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International Journal of Toxicological and Pharmacological Research 2010; 2(2) ISSN: 0975-5160
Research Article
Acute and Sub acute Toxicity Study of Ayurvedic Formulation
(AYFs) Used for Migraine Treatment
Prakash Vaidya Balendu 1*, Saraf Madhusudan 2, Chandurkar Nitin 3
1 Ipca Traditional Remedies Pvt. Ltd., Mumbai, India
2 Bombay College of Pharmacy, Mumbai, India
3 Medical Affairs and Clinical Research, Ipca Laboratories Ltd., Mumbai, India
ABSTRACT:
Propose: A combination of five classical ayurvedic formulations (Narikela Lavana, Sootashekhara Rasa, Sitopala
d
Churna, Rason Vati and Godanti Mishran) has been employed as prophylactic remedy for migraine. These ayurvedi
c
formulations (AYFs) contain certain Bhasma and plant materials. An investigation was initiated to evaluate safety profil
e
of these AYFs in Sprague Dawley rats and Swiss Albino mice following OECD guidelines. Material and Metho
d
Acute toxicity studies were done after ingestion of 5 g/kg of AYFs in a day in both the animal species. Sub acute toxicit
y
studies were carried in five different groups in which AYFs was administrated in various doses ranging from 1.47 – 6.4
8
g/kg for mice and 0.7 – 7.45 g/kg for rats. The highest dose were 10 times higher that the recommended human dos
e
Detailed hematological, biochemical, necropsy and histopathological evaluation of organs was performed for all animal
s
Results: The AYFs was well tolerated and no toxic manifestations were seen in any animal. Mortality observed in hig
h
dose groups; 4% in rats and 6% in mice was not related to treatment. Conclusion: The AYFs was found to be safe i
n
animals. However, chronic toxicity studies are required to know the long term safety of these AYFs.
INTRODUCTION
In the recent years complementary and alternative
medicine (CAM) has upsurge globally for the treatment
and prevention of many aliments which are non-
communicable and chronic in nature 1. Most surveys agree
that herbal remedies are amongst the most prevalent
therapies and that headache/migraine is one of the most
frequent reasons for trying plant-derived medications 2.
CAM is often perceived by the public to be more helpful
than conventional care for the treatment of headache 3.
However, there is always apprehension about their safety,
efficacy, toxicity and reproducibility of CAM therapies.
Ayurveda the traditional system of medicine of India
was initially taught and practiced in a Guru-Shisya
Parampara has now been institutionalized. Ayurveda is
largely practiced in India under the patronage of Central
and Provincial Government. An Ayurvedic Treatment
Protocol (AYTP) developed by the principal author was
tried for migraine treatment with encouraging results 4, 5
this AYTP consist of a herbo-mineral combination of five
classical ayurvedic formulations (Narikela Lavana,
Sootashekhara Rasa, Sitopaladi Churna, Rason Vati and
Godanti Mishran) 6 along with regulated diet and lifestyle
modification. The same AYTP was used by other
ayurvedic physicians with similar results7.
Medicines of AYTP were derived from Rasa Sashtra
(the science of Ayurvedic Pharmaceutics) and contain
substances which are moderate to severely toxic in the raw
form. Substances of plant, animal and mineral origin are
routinely used in manufacturing of ayurvedic medicines 8.
However, the intrigue phenomenon of its manufacturing
converts these into complex mineral forms which are
effective and non toxic. However, improper
processing/manufacturing of ayurvedic medicines may
result into severe toxicity 9. Recently, heavy metal
contamination was also reported in some ayurvedic
medicines sold in USA 10. This raised concerns regarding
safety of such products for human use as medicines.
Hence, the present investigation was undertaken to assess
the safety profile of the ayurvedic formulations (AYFs)
used in the AYTP for migraine treatment in animal models
using OECD guidelines.
MATERIALS AND METHODS
Animals: Six to eight weeks old male Sprague Dawley rats
weighing 170 to 210 gms and female rats weighing 150 –
170 gms and Swiss Albino mice of either sex weighing
between 18 - 22 gms were selected for the present study.
The animals were kept in polypropylene cages with
stainless lid with rice husk bedding. Individual animal was
identified by specific marking and cages were identified
with label pasted on cages with relevant information.
Animals were housed at a temperature of 24 ± 2oC and
relative humidity of 30 to 70 %. A 12:12 light: dark cycle
was followed. All animals had free access to water and
standard pelleted laboratory animal diet. The animals were
acclimatized for 7 days before starting the experiment.
Ethical Clearance: This study was approved by the
Institutional Ethics Committee of the Bombay College of
Pharmacy, Mumbai.
*Author for Correspondence: balenduprakash@gmail.com
Study Drugs: The recommended daily human dose of
AYFs is 7.3 gm/day. The break up of which as follows:
Narikela Lavana 2.0 gm; Sootshekhara Rasa 0.375 gm;
Sitopiladi Churna 1.425 gm; Rason Vati 3.0 gm; Godanti
Mishran 0.5 mg. The ayurvedic formulations were
manufactured at Bharat Bhaishajya Shala Pvt Ltd,
Dehradun, India. Based on the doses of these individual
formulations the equivalent animal dose were calculated
for the mixture of AYFs in the same proportion these are
used in human being.
Dose administration: AYFs were triturated to get fine
powder and were mixed uniformly in the required
proportion. The suspension for low, medium and high
doses were prepared in 0.5% Carboxy Methyl Cellulose in
distilled water and administered to animals orally with the
help of gastric catheter.
Acute Toxicity study: Six female rats and mice were
orally administered 5 g/kg suspension of the mixture of
AYFs in three divided doses, at an interval of 30 minutes
in a day. The equivalent human dose per day of these
AYFs in rat and mice was calculated to be 0.7 g/kg and
1.47 g/kg respectively. The dose administered in rat and
mice was approximately 7 times and 3.4 times higher than
the normal human dose respectively. The animals were
observed for first 4 hours of treatment to next 14 days. The
evaluated parameters were mortality, signs and symptoms
of toxicity, body weight, food consumption and necropsy
observations.
Sub Acute Toxicity study: The animals were divided into
5 groups. There were 10 animals (5 males and 5 females)
in each group. The group I served as vehicle control.
Group II, III and IV received low, medium and high dose
of medicine respectively for 28 days. All the animals of
group I – IV were sacrificed on 29th day. However, group
V received high dose medicine for 28 days, and then they
were further observed for next 2 weeks and sacrificed on
43rd day. The dose was calculated taking into account the
difference in surface area: body weight ratios between
species. The details of dosing are given in the box below:
Study parameters: Toxic manifestations like alteration in
water or food intake, weight loss, respiration pattern,
mobility, response to handling, salivation, piloerection,
bizarre behavior were studied on day 0 and every week
thereafter. Audio-visual reflexes and grip strength
determination was performed in all animals at 4th week of
study. Opthalmological observation was done on day 1 and
day 28. Parameters like pupil size, redness of eye and
lacrimation were checked and scored. Blood samples were
collected from all animals after terminal sacrifice.
Hematological and biochemical parameters viz.
hemoglobin (Hb), red blood corpuscles (RBC), white
blood corpuscles (WBC) and platelets (Plt), reticulocyte
(Reti), packed cell volume (PCV), prothrombin time (PT),
mean corpuscular volume (MCV), mean corpuscular
hemoglobin (MCH), mean corpuscular hemoglobin
concentration (MCHC) , Alanine transaminase (SGPT),
Aspartate transaminase (SGOT), alkaline phosphatase
(ALP), total protein (TP), albumin (ALB), blood urea
nitrogen (BUN), creatinine (CRE), random blood sugar
(RBS), sodium (Na+), potassium (K+), calcium (Ca++),
phosphorus (P), chloride (Cl) and cholesterol (CHO).
Biochemical parameters were studied using commercially
available kits, Span autochem 2011 autoanalyzer and flame
photometry.
Necropsy: Body orifices and organs of all animals were
carefully observed after dissection for morphological and
pathological changes. Wet weight of liver, kidney, spleen,
brain, heart, adrenals and gonads (testes/ovaries) were
recorded for all animals.
Histopathology: Tissue samples from various organs of all
animals were preserved in 10% formalin saline and were
studied for pathological changes.
Statistical analysis: The data was analyzed by using SPSS
software (version 12.0, SPSS, Chicago, IL, USA). The
results are presented as mean ± s.d. and statistical
significance between the groups was analyzed by means of
an analysis of variance (ANOVA).
RESULTS
Acute Toxicity study: There were no signs of any toxicity
in animals of both the species after the administration of
the test dose of 5 g/kg suspension of the mixture of AYFs.
All the animals showed similar food intake, body weight
gain and clinical signs as that of the control group. No
morbidity or mortality was observed in the treated animals.
The necropsy studies did not detect any abnormality.
Sub Acute Toxicity study: The animals from both the
species showed normal body weight gain throughout the
dosing period. No significant change in weight was noted
in any of the groups. There was no difference in the food
intake noted in rats. However, significant reduction in food
consumption in mice was noted from second week of study
in the medium dose, high dose and satellite treatment
group in comparison to control group.
One rat each from group IV and V died on 4th and 5th
week of study. Three mice died during the study period.
Two mice from group IV and V died in the 1st week of
study. One mouse died at the 4th week of treatment from
group V.
Rat
Mice
Group Dose
(g/kg body
weight/ day)
Treatment
(days) Day of
sacrifice
I Control -- -- 28 29
II Low 0.7 1.47 28 29
III Medium 2.23 3.04 28 29
IV High 7.45 6.48 28 29
V High 7.45 6.48 28 43
54
Animals from all treated groups in both the species
showed a normal hematological profile except for the
MCH value of the medium dose group in rats (Table 1) and
satellite group in mice (Table 2) was significantly higher
from that of the control group. The reticulocyte count in
the satellite group was also significantly higher than the
control group in rats.
Animals from all treated groups in both the species
showed a normal biochemical profile including electrolyte
levels (Table 3, 4, 5 & 6) except for the albumin levels in
the satellite group in mice which was significantly higher
from that of the control group (Table 5).
Treated animals from both the species showed organ
weights as well as organ to body weight ratio comparable
to control group.
Table 1: Comparison of Group Mean Hematological Investigations in Rats
Group
Control
Low dose
Medium dose High Dose Satellite high dose
Hemoglobin
(gm/dl) 12.1± 1.0 12.4± 0.98 12.5 ± 0.96 12.9 ± 0.87 13.0 ±0.74
RBC
(x 106/cmm) 7.27 ±0.52 7.09 ±
0.58 6.83 ± 0.64 7.5 ±0.57 7.56 ± 0.53
WBC
(x 103 /cmm) 9.360 ± 2.249 8.950
±2.089 7.970 ±1.567 6.690
±2.060 8.090 ±1.491
Platelets
(x 105 /cmm) 9.472±6.8171 9.788 ±
4.738 10.242±5.859 10.120±
1.206 9.094±5.607
Reticulocytes
(%) 2.46 ±0.49 2.79 ±
0.78 2.56 ± 0.55 2.24 ± 0.69 1.36 ± 0.56*
PCV
(µ m3) 37.7 ± 3.84 37.6 ±
4.03 38.0 ± 3.33 39.8 ± 2.88 41.2 ± 3.47
MCV
(µ m3) 52.5 ± 3.6 52.9 ±3.33 55.6 ± 2.75 52.6 ± 4.01 55.3 ± 2.46
MCHC
(gm/dl) 32.5 ± 2.03 33.0 ±
0.95 33.1 ± 0.96 32.4 ± 1.24 31.6 ± 1.03
MCH
(pg) 16.7 ± 0.89 17.5 ±
0.97 18.4 ± 1.11* 17.2 ± 1.38 17.5 ± 0.76
Values are mean of 10 animals ± S.D.
* Significant at 95 % level of confidence (p<0.05) vs. control group.
Table 2: Comparison of Group Mean of Hematological Investigations in Mice
Group Control
Low dose
Medium dose Satellite high
dose
High Dose
Hb
(gm/dl) 13.87± 0.8 13.56± 0.6 14.56± 0.5 12.97± 3.3 14.18± 0.6
RBC
(x 106/) 9.2 ± 0.8 9.1± 0.5 9.3± 0.6 8.23±1.3 7.9± 0.6
WBC
(x 103 /cmm) 8.700 ±2740 7.580± 1452 8.210± 1733 8.240± 3714 9.550± 1676
Platelets
(x 105 /cmm) 7.970 ±4478 8.389± 7440 8.934 ±9531 8.547 ± 9856 7.992±19330
Reticulocytes
(%) 0.9±0.35 0.9± 0.31 1.0 ± 0.22 1.0 ± 0.22 1.0 ± 0.29
PCV
(µ m3) 45.4 ± 4.5 44.1 ± 3.9 45.8 ± 2.9 39.9 ± 9.8 42.1 ± 2.7
MCV
(µ m3) 50.15 ± 5.1 48.85 ± 3.6 49.0 ± 1.5 49.53 ± 6.3 52.8 ± 6.7
MCHC
(gm/dl) 30.4± 1.9 30.7 ± 2.0 31.91± 1.5 32.18 ± 0.4 32.7 ± 0.4
MCH
(pg) 15.2 ± 1.2 15.1 ± 0.6 15.6 ± 0.7 15.7 ± 1.9 17.6 ± 0.8*
Values are mean of 10 animals ± S.D.
* Significant at 95 % level of confidence (p<0.05) vs. control group.
55
Animals from high dose treatment group and satellite
group of both the species showed decreased motor activity
(reduced alertness, reduced exploratory behavior). The
effect lasted for approximately two hours post dose
administration. However, the animals responded
comparably to control group in the functional test on
retard. All the other treatment animals were found to be
free of any intoxicating sign.
Treated animals of both the species showed normal
ophthalmologic and audio-visual reflex at the end of study
period. Gross pathological examination of all animals did
not reveal any abnormality attributable to the treatment in
both the species. No significant histopathological changes
were noted in different organs that were examined.
DISCUSSION
In the present investigation we tried to assess the
safety profile of AYFs. No Adverse Effect Level
(NOAEL) could be established for all the dose range tested
in both the animal models. The higher dose range tested
was well above and at an adequate safety distance of the
recommended dose in humans. Though the animals treated
with high dose of medicines showed reduced alertness and
decreased motor activities, they responded comparably to
control group in the functional test on rotarod. It may be
assumed that the AYFs at high dose may have mild CNS
depressant activity. Mortality observed was not found to be
related to treatment.
Table 4: Comparison of Group Mean of Electrolyte
Levels in Rats
Group
Control
Low
dose
Medium
dose High
Dose
Satellite
high
dose
Na 132.4 ±
41.7 147.0
± 4.98 147.4 ±
5.39 147.7
± 3.97 145.9 ±
3.9
Ca 8.5 ±
2.54 9.58 ±
1.16 9.43 ±
1.22 9.25 ±
0.82 8.98 ±
1.6
P 5.5 ±
0.77 5.7 ±
0.5 6.1 ±
1.22 5.9 ±
1.32 6.8 ±
1.84
K 4.58 ±
0.36 4.83 ±
0.47 4.71 ±
0.47 4.74 ±
0.39 4.52 ±
0.43
Cl 105.8 ±
2.61 107.8
± 4.15 107.1 ±
5.56 107.1
± 5.56 107.3 ±
3.62
Values are mean of 10 animals ± S.D. No significant
difference was observed in any parameter.
The present investigation indicated that the ayurvedic
formulations did not produce any adverse toxicity in both
Table 3: Comparison of Group Mean of Biochemical Investigations in Rats
Group
Control
Low dose
Medium dose High Dose Satellite high dose
AST (IU/L) 143.5 ± 22.4 136.5 ± 8.7 144.6 ± 13.4 160.2 ± 18.8 127.6 ± 5.2
ALT (IU/L) 49.3 ±10.1 50.3 ±11.3 39.0 ± 6.9 43.0 ± 10.9 56.4 ± 11.8
ALP (IU/L) 388.3 ± 90.1 458.5 ± 84.6 333.4 ± 32.7 364.4 ± 87.1 394.1 ± 65.4
Protein (g/dl) 5.87 ± 0.35 5.86 ± 0.47 6.03 ± 0.82 6.18 ± 0.36 6.59 ± 0.36
Albumin (g/dl) 4.07 ± 0.37 4.7 ± 0.88 4.59 ± 0.83 4.26 ± 1.04 3.7 ± 0.44
BUN (mg/dl) 39.9 ± 10.2 37.0 ± 9.5 35.7 ±12.6 38.0 ± 9.01 28.9 ±8.02
CRE (mg/dl) 0.33 ± 0.27 0.71 ± 0.31 0.33 ± 0.41 0.43 ± 0.43 0.67 ± 0.72
RBS (mg/dl) 117.8 ± 30.5 129.1 ± 2.55 113.8 ± 16.5 121.2 ± 28.2 119.2 ± 25.3
CHO (mg/dl) 137.7 ± 4.1 145.6 ± 21.5 153.5 ± 46.7 152.6 ± 21.9 141.9 ± 25.7
BIL (mg/dl) 0.53 ± 0.14 0.5 ± 0.12 0.56 ± 0.17 0.45 ± 0.1 0.5 ± 0.13
Values are mean of 10 animals ± S.D. No significant difference was observed in any parameter.
56
Table 5: Comparison of Group Mean Biochemical
Investigations in Mice
Group
Control
Low
dose
Medium
dose High
Dose
Satellite
high
dose
AST
(IU/L) 95.5 ±
10.7 103.6
±
18.8
97.2
±16.86 160.6
±
17.1
93.1
±15.36
ALT
(IU/L) 44.4 ±
10.16 54.0
±
16.5
56.5 ±
13.9 52.0
±
23.7
53.0
±13.6
ALP
(IU/L) 286.5 ±
47.4 292.5
±
22.5
332.9 ±
42.9 295.4
±
46.5
283.8 ±
28.8
Protein
(g/dl) 7.01±
0.7 7.1 ±
0.6 6.94 ±
0.7 7.08
±0.7 6.97 ±
0.3
Albumin
(g/dl) 2.83 ±
0.4 2.75
± 0.3 2.85 ±
0.4 3.06
± 0.2 3.47 ±
0.2*
BUN
(mg/dl) 54.0 ±
20.1 53.3
±
11.4
65.1 ±
21.2 82.1
±
41.6
77.1 ±
13.0
CRE
(mg/dl) 1.5 ±
0.06 0.6 ±
0.45 1.2 ±
0.67 0.81
±
0.55
0.7 ±
0.41
RBS
(mg/dl) 124.2 ±
41.6 152.6
±36.0 121.8 ±
25.4 122.6
±
50.7
148 ±
27.2
CHO
(mg/dl) 124.5 ±
17.2 126.0
±
25.1
123.5 ±
22.0 131.6
±
22.2
120.2 ±
15.1
BIL
(mg/dl) 0.44 ±
0.1 0.92
±
1.43
0.48 ±
0.12 0.46
±
0.12
0.51 ±
0.16
Values are mean of 10 animals ± S.D.
* Significant at 95 % level of confidence (p<0.05) vs.
control group.
the animal models studied. There was no pathological
evidence of toxicity in kidney, liver, spleen, heart and
brain. Mortality observed in the treatment groups was
not found to be dose related. The animals that died did not
show any signs of morbidity and necropsy. The
histopathology findings did not indicated any toxic
changes in these animals.
The presence of metals and minerals in food /
ayurvedic medicines is a matter of great concern for human
health 11. The AYFs used in the present study is a
combination of 5 ayurvedic formulations derived from
Rasa Aushadi and contained herbs, metals and minerals.
However, classic Rasa text claims the intrigue processing
of metals and final composition of its formulations are safe
for human consumption and therapeutically effective. The
preliminary observational research indicates that AYFs are
significantly effective in the prevention of migraine and
need not produce any noticeably side effect among
migraineurs 4, 5.
Normally, heavy metals produce nephrotoxicity and
blood disorders. However, these heavy metals after
subjecting to specific and proper processing are
transformed into Bhasma12 and used in treatment of
various ailments and are considered as nontoxic in
prescribed dose. Bhasma along with appropriate herbs are
used for the treatment of critical ailments. The procedures
for preparing these medicines are stringent, time-
consuming and complicated 13. Metals are triturated and
burnt several times 14 with herbs juices / decoction thereby
converting them into non toxic form and suitable for
clinical usage. It is estimated that 35% to 40% of the
approximately 6000 medicines in the ayurvedic formulary
intentionally contain at least one metal. Metal-containing
herbal medicine products are purportedly "detoxified"
through multiple heating/cooling cycles and by the
addition of specific herbs 15.
Ayurvedic is largely practiced using ancient protocols
and parameters. Though there is a need of scientific
scrutiny of its principles of treatment, very few attempts
have been made for its scientific and systemic validation.
The present study indicates that the AYFs that are clinical
used are also safe in animals in dose that was 4 - 10 times
higher the human equivalent dose. However, further
studies are required to know the long term chronic toxicity
of these AYFs.
Table 6: Comparison of Group Mean of Electrolyte
Levels in Mice
Group
Control
Low
dose
Medium
dose High
Dose
Satellite
high
dose
Na 142.8 ±
5.3 142.3
± 3.4 142.4 ±
4.3 142.5
± 4.05 144.9 ±
5.04
Ca 8.27 ±
2.6
9.09
±
1.18
9.4 ±
1.74 9.72 ±
0.75 9.54 ±
0.85
p 7.63 ±
1.43 8.19
± 1.3 7.32 ±
0.69 7.47 ±
1.3 8.23 ±
0.97
K 4.64 ±
1.33 4.83
± 0.7 4.49 ±
0.84 4.49 ±
0.66 4.23 ±
0.94
Cl 105.8 ±
2.61
107.2
±
4.23
107.4 ±
4.16 108.3
± 5.85 107.2 ±
4.21
Values are mean of 10 animals ± S.D. No significant
difference was observed in any parameter.
ACKNOWLEDGEMENTS
Authors are grateful to Ipca Laboratories Limited,
Mumbai for providing fundamental assistance and
financial support to carryout this study. We like to thank
Dr. Sanjoy Kumar Pal of Ipca Traditional Remedies Pvt.
Ltd. for drafting this manuscript.
REFERENCES
57
1. Astin JA (1998) Why patients use alternative
medicine. Results of a national study. J Am Med
Assoc 279: 1548 – 1553.
2. MacLennan AH, Wilson DH, Taylor AW (1996)
Prevalence and cost of alternative medicine in
Australia. Lancet 347: 569 - 573.
3. Evans RW, Taylor FR (2006) Natural or Alternative
Medications for Migraine Prevention. Headache 46:
1012 - 1018.
4. Prakash VB, Pareek A, Bhat V, Chandurkar N,
Babu R, et al. (2007) Response to ayurvedic
treatment in prevention of migraine: An update of
multicentric observational clinical study.
Cephalalgia 27: 745.
5. Prakash VB, Pareek A, Narayan JP (2006)
Observational study of ayurvedic treatment on
migraine without aura. Cephalalgia 26: 1317.
6. Acharya Jadavji Trikramji. Siddha Yogya Sangrah
[Hindi]. Jhansi, India: Baidyanath Bhawan, XIIIth
edition, 1935.
7. Prakash VB, Babu SR, Sureshkumar K (2010).
Response to Ayurvedic therapy in the treatment of
migraine without aura. International J Ayurveda Res
1 (1): 30-36.
8. Centers of Disease Control and Prevention (2004)
Lead poisoning associated with use of Ayurvedic
medications—five states, 2000-2003. MMWR Morb
Mortal Wkly Rep 53: 582 - 584.
9. Saper RB, Kales SN, Paquin J, Burns M J, et.al.
(2004) Heavy Metal Content of Ayurvedic Herbal
Medicine Products. JAMA 292: 2868 - 2873.
10. Saper RB, Phillips RS, Sehgal A, et.al. (2008) Lead,
Mercury, and Arsenic in US - and Indian-
Manufactured Ayurvedic Medicines Sold via the
Internet. JAMA 300: 915 - 923.
11. Kari SK, Saper R B, Kales SN (2008) Lead
encephalopathy due to traditional medicine. Curr
Drug Saf 3: 54 – 59.
12. Bhasma. Wikipedia.
http://en.wikipedia.org/wiki/Bhasma [Assessed on
20.05.2010].
13. Chanddra D, Mandal AK (2000) Toxicological and
pharmacological study of Nabal Rasayan – A metal
based formulation. Indian J Pharmacol 32: 369 -
371.
14. Letter to JAMA.
http://www.ayurvedicure/heavymetal.html [assessed
on 20.05.2010].
15. Gogtay NJ, Bhatt HA, Dalvi SS, Kshirsagar NA
(2002) The use and safety of non-allopathic Indian
medicines. Drug Saf 25: 1005 - 1019.
58
