DOI: http://dx.doi.org/10.22159/ajpcr.2020.v13i7.37803
Research Article

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Mishra et al.
emulsion with trituration to produce the required volume. Next, the
mixture was stirred thoroughly so as to form a uniform emulsion. It was
then transferred and stored in a cool and dry place [12].
Experimental animals
Ten weeks old healthy Swiss albino mice were selected in this study
(n=4), weighing around 20–25 g was purchased from disease-free
small animal house, Kerala Veterinary College, Mannuthy. Animals were
housed separately in groups of 4 per cage under laboratory conditions
with alternating light and dark cycle of 12 h each having free access
to food and water. The animals were kept fasting 2 h before and 2 h
after drug administration. The animals were acclimatized for at least
5 days before the actual dosing schedule started between 09:00 and
17:00 h. The laboratory animals for using in this experimental study
were approved by the Institutional Animal Ethics Committee of NCP
(Registration No. 1411/PO/Re/S/11/The Committee for the Purpose
of Control and Supervision of Experiments on Animals).
Acute toxicity study
Acute oral toxicity study for the extract was conducted in accordance
with the Organization for Economic Cooperation and Development
(OECD) guidelines No. 423. The female mice were used for the study.
Sixteen female mice were divided into four groups, with each containing
four animals. Group I received 1% Tween 20 and dimethyl sulfoxide.
Group II, III, and IV received CP seed oil orally, at doses of 300, 2000, and
5000 mg/kg body weight, respectively. After dosing, each animal was
observed carefully, at least once during the first 30 min, periodically
during the first 24 h. Special attention was given during the first 3 h
and daily thereafter for a total of 14 days [13,14]. Behavioral profile
(alertness, restlessness, irritability, and fearfulness), autonomic profile
(defecation and urination), neurologic profile (locomotion, reactivity,
touch, and pain response), physical states such as changes in skin, fur,
eyes, mucous membranes, including respiratory, circulatory, central
nervous systems, and somatomotor activity, and any lethality or death
were also considered during observation. Animals were also carefully
observed for tremors, convulsions, salivation, diarrhea, lethargy, sleep,
and coma. The body weight, food, and water intake were monitored
weekly.
Subacute toxicity study
The repeated dosing in oral toxicity study has been conducted as the
bedside observation as per the OECD 407 for 28 days. Albino young,
healthy male mice and non-pregnant female mice were used in this
study. Animals were divided into four groups with four animals in each
group (n=16; 8 female and 8 male). Group I received 1% Tween 20 and
dimethyl sulfoxide. Group II, III, and IV received CP seed oil orally at
doses of 300, 2000, and 5000 mg/kg body weight, respectively. Clinical
signs were observed daily during the 28 days of the treatment period.
Body weights were measured once a week. On the 29th day, the animals
were kept to overnight and blood samples were collected via retro-
orbital puncture for biochemical analysis.
Mortality rate and sign of toxicity of CP
The observations after the 28 days study have been made to record the
rate of mortality and any other sign of toxicity than the signs observed
in acute and subacute toxicity studies for the groups received CP seed oil
orally at doses of 300, 2000, and 5000 mg/kg body weight, respectively.
Effect of CP on mean body weight changes in mice
The change in body weight is a very important and conclusive sign of
the toxicity caused to the animals by any of the doses. A dose with 10%
or more reduction in the body weights declared to be a toxic dose. The
animals treated with CP will be observed for their growth pattern and
body weight after oral administration of the 2000 mg/kg extract.
Statistical analysis
Data were presented as mean ± SEM. Two way ANOVA followed by
Tukey–Kramer post-tests was applied on GraphPad Prism version 5.
p<0.001 was considered statistically significant.
RESULTS
Preliminary phytochemical screening
Preliminary phytochemical screening of CP seed oil was performed and
the observations are summarized in Table 1:
Acute toxicity study
As per the OECD guidelines, to establish the safety and efficacy of a
new drug, toxicological studies are crucial in animals like mice, rat,
guinea pig, dog, rabbit, and monkey under various conditions of the
drug employed. Toxicological studies aid in deciding whether a new
drug should be adopted for clinical use or not. OECD guidelines – 401,
423, and 425 intercepts the use of drugs clinically without its clinical
trial and the related toxicity studies. Depending on the duration of drug
exposure to animals, toxicological studies are of three types: Acute,
subacute, and chronic toxicological studies.
The present study has been undertaken to estimate the toxic effects of
CP in Swiss albino mice (female) for a period of 14 days using OECD
423 (acute toxic class method) as per the method discussed above in
acute toxicity study. Their results and observations were recorded
accordingly [13,14].
Mortality rate and sign of toxicity of CP
Mortality rate and sign of toxicity of CP were carried out conforming to
OECD guidelines – 423 as per the method discussed above. No symptoms
of toxicity, morbidity, or mortality were noted in animals during the 14
days period following single oral administration at a selected dose level
of CP (2000 mg/kg). The CP oil was safe until 2000 mg/kg body weight
and its lethal dose would be greater than that of the test doses. The
results were summarized in Tables 2 and 3:
Effect of CP subacute toxicity study
The repeated dosing oral toxicity study been conducted for 28 days. The
effect of CP on cage-side observations was carried out as per the method
discussed in subacute toxicity in the study. After oral administration
of vehicle and extract, animals were observed continuously during
the first 30 min after dosing and observed periodically (with special
attention given during the first 4 h) for the next 24 h and then daily
thereafter, for 28 days. All observations were systematically recorded,
with individual records being maintained for each animal. Observations
include changes in skin and fur, eyes, mucous membrane, and
behavioral pattern. Attention was given for monitoring of tremors,
convulsions, salivation, diarrhea, lethargy, sleep, coma, and mortality.
Further individual body weights of animals were recorded before the
Table 1: Observations of the preliminary phytochemical
screening
Test Observation
Test for alkaloids
a. Mayer’s test Negative
b. Dragendorff’s test Negative
c. Wagner’s test Negative
Test for carbohydrates
a. Molisch’s test Negative
b. Benedict’s test Negative
c. Fehling’s test Negative
Test for proteins
a. Millon’s test Negative
b. Biuret test Negative
Test for saponins glycoside
Foam test Negative
Test for tannins Negative
Test for flavonoids
Shinoda test Negative
Test for steroids
Liebermann–Burchard reaction Positive
Test for terpenoids Negative
Test for glycosides Negative

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administration of drug on 1st day of the study and thereafter on the
7th and 14th day of the experiment. Changes in the weight of individual
animals were calculated. The results were summarized in Table 4:
Effect of CP on mean body weight changes in mice
A dose which produces 10% or more reduction in the body weights was
considered to be a toxic dose. The effect of CP on body weight changes
in mice was observed as per the method discussed above. The animals
treated with CP showed a normal growth pattern and body weight after
oral administration of 2000 mg/kg extract.
It indicates that the administration of the extract does not affect the
normal growth of the animals. The results were summarized in Fig. 1.
No significant changes were observed in body weight when compared
with control. The values are expressed as mean ± SD, n=4. The statistical
analysis was carried out using multiple t-test followed by Tukey’s
multiple comparison test.
DISCUSSION
Plant origin drugs are known to play a vital role in the management
of various chronic diseases and alternative sources to allopathic
pharmaceutical drugs in recent times. The herbal products ensure
safety in contrast to the synthetics that are regarded as unsafe to
humans and environment. However, the use of these products should be
based on the scientific origin; or else they may be futile and unreliable.
Furthermore, the irrational use of this phytotherapy may cause serious
toxicity in humans. Unfortunately, a large proportion of the population
of humans underestimates the toxicity of natural products and does not
realize that these agents could be as toxic or more than synthetic drugs.
Toxicity testing is salient in the screening of newly developed drugs
before it is marketed for human use. The guiding principles of toxicity
testing are to check the effect of the test substances on laboratory
animals and its direct toxic effect on humans and furthermore the
exposure of laboratory animals to high doses to evaluate its possible
hazard on humans that are exposed to a much lower dose.
Toxicity testing employs an array of test in different species of
animals with long-term administration of drug, regular monitoring of
physiological, biochemical abnormalities, and detailed post mortem
examination toward the end of the trial to detect gross or histological
abnormalities.
In acute toxicity studies, a single dose of the drug is given in large
quantities on a particular animal species to determine the immediate
toxic effect. It is used to determine LD50 of drugs or chemicals and
natural products.
As per the OECD guidelines, to establish the safety and efficacy of a
new drug, toxicological studies are crucial in animals such as mice, rat,
guinea pig, dog, rabbit, and monkey under various conditions of the
drug employed. Toxicological studies aid in deciding whether a new
drug should be adopted for clinical use or not. OECD 401, 423, and 425
intercepts the use of drug clinically without its clinical trial and the
related toxicity studies. Depending on the duration of drug exposure to
animals, toxicological studies are of three types: Acute, subacute, and
chronic toxicological studies.
The acute toxicity study of CP was carried out as per OECD – 423
guidelines. On the basis of literature reviews, it has been reported
that CP seems to be safe at a dose level of 2000 mg/kg, and the LD50 is
considered to exceed 2000 mg/kg [13,14]. A single dose of CP seed oil
when administered orally and observed for a period of 14 days did not
Table 4: Effect of Celastrus paniculatus subacute toxicity study at bedside
Parameters Control Celastrus paniculatus
at 5 mg/kg
Celastrus paniculatus
at 50 mg/kg
Celastrus paniculatus at
300 mg/kg
CP at
2000 mg/k g
Skin and fur Normal Normal Normal Normal Normal
Eye lacrimation Normal Normal Normal Normal Normal
Salivation Normal Normal Normal Normal Normal
Diarrhea Nil Nil Nil Nil Nil
Lethargy Nil Nil Nil Nil Nil
Respiration Normal Normal Normal Normal Normal
Tremors Nil Nil Nil Nil Nil
Convulsions Nil Nil Nil Nil Nil
Coma Nil Nil Nil Nil Nil
Locomotor activity Nil Nil Nil Nil Nil
Excitement Nil Nil Nil Nil Nil
Other symptoms Nil Nil Nil Nil Nil
Morbidity Nil Nil Nil Nil Nil
Mortality Nil Nil Nil Nil Nil
Table 2: Observations of acute toxicity study
S. No. Parameters Observations
2 h 12 h 24 h 72 h
1 Behavioral profile
a. Alertness Normal Normal Normal Normal
b. Restlessness Normal Normal Normal Normal
c. Irritability Normal Normal Normal Normal
d. Fearfulness Normal Normal Normal Normal
2 Autonomic profile
a. Defecation Normal Normal Normal Normal
b. Urination Normal Normal Normal Normal
3 Neurologic profile
a. Locomotion Normal Normal Normal Normal
b. Reactivity Normal Normal Normal Normal
c. Touch response Normal Normal Normal Normal
d. Pain response Normal Normal Normal Normal
4 Physical profile
a. Texture of fur Normal Normal Normal Normal
b. Nasal secretions Normal Normal Normal Normal
c. Ear secretions Normal Normal Normal Normal
d. Color and texture
of feces
Normal Normal Normal Normal
5 Lethality Absent Absent Absent Absent
Table 3: Mortality rate and sign of toxicity of Celastrus
paniculatus
Groups Dose (mg/kg) Sign of toxicity
(ST/NB)
Mortality
(D/S)
Group I 5 mg/kg 0/3 0/3
Group II 50 mg/kg 0/3 0/3
Group III 300 mg/kg 0/3 0/3
Group IV 2000 mg/kg 0/3 0/3
The values are expressed as number of animals (n=4), where ST: Sign of toxicity,
NB: Normal behavior, D: Animals died, S: Animals survived

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exhibit any mortality or toxic symptoms (Table 2). Acute oral toxicity
studies aid in evaluating the intrinsic toxicity of the substances and
effect on the living tissues [15].
Cage side observations such as behavior, breathing, skin fur,
water consumption, and food intake were found to be normal. All
treated animals were in live condition after the administration
of CP. Mortality was absent in both treatment and control groups
(Table 2). This indicates that there were no disturbances in
carbohydrate, protein, or fat metabolism. All the treated mice did
not exhibit any significant weight loss throughout the experiment,
suggesting that the animals were free from wasting syndrome. They
presented normal growth pattern and body weight which is similar
to the normal control. Decrease or increase in the body weights was
associated with toxic effects of chemicals and drugs accompanied
with the accumulation of fats and physiological adaptation
responses to the plant extracts rather than to the toxic effects of
chemicals or drugs that lead to decrease appetite and, hence, lower
caloric intake by the animal. Thus, the treatment with CP did not
affect the normal health status of animals. It is suggested that CP is
considered safe or practically non-toxic. Any pharmaceutical drug
or compound with the oral LD50 higher than 1000 mg/kg could be
considered safe and low toxic. This suggests that CP is practically
non-toxic in a single dose level of 2000 mg/kg body weight [13,14].
These results further open the scope for further research on the
effects of the plant at the genetic level [16].
CONCLUSION
The acute toxicity study of CP was carried out as per OECD – 423
guidelines. On the basis of literature reviews, it has been reported
that CP seems to be safe at a dose level of 2000 mg/kg, cage side
observations indicated that there were no signs of toxicity or changes
in physical appearances such as skin, fur, eyes, mucous membrane,
behavioral pattern, salivation, and sleep of the treated as well as the
control animals were found to be normal. Tremors, lethargy, diarrhea,
coma, and lethality did not occur in any of the animal at the end of 14
and 28 days of the observation period.
AUTHORS’ CONTRIBUTIONS
Conceptualization: Bharat Mishra, Data Collection: Elezabeth John,
Krupamol Joy, and Bharat Mishra Formal Analysis: Badmanaban R,
Aleesha R, and Bharat Mishra Funding Acquisition: Bharat Mishra
and Elezabeth John, Methodology: Badmanaban R, Aleesha R, and
Bharat Mishra Project Administration: Bharat Mishra and Elezabeth
Visualization: Bharat Mishra and Elezabeth Writing – Original Draft:
Bharat Mishra, Elezabeth, and Krupamol Joy Writing – Review and
Editing: Bharat Mishra, Elezabeth, and Krupamol Joy.
CONFLICTS OF INTEREST
The authors declared no conflicts of interest.
AUTHORS’ FUNDING
The project was self-funded.
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Fig. 1: Effect of Celastrus paniculatus on mean body weight
changes in mice