Acute and Sub-acute Toxicity study of Amrtadi Churna

 

Sangeeta Mukhi1, Anindya Bose1, Dilip Kumar Das2, Sitansu Kumar Panda3,

Debahuti Mohapatra4, S. Latha5, Ashok Kumar Balaraman6

1Department of Pharmaceutical Analysis, School of Pharmaceutical Sciences,

Siksha ‘O’ Anusandhan (Deemed to be University), Kalinga Nagar, Bhubaneswar - 751003, Odisha, India.

2Department of Pathology, Hi-Tech Medical College, Bhubaneswar, Odisha, India.

3Department of Anatomy, Institute of Medical Sciences and SUM Hospital,

Siksha ‘O’ Anusandhan (Deemed to be University), Bhubaneswar, Odisha, India.

4Department of Pathology, Institute of Medical Sciences and SUM Hospital,

Siksha ‘O’ Anusandhan (Deemed to be University), Kalinga Nagar, Bhubaneswar, Odisha, India.

5Department of Pharmaceutical Technology, Centre for Excellence in Nanobio Translational Research,

Anna University, Bharathidasan Institute of Technology Campus, Tiruchirappalli, Tamil Nadu, India.

6Faculty of Pharmaceutical Sciences, UCSI University, Kuala Lumpur, Malaysia.

*Corresponding Author E-mail: anindyabose_in@yahoo.com

 

ABSTRACT:

Amrtadi Churna is an Ayurvedic polyherbal formulation containing three herbs viz., Amalaki (Emblica officinalis), Gokshur (Tribulus terrestris) and Guduchi (Tinospora cordifolia). It is prescribed in India for immunomodulation and treating hyperacidity. The present work reports the acute and sub-acute toxicity assessment of Amrtadi Churna on experimental animals to rule whether it might produce toxicity on herb-herb interactions by combining its ingredient. The results showed that, the single administration of high dose (5000 mg/kg) of the Churna neither induced mortality nor any adverse toxicity signs in rats, suggesting its practically non-toxic nature in the therapeutic doses. Sub-acute toxicity testing results of hematology, serum biochemistry and organ histology showed that the product did not induce any toxic signs at the tested dose levels. However, it produced an apparently harmless hyperbilirubinemia without any signs of liver damage. There were no major gender specific variations except a few hematological parameters. It was concluded that, Amrtadi Churna could be relatively safe at therapeutic dose levels, ruling out any serious side effects by the interaction of its three herbal ingredients.

 

KEYWORDS: Churna, herb-herb interaction, HPLC fingerprinting, hematology, serum biochemistry, histopathology.

 

 

INTRODUCTION:

Simultaneous administration of two or more drugs, resulting some pharmacodynamics, pharmacokinetics or clinical response, which differ from the known effect of each of these drugs taken separately, is called drug-drug interaction. In different traditional and complementary medicines, herbal products are administered as a single herb, a combination of herbs, or combination of herbs and minerals.

 

 

When herbs are used in combination as polyherbal formulation, various types of interactions can occur among its individual ingredients. The most desirable interactions are those which results in additional therapeutic benefits. A combination of herbs may also improve the pharmaceutical properties like taste, odour and stability. However, the presence of multiple herbs in herbal products may result in unpredictable and complicated herb-herb interactions leading to severe toxic effects. Till date, much less information is available about the herb-herb interaction in polyherbal formulations1.

 

 

 

Amrtadi Churna, an Ayurvedic Polyherbal powder, is consumed for treating hyperacidity and for immunomodulation2,3. It contains three ingredients, viz., Amalaki (Emblica officinalis), Gokshur (Tribulus terrestris) and Guduchi (Tinospora cordifolia) mixed in equal proportion Although, the safety profile of the ingredients of the said Churna is already reported in animal models, the toxicology study of this polyherbal formulation has not been investigated7-7. The present work, therefore presents the acute and sub-acute toxicity evaluation of Amrtadi Churna on experimental animals to rule whether it might produce toxicity on herb-herb interactions by combining its ingredient.

MATERIALS AND METHODS:

Plant materials:

The ingredient herbs of Amrtadi Churna were purchased from Bhubaneswar, India. They were authenticated by Miss. Rashmibala Sahoo, Scientific officer, Department of Botany, State Drug Testing and Research Laboratory (ISM), Bhubaneswar, India.

 

Preparation of Amrtadi Churna:

The Amrtadi Churna was prepared according to the standard method of churna preparation8. In brief, equal amount of ingredients were separately shade dried, powdered, passed through 80 mesh sieve, and mixed together uniformly to obtain the product.

Extraction of phytoconstituents:

250g of the Churna was separately extracted in 1 liter each of distilled water and methanol to obtain solvent extracts with yields of 13.2% and 24.7% respectively. The extracts were filtered, concentrated, lyophilized and stored at -20°C until use.

HPLC fingerprinting of Amrtadi Churna extracts:

The HPLC fingerprints for methanol as well as water extracts of the Churna were recorded using a (Shimadzu UFLC) Prominence CBM-20A Bus Module system consisting of prominence SPD-M20A diode array detector, promence LC20AT pump, CTO-10AS column oven, Prominance- SIL auto sampler and Phenomenex Luna C18 column (250Χ4.6 mm2 i.d.; 5μm, 100A). The extracts were defeated with equal proportion of n-hexane. After injection of individual samples (10μl), the gradient elution was performed using a binary solvent system comprising of Solvent A (Methanol) and Solvent B (Acetic acid in water in 1:25 ratio). The program started with 100% solvent B for the first 4 minutes, then solvent A was increased to 50% for the next 6 minutes; thereafter the solvent A was increased to 80% for the next 10 minutes and then reduced to 50% again in the next 2 minutes. The initial conditions were restored after the total runtime of 25 min. The flow rate was kept at 1 ml/min for the entire run and detection was done at 240 nm.

Experimental Animals:

Healthy Wistar rats of either sex, weighing 150-180g, were obtained from the animal house of the School of Pharmaceutical Sciences, Siksha ‘O’ Anusandhan, India. Animals were maintained at 22±2°C temperature with an equal light-dark cycle and free access to food and water. Animal experimental procedures were approved by the institutional ethical committee on animal experimentation of our institute (Protocol number: 03/15/IAEC/SPS/SOAU).

Acute oral toxicity:

Amrtadi Churna, suspended in distilled water, was orally administered to a female rat at a dose level of 5000 mg/kg. After survival of treated animal, the same dose was administered to five female rats at intervals of 48 hours, totaling six animals treated with the tested dose. A negative control group treated with distilled water, constituting of six female rats, were also maintained in parallel9. The animals were observed individually for 4 h after dosing and daily once in next 14 days for mortality, changes in general behavior, clinical signs of toxicity, food and water intake. Body weights were also measured in 0, 4, 7, 10 and the 14th day of the administration. After fourteenth days, animals were sacrificed under high dose of carbon dioxide anesthesia and the major organs were removed, weighed and examined for macroscopic changes9.

 

Sub-acute oral toxicity:

Sub-acute oral toxicity evaluation was performed as per the OECD guideline no. 40710. The animals were segregated into four groups of 12 animals (six males and six females). The Group 1 animals received only the distilled water vehicle (10ml/kg body weight) and served as controls. Groups 2, 3 and 4 were administered orally with the Churna daily for 28 days at the doses of 300, 600 and 1800mg/kg body weight, respectively. During the treatment period, animals were observed once daily to detect signs of toxicity, and weighed weekly. After completion of the observation period, all animals were fasted overnight (12h) and sacrificed by high dose of carbon dioxide anesthesia. Blood samples were obtained by cardiac puncture. A part of the blood sample was collected in EDTA-coated tubes for hematological analysis. The routine hematological analysis including hemoglobin (HGB), white blood cell count (WBC), lymphocytes (Lym), monocytes (Mon), granulocytes, red blood cell (RBC) count, hematocrit (Ht), Eosinophil (Eos), basophile (Bas), abnormal cells, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and platelet count were performed using an ABX Micros60 Automated Hematology cell counter. The other part of the blood sample was collected in dry tubes for separation of serum for biochemical analysis. Triglycerides (TG), total cholesterol (TC), high density lipoprotein (HDL), alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphate, total bilirubin, creatinine and total proteins were measured by an automatic biochemical analyzer (Microlab 300, Merck). Low density lipoprotein was estimated using the Friedewald formula11-13.

 

After collecting blood, the abdomen was opened through a midline incision to detect autopsy changes followed by dissection of major organs namely the brain, heart, lung, kidney, liver, stomach and spleen, testis or ovary. After weighing and observation of gross lesion and ponderal changes, organs were fixed with 10% v/v formalin solution. Thereafter, the organs were subjected to dehydrating, wax embedding, sectioning, and staining with heamatoxylin and eosin for histological evaluation under light microscopy.

 

Statistical analysis:

Results were expressed as mean ± standard error of the mean (SEM)14. The results of toxicity testing were compared by one-way ANOVA followed by post-hoc Tukey HSD (honestly significant difference) test. The level of significance for acceptance was taken as p<0.05 for all experiments.

 

RESULTS AND DOSCUSSION:

HPLC fingerprinting of methanol and water extracts of Amrtadi Churna:

RP-HLPC chromatograms were successfully developed for water and methanol extracts of the Churna. The chromatograms could resolve 37 and 34 peaks from water extract and methanol extract respectively (Figure 1). While comparing, 7 peaks were identified to have similar retention times in both chromatograms with approximate retention time (RT) of 8.2 min, 8.9 min, 9.2 min, 9.6 min, 10.02 min, 10.3 min and 10.9 min with 8.9min peak RT being the most prominent peak in both chromatograms.

 

The HLPC chromatograms of water and methanol extracts of Amrtadi Churna suggest that mostly there is the presence of a few common compounds along with many different phytoconstituents between aqueous and methanol extracts due to polarity difference of these two solvents. By combining these two chromatograms we may have obtained the almost entire HPLC fingerprint profile of Amrtadi Churna containing both polar and nonpolar constituents, which are expected to be absorbed by the oral administration of the Churna. The combined HPLC fingerprint obtained represent the chemical constituents present in Amrtadi Churna exerting its pharmacological as well as toxicological effects (if any). This may be used for authentication or quality evaluation of the product Amrtadi Churna. Moreover, deviation from authentic fingerprint may be indicative of adulteration or deterioration leading to compromised efficacy and/or safety.

 

 

Water Extract

 

Methanolic Extract

Figure 1: HPLC fingerprints of the water and methanolic extract of Amrtadi Churna at 240nm

 

Acute toxicity:

The preclinical toxicity studies on different biological systems are employed to reveal the species, organ and dose specific toxic effects of a drug product. Rodents are widely used in preclinical toxicity testing as predictors to access the toxicity in humans, thereby saving time, resources and efforts15. Rats with a body volume nearly 10 times larger than mice are preferred over mice to satisfy the requirement of a sufficient volume of blood to perform a series of laboratory examinations. In our study, the acute toxicity of Amrtadi Churna was carried out in Wistar rats to record adverse signs and symptoms on single dose administration of the drug at a dose level of 5000mg/kg that is several folds higher than the therapeutic equivalent dose.

 

Mortality is an important criterion of toxicological evaluation16. As the tested dose of Amrtadi Churna (5000mg/kg) did not induce any mortality in testing animals during the 14-day observation period, its LD50 is estimated to be greater than 5000mg/kg17. Thus, Amrtadi Churna comes under the category 5 or unclassified in the Globally Harmonized Classification System for Chemical Substances and Mixtures (GHS). Moreover, no indications of any behavioral changes of the animals were observed. In addition, necropsy was unable to reveal any gross pathological signs with the absence of significant differences in relative weights of brain, lung, liver, heart, kidney, spleen, stomach and testis or ovary (Table 1). The Churna can therefore be considered as practically non-toxic in accordance with previous reported literatures18,19.

 

Table 1: Relative organ weight (g/100 g of body weight) of rats orally treated with Amrtadi Churna

Acute toxicity

Sub-acute toxicity

 

Control

5000 mg/kg

Control

300 mg/kg

600 mg/kg

1800 mg/kg

Female

 

 

 

 

 

 

Brain

0.745±0.042

0.735±0.061

0.740±0.041

0.736±0.042

0.818±0.036

0.758±0.040

Heart

0.271±0.02

0.281±0.06

0.261±0.013

0.282±0.015

0.302±0.009

0.292±0.011

Lungs

0.841±0.030

0.805±0.015

0.811±0.047

0.760±0.021

0.885±0.050

0.725±0.033

Kidney

0.609±0.008

0.691±0.012

0.647±0.020

0.679±0.024

0.636±0.029

0.639±0.019

Liver

2.91±0.24

2.84±0.38

3.066±0.141

2.964±0.152

3.129±0.057

3.059±0.165

Spleen

0.292±0.002

0.288±0.014

0.288±0.016

0.314±0.020

0.333±0.025

0.286±0.012

Stomach

0.66±0.021

0.65±0.050

0.654±0.007

0.629±0.017

0.610±0.023

0.641±0.027

Ovary

0.864±0.018

0.942±0.044

0.869±0.040

0.946±0.059

0.9115±0.036

0.951±0.035

Male

 

 

 

 

 

 

Brain

NP

NP

0.6408±0.037

0.626±0.019

0.697±0.041

0.641±0.025

Heart

NP

NP

0.307±0.016

0.297±0.016

0.318±0.014

0.322±0.016

Lungs

NP

NP

0.775±0.037

0.768±0.026

0.805±0.029

0.763±0.035

Kidney

NP

NP

0.654±0.039

0.624±0.021

0.654±0.029

0.668±0.031

Liver

NP

NP

2.869±0.102

2.830±0.163

3.090±0.127

2.975±0.150

Spleen

NP

NP

0.301±0.015

0.262±0.022

0.305±0.011

0.286±0.013

Stomach

NP

NP

0.645±0.091

0.652±0.048

0.650±0.030

0.657±0.023

Testis

NP

NP

1.063±0.056

1.194±0.056

1.151±0.043

1.304±0.076*

Values expressed as mean ± SEM, n = 6, p ˂ 0.05, NP = Not performed.

 

Sub-acute oral toxicity:

Sub-acute toxicity testing, generally performed in rodents, is used to assess the adverse effect which a drug might exert on a patient with repeat exposure for a minimum of 28 days, including any compound toxicity. Baseline parameters such as the behavioral and biochemical parameters of the animals are recorded in these studies to predict the human safety profile in the repeated dose administration20. In Indian classical Ayurvedic texts, the human intended dosage for Amrtadi Churna is mentioned as 3-6g/dose, up to thrice daily. Based on a surface body ratio between human and rat, three dosing levels (300, 600 and 1800mg/kg) of the Churna were assigned for 28 days treatment to establish the safety of the drug against cumulative toxicity21. The animal groups of both sexes were used separately at each dosage level to evaluate the differences in gender-based response.

 

General signs and mortality:

No exterior sign of toxicity was observed in rats by any of the three tested dosage levels of the Churna. All animals (both female and male) survived during the entire 28 days of treatment without any significant behavioral changes between the control and treatment groups. This corroborated the hypothesis of the low toxicity of the Churna after sub-acute exposure, similar to its acute toxicity results.

 

 

 

Changes of body weights:

The significant changes in body weight can indicate physiological disorders such as hormonal variations, liver disorders and decreased absorption of nutrients22. The reduction in relative internal organ weight is also a simple and sensitive index of toxicity after exposure to toxic compounds23,24.

 

Overall observation on the body weights did not reveal any significant difference between the tested groups during the 28day treatment period. All the animal groups consumed substantial amounts of feed. The average body weights increased constantly in all the groups, irrespective of administering doses. The absence of any significant changes in body weight or relative organ weights of animals at any dosage levels after 28-day oral treatment of the Churna, suggested that the drug did not affected animal’s normal growth.

 

Hematological parameters:

A significant decrease of granulocytes was observed in females treated with the Churna at 600mg/kg and 1800 mg/kg dosage levels. Moreover, females treated with 1800mg/kg of Churna showed a significant reduction on eosinophil. A non-significant tendency of reduction of WBC was observed in drug treated animals, more prominently in females. No other alterations in male and females hematological parameters were noticed (Table 2).

 

Table 2: Haematological parameters of rats orally treated with Amrtadi Churna in Sub-acute toxicity evaluation

 

Control

300 mg/kg

600 mg/kg

1800 mg/kg

Female

 

 

 

 

WBC

7833±65.81

7567±93.46

6983±115.156

6883±108.771

Lymphocytes (%)

40.166±1.49

35.83±0.70

41.833±1.35

41.66±1.64

Monocytes (%)

0.666±0.21

0.5±0.22

0.833±0.16

0.666±0.21

Granulocytes (%)

55.83±1.077

55.66±1.72

50.33±1.22*

50.66±0.88*

Eosinophils (%)

3.58±0.20

2.66±0.21

3.33±0.21

1.66±0.33**

Basophils (%)

BDL

BDL

BDL

BDL

Abnormal cells (%)

BDL

BDL

BDL

BDL

RBC

5.003±0.095

5.013±0.104

5.351±0.063*

4.97±0.073

Hematocrit (%)

44.51±0.64

43.366±0.35

44.883±0.83

43.05±1.17

Haemoglobin (g/dL)

14.2±0.38

14.53±0.37

15.15±0.41

13.98±0.34

MCV (fL)

86.066±0.48

85.01±0.53

87.11±0.66

85.93±0.56

MCH (pg)

28.13±0.13

27.78±0.24

28±0.14

28.1±0.13

MCHC (g/dL)

32.68±0.15

32.85±0.29

32.36±0.16

32.76±0.32

Platelets

2.87±0.047

2.85±0.054

2.901±0.049

2.94±0.064

Male

 

 

 

 

WBC

7450±76.065

7150±73.66

7083±66.99

7167±35.86

Lymphocytes (%)

40.33±1.28

40.66±1.56

39.66±1.56

40.33±1.30

Monocytes (%)

0.833±0.16

0.666±0.21

0.833±0.16

0.833±0.16

Granulocytes (%)

52.33±1.05

55.166±0.94

55.66±1.58

53.66±0.98

Eosinophils(%)

3.16±0.30

3.83±0.16

3.16±0.16

2.83±0.16

Basophils (%)

BDL

BDL

BDL

BDL

Abnormal cells (%)

BDL

BDL

BDL

BDL

RBC

4.96±0.08

5.073±0.127

4.961±0.124

5.231±0.15

Hematocrit (%)

44.366±1.05

46.95±1.18

43.71±1.047

46.61±1.146

Haemoglobin (g/dL)

13.35±0.42

14.71±0.61

13.93±0.30

15.06±0.57

MCV (fL)

87.18±0.53

85.86±0.69

85.2±0.35

86.11±0.77

MCH (pg)

27.96±0.17

28.06±0.13

28.05±0.23

28.35±0.061

MCHC (g/dL)

32.25±0.20

32.75±0.160

32.6±0.414

32.56±0.20

Platelets

2.83±0.034

2.89±0.052

2.95±0.060

3.001±0.052

Values expressed as mean ± SEM, n = 6. * p< 0.05 and ** p < 0.01 compared with control group. BDL = Below detection limit.

 

The hematological system is considered to be a highly sensitive target for toxic compounds and an important indicator of physiological and pathological state25. The effect of Amrtadi Churna on hematological parameters showed some variations between the dose and gender. Significant decrease of granulocytes observed in females may be contributed by the tendency of reduction of eosinophil (although non-significant). Eosinophil has a prime role in regulating local immune and inflammatory responses, and its accumulation in the blood and tissue (eosinophilia) is associated with several inflammatory and infectious diseases. Lowering of eosinophil may help in controlling various diseases, including asthma and allergy. Hence the observed lowering of eosinophil by Amrtadi Churna can be accepted as a medical benefit rather than its adverse effect.

 

A low WBC levels induced by the Churna apparently indicate temporary disruption of its production in the bone marrow. However, various drugs, particularly chemotherapeutic drugs have a history of similar leukocyte lowering effect resulting immune suppression. However, in our studies, the WBC lowering effect of Amrtadi Churna was not established statistically. The other hematological levels were found within the normal ranges for the groups of both males and females. Moreover, the changes did not reflect any clear dose-response relationship and can be assumed to be clinical non-significant. Hence, the differences between the results of the hematological analysis of males and females can be due to the influence of sex hormones, especially the estrogen and its effect on B and T lymphocytes cells equilibrium and cytochrome P450 enzymes metabolism. Similar observations have been reported in several other clinical and experimental studies26-28.

 

Biochemical parameters:

There was a significant decrease in triglyceride level for the highest dose group of male animals (1800mg/kg).  A significant increase of bilirubin level in female rats was observed for all tested dosage levels of the Churna. Similar significant elevation of bilirubin was also shown in male rats in exception of the 600mg/kg animal group, whose bilirubin level remained unaffected. Rats of both sexes showed a decrease of total serum protein concentration (P˂0.05) at the lowest dose level (300 mg/kg) as well as in the 1800mg/kg treated male group. No other abnormalities in biochemical parameters were detected in testing rats (Table 3).

 

 

 

Table 3: Biochemical parameters of rats orally treated with Amrtadi Churna in Sub-acute toxicity evaluation

 

Control

300 mg/kg

600 mg/kg

1800 mg/kg

Female

 

 

 

 

Triglycerides (g/L)

80.33±5.78

100.66±7.60

97.66±5.18

93.16±4.83

TC (g/L)

83.83±1.97

89.66±3.42

81±2.08

77.5±4.81

HDL (g/L)

21±0.73

22.16±1.13

21.5±0.99

19.83±1.10

LDL (g/L)

49.66±3.24

55.16±5.71

45.66±3.27

47.33±2.71

VLDL (mg/dL)

14.83±0.98

19.33±1.64

18.66±1.62

16.83±1.01

Creatinine (mg/dL)

0.833±0.042

0.816±0.03

0.733±0.042

0.83±0.061

Total bilirubin (mg/dL)

0.393±0.012**

0.516±0.017**

0.536±0.013**

0.518±0.005**

Total protein (g/L)

7.28±0.083

6.68±0.22*

6.93±0.095

7.16±0.08

ALT (U/L)

55.33±5.71

52.16±4.15

64.5±8.42

52.33±4.52

AST (U/L)

51.33±4.40

46.33±4.58

62.33±7.33

47.33±3.09

Alkaline phosphate (U/L)

135.33±7.18

137.5±8.70

123.66±8.29

142.16±12.40

Male

 

 

 

 

Triglycerides (g/L)

76.66 ±1.68

76.33±3.37

81.66 ±1.17

92.5±6.28*

TC (g/L)

84.33±1.20

82.33±1.74

80.83±3.50

87.66±4.48

HDL (g/L)

21.16±0.60

20.83±0.6

20.66±0.76

22.16±1.4

LDL (g/L)

51.66±1.17

49.16±2.85

53.5±3.82

56.66±1.52

VLDL (mg/dL)

14.66±1.22

13.33±0.80

14.83±1.55

18.83±1.51

Creatinine (mg/dL)

0.81±0.047

0.81±0.03

0.85±0.034

0.88±0.03

Total bilirubin (mg/dL)

0.42±0.008

0.44±0.009

0.508±0.01**

0.488±0.006**

Total protein (g/L)

7.68±0.13

6.68±0.183**

7.36±0.21

6.68±0.13**

ALT (U/L)

57.33±5.26

59.16±6.405

63.5±7.093

53.33±4.31

AST (U/L)

49.66±6.15

60.66±3.66

68.66±6.80

46.66±4.61

Alkaline phosphate (U/L)

130.5±8.48

128.33±11.70

138.16±11.61

122.5±7.64

Values expressed as mean ± SEM, n = 6. * p< 0.05 and ** p < 0.01 compared with control group.

 

Triglyceride levels in our body may vary with age and sex. A high triglyceride level combined with low HDL cholesterol or high LDL cholesterol is suggestive of atherosclerosis that increases the risk for heart attack and stroke. In the sub-acute toxicity studied of the Churna, a tendency of increase of triglyceride level was observed in the experimental animals. However the difference in triglyceride level was clinically significant only for the highest dose group (1800mg/kg) male animals, although without any significant elevation of total and LDL cholesterol. Hence, the change seemed to be not indicative of any health hazards.

 

Several toxic compounds are accumulated in liver for detoxification29. ALT is exclusively present in the cytoplasm of hepatocytes and a majority of AST is found in liver, cardiac muscle and kidneys30. Acute or chronic liver injury can cause the elevation of ALT and AST in the bloodstream31,32. Hence, elevated serum ALT and AST levels are considered as the sensitive markers of possible liver tissue damage, particularly by liver toxicity30. Bilirubin is produced by the normal breakdown of pigment containing proteins, especially hemoglobin from senescent red blood cells and myoglobin from muscle breakdown. After bonding with albumin, bilirubin circulates in the blood and is taken up by the liver hepatocytes. Within hepatocytes, bilirubin is conjugated with glucuronic acid catalyzed by uridine diphosphate glucuronosyltransferase (UDP-GT) and secreted into the bile for excretion through the gut. As a result, the plasma unconjugated bilirubin concentration remains low. Gilbert syndrome is an apparently harmless condition characterized by intermittent unconjugated hyperbilirubinemia in the absence of hepatocellular disease or hemolysis. The syndrome can be produced by various reasons such as action of certain drugs which inhibit UDP-GT activity (like gemfibrozil, irinotecan, atazanavir, indinavir etc.) or by alteration of sex hormonal levels. The cause of hyperbilirubinemia is diagnosed as the Gilbert syndrome only after excluding the possibilities of other liver and hematologic disorders33. As the Churna treated test animals showed signs of hyperbilirubinemia without any abnormal liver enzyme (ALT or AST) or lowering of hemoglobin, it can be hypothesized that the Churna may have been inhibiting UDP-GT activity or producing alteration of hormonal levels in the test animals to exert an effect clinically similar to Gilbert syndrome. The genotoxic and endocrine disrupting potential of T. terrestris, one of the constituent of Churna, is already reported previously34. Hence T. terrestris alone or by interaction with the other ingredients of Amrtadi Churna might be responsible for this observed hyperbilirubinemic activity34.

 

The substance that reaches the systemic circulation also reaches kidney, making it frequent targets of toxicity35. The serum creatinine level is an indicator of renal function as its high serum levels are associated with a marked failure of neurons function36. A non-significant trend (P>0.05) of increase in serum creatinine levels in Churna treated male rats can be predicted as indicator of slowing of kidney function. However, in this case there was the absence of any significant change of kidney weight of those animals. The histological analysis of the kidneys also revealed the absence of any abnormality in the said organs, overruling the possibility of kidney damage.

 

A low total serum protein level can suggest a liver or kidney disorder. In sub-acute toxicity evaluation, there was significant (P˂0.05) lowering of serum protein levels in some of the Amrtadi Churna treated groups (i.e. 300mg/kg male and female as well as 1800mg/kg male group). However, as mentioned earlier, the drug did not cause any significant alterations in serum levels of ALT and AST. Any tissue changes visible in the histological analysis of the liver or kidney were also absent. Hence, these findings are suggestive of isolated cases of change in protein metabolism or protein absorption of rats without any liver or kidney damage. All other biochemical parameters remained unaffected providing strong evidence for biosafety and high tolerance of repeated administration of Amrtadi Churna in mammalian systems.

 

 

 

 

Organ weight measurements, Necropsy and Histopathology:

Drugs which apparently do not cause immediate adverse physiological effects, may damage subcellular components on bioaccumulation due to repeated dosing or by exerting delayed effects. Hence, the assessment of pathological changes in the organs of the treated animals of repeated toxicity, both macro and microscopically, is considered as a gold standard for safety assessment37.

 

Gross necropsy of the organs examined after dissection on 29th day showed no gross pathological lesions.  Relative organ weights (g/100g of body weight) calculated on the basis of the absolute organ weights of brain, lung, liver, heart, kidney, spleen, stomach, testis or ovary were very similar to that of the respective control groups (Table 1).  Histological examination performed on the highest dose treated (1800mg/kg) male and female animals exhibited no apparent pathological alterations in any of the tested organs, suggesting tissue integrity of these organs independent of the drug treatment (Figure 3). These results were consistent with reports of biochemical analyses, confirming the safety of Amrtadi Churna on long term oral administration.

Figure 3: Histological sections from different organs of control rats and Amrtadi Churna (1800 mg/kg) treated rats in the sub-acute toxicity testing

Male Rats

Female Rats

 

 

 

CONCLUSION:

In Conclusion, the administration of 5000 mg/kg body weight of Amrtadi Churna, in acute toxicity evaluation, did not induce mortality or any adverse toxicological signs in rats suggesting that it’s oral LD50 is higher than 5000 mg/kg. The results of sub-acute toxicity showed that the Churna can be relatively safe at therapeutic dose levels in rats, although it produced an apparently harmless hyperbilirubinemia without any signs of liver damage. The presence of T. terrestris in Amrtadi Churna alone or by interaction with the other ingredients of the churna might be responsible for this observed activity. Moreover, there were no major gender specific variations except some hematological parameters probably due to hormonal variation between males and females, especially the estrogen. The developed HPLC fingerprints of methanol and aqueous extracts of Amrtadi Churna may be useful for its quality evaluation as well as detection of adulteration or deterioration, which can lead to the reduced efficacy and risk of toxicity of the product. Further studies like chronic toxicity, reproductive toxicity, etc. should be performed in order to evaluate the complete safety of this polyherbal formulation in humans.

 

REFERENCES:

1.      Che CT, Wang ZJ, Chow MSS, Lam CWK. (2013) Herb-Herb Combination for Therapeutic Enhancement and Advancement: Theory, Practice and Future Perspectives. Molecules. 2013; 18(5): 5125-5141.

2.      Anonymous. Ayurvedic Formulary of India Part 3. New Delhi: Ministry of Health and Family Welfare, Department of AYUSH, Government of India. 2011; 1st ed.

3.      Mukhi S, Bose A, Ray A, et al. Analytical Standards of Amrtadi Churna: A Classical Ayurvedic Formulation. Indian Journal of Pharmaceutical Sciences. 2017; 79(2): 227-240.

4.      Abudayyak M, Jannuzzi AT, Ozhan G, et al. Investigation on the toxic potential of Tribulus terrestris in vitro. Pharmaceutical Biology. 2015; 53(4): 469-476.

5.      Hemalatha S, Hari R. Acute and Sub-acute Toxicity Studies of the Saponin Rich Butanol Extracts of Tribulus terrestris Fruits in Wistar Rats. International Journal of Pharmaceutical Sciences Review and Research. 2014; 27(2): 307-313.

6.      Jaijoy K, Soonthornchareonnon N, Lertprasertsuke N, et al. Acute and chronic oral toxicity of standardized water extract from the fruit of Phyllanthus emblica Linn. International Journal of Applied Research in Natural Products. 2010; 3(1): 48-58.

7.      Devbhuti D, Gupta JK, Devbhuti P, et al. Phytochemical and acute toxicity study on Tinospora tomentosa Miers. Acta Poloniae Pharmaceutica. 2009; 66(1): 89-92.

8.      Kansabanik S, Mukhi S, Das C, et al. Establishment of Quality Control Parameters of Nagarmotha Churna. Research Journal of Pharmacy and Technology. 2019; 12(12): 5967-5971.

9.      Anonymous. OECD guidelines for testing of chemicals test no. 425: Acute oral toxicity – up-and-down procedure. OECD Publishing, Paris. 2008.

10.   Anonymous. Guidelines for testing of chemical. Repeated dose 28-day oral toxicity study in rodents. OECD Publishing, Paris. 2008.

11.   Parimalakrishnan S, Sundar VD, Ulaganathan I, et al. Preliminary Toxicity Evaluation of Bidens pilosa Extracts Using Experimental Mice. Research Journal of Pharmacognosy and Phytochemistry. 2010; 2(5): 397-402.

12.   Anthony OE, Comfort MC, Chuka BD. Phytochemical Screening and Acute- and Organ- Toxicity Evaluation of Telfairia occidentalis Root Aqueous Extract on Normal Wister Rats. Research Journal of Pharmacognosy and Phytochemistry. 2010; 2(5): 417-420.

13.   Hassan S., El-Twab SA, Hetta M, et al. Improvement of lipid profile and antioxidant of hypercholesterolemic albino rats by polysaccharides extracted from the green alga Ulva lactuca Linnaeus. Saudi Journal of Biological Sciences. 2011; 18(4): 333–340.

14.   Baghel SS, Dangi S, Soni P, et al. Acute Toxicity Study of Aqueous Extract of Coccinia indica (Roots). Asian Journal of Pharmaceutical Sciences. 2011; 1(1): 23-25.

15.   Kramer JA, O’Neill E, Phillips ME, et al. Early toxicology signal generation in the mouse. Toxicologic Pathology. 2010; 38(3): 452–471.

16.   Asare GA, Gyan B, Bugyei K, et al. Toxicity potentials of the nutraceutical Moringa oleifera at supra-supplementation levels. Journal of Ethnopharmacology. 2012; 139(1): 265–272.

17.   Anonymous. Harmonised integrated classification system for human health and environmental hazards of chemical substances and mixtures. Chapter 2.1, adopted 14th August 2001. OECD Publishing, Paris. 2001.

18.   Kennedy GL, Ferenz RL, Burgess BA. Estimation of acute oral toxicity in rats by determination of the approximate lethal dose rather than the LD50. Journal of Applied Toxicology. 1986; 6(3): 145–148.

19.   Ateba SB, Simo RV, Mbanya JC, et al. Safety profile and gender specific differences of a methanol extract of Eriosemala urentii (Leguminosae) in acute and subchronic (28 days) oral toxicity studies in Wistar rats. Food and Chemical Toxicology. 2014; 65: 27–32.

20.   Parasuraman S. Toxicological screening. Journal of Pharmacology and Pharmacotherapeutics. 2011; 2(2): 74–79.

21.   Nair AB, Jacob S. A simple practice guide for dose conversion between animals and human. Journal of Basic and Clinical Pharmacy. 2016; 7(2): 27-31.

22.   Lee MY, Shin IS, Seo CS, et al. Sub chronic oral toxicity studies of the traditional herbal formula Bangpunglongseong-an in Crl:CD (SD) rats. Journal of Ethnopharmacology. 2012; 144(3): 720–725.

23.   Teo S, Strlig D, Thomas S, et al. A 90 days oral gavage toxicity study of D-methylphenidate and D, L-methylphenidate in Sprague-Dawley rats. Toxicology. 2002; 179(3): 183–196.

24.   Thanabhorn S, Jaijoy K, Thamaree S, et al. Acute and sub-acute toxicity study of the ethanol extract from Locinera japonica Thunb. Journal of Ethnopharmacology. 2006; 107(3): 370–373.

25.   Mukinda JT, Syce JA. Acute and chronic toxicity of the aqueous extract of Artemisia afrain rodents. Journal of Ethnopharmacology. 2007; 112(1): 138-144.

26.   Angele MK, Ayala A, Cioffi WG, et al. Testosterone: the culprit for producing splenocyte immune depression after trauma hemorrhage. American Journal of Physiology. 1998; 274(6): 1530-1536.

27.   Ahmed SA, Hissong BD, Verthelyi D, et al. Gender and risk of autoimmune diseases: possible role of estrogenic compounds. Environmental Health Perspectives. 1999; 107(Suppl 5): 681–686.

28.   Sin DD, Cohen SBZ, Day A, et al. Understanding the biological differences in susceptibility to chronic obstructive pulmonary disease between men and women. Proceedings of the American Thoracic Society. 2007; 4(8): 671–674.

29.   Clarke EGC and Clarke ML. Veterinary Toxicology. Cassel and Collier Macmillan Publishers. 1997.

30.   Ramaiah SK. A toxicologist guide to the diagnostic interpretation of hepatic biochemical parameters. Food and Chemical Toxicology. 2007; 45(9): 1551–1557.

31.   Han YD, Song SY, Lee JH, et al. Multi enzyme-modified biosensing surface for the electrochemical analysis of aspartate transaminase and alanine transaminase in human plasma. Analytical and Bioanalytical Chemistry. 2011; 400(8): 797–805.

32.   Ozer J, Ratner M, Shaw M, et al. The current state of serum biomarkers of hepatotoxicity. Toxicology. 2008; 245(3): 194-205.

33.   VanWagner LB, Green RM. Evaluating Elevated Bilirubin Levels in Asymptomatic Adults. Journal of the American Medical Association. 2015; 313(5): 516–517.

34.   Abudayyak M, Jannuzzi AT, Ozhan G, et al. Investigation on the toxic potential of Tribulus terrestris in vitro. Pharmaceutical Biology. 2015; 53(4): 469-476.

35.   Dekant W, Vamvakas S. Biotransformation and membrane transport in nephrotoxicity. Critical Reviews in Toxicology. 1996; 26(3): 309–334.

36.   Lameire N, Van Biesen W, Vanholder R. Acute renal failure. Lancet. 2005; 365(9457): 417–447.

37.   Kulkarni YA, Veeranjaneyulu A. Toxicological evaluation of the methanol extract of Gmelina arborea Roxb. Bark in mice and rats. Toxicology International. 2012; 19(2): 125-131.

 

 

 

Received on 05.06.2020           Modified on 15.07.2020

Accepted on 10.08.2020         © RJPT All right reserved

Research J. Pharm. and Tech. 2021; 14(6):3111-3118.

DOI: 10.52711/0974-360X.2021.00543