Genotoxicity Testing of Lipovedic- A Polyherbal Anti-hypercholesterolemic Drug

 

Meera Sumanth¹, Swetha S.¹, Narasimharaju K.², Anusha¹ and Natesh T.S.¹

¹Department of Pharmacology, Visveswarapura Institute of Pharmaceutical Sciences, 22^nd main, 24^th cross, Banashankari IInd Stage, Bangalore, 560 070   Karnataka, INDIA

²Research Associate, Syngene Intl Ltd, Plot no: 2 and 3, Bommasandra Industrial area, Bangalore 560 100

*Corresponding Author E-mail: meerasumanth@gmail.com

ABSTRACT:

Present study was taken up to evaluate genotoxicity of Lipovedic- a polyherbal formulation, by mouse bone marrow micronucleus test (MNT) and chromosomal aberration test (CAT). Since Lipovedic is a drug intended for lifelong use in treatment of hypercholesterolemia it is necessary to evaluate genotoxicity of the drug. Cyclophosphamide (100 mg/kg, i.p) was used as a genotoxic challenge and bone marrow of control and Lipovedic (260mg/kg p.o.× 7 days) treated mice was collected after 24, 48 and 72 h, respectively, after clastogenic challenge. In MNT, the bone marrow smears were stained with May-Grunwald’s followed by Giemsa stain. Polychromatic and Normochromatic erythrocytes were counted and P/N ratio was calculated. Number of micro nucleated PCE and NCE were counted per 2000 erythrocytes. In CAT, Colchicine, 4mg/kg, i.p, was administered 90 min before sacrifice. Bone marrow was collected and smears were prepared, stained with Giemsa stain and observed under 100X for different types of chromosomal aberrations (rings, exchanges, breaks and minutes). Mitotic index was calculated. Lipovedic did not show any mutagenicity, but there was no antimutagenic effect against cyclophosphamide induced clastogenicity. Similarly, chromosomal aberration assay did not reveal any significant alterations. In conclusion, Lipovedic is does not provide protection against mutagenicity, but it is free of genotoxicity. Hence Lipovedic can be used for a long time without any alterations in the genetic apparatus.

 

KEYWORDS: Genotoxicity, Chromosomal aberration, Micronucleus, Lipovedic, Anti- hypercholesterolemia.

 

 

INTRODUCTION:

Genotoxicity refers to alteration in gross structure or content of chromosomes (Clastogenicity) or base pair sequencing of DNA (Mutagenicity) by exposure to toxic agents.¹ Toxicological studies have undergone a significant evolution during the past decade, with much greater emphasis being placed on chronic toxicity, carcinogenicity, teratogenicity and mutagenicity.² Mutagenicity testing is an important part of the regulatory hazard assessment of chemicals. It is undertaken for two main reasons: a) to detect chemicals that might cause genetic damage in germ cells, and thus increase the burden of heritable (genetic) disease in the human population; and b) to detect chemicals that might be Carcinogenic.³

 

Mutagens are not only involved in genotoxicity and carcinogenesis but also involved in the inception and pathogenesis of several chronic degenerative diseases including hepatic disorders, neurodegenerative disorders, cardiovascular disorders, diabetes, arthritis, chronic inflammation and in the process of ageing.⁴  Micronucleus test (MNT) and chromosomal aberration test (CAT) using Mouse bone marrow test system is extensively used cytogenetic tests for detection of anticlastogenetic agents.²

 

Lipovedic, manufactured by Vedic Bio labs, Bangalore, is a polyherbal formulation containing Guggulu, Triphala, Trikatu, Nagakesara, Chitraka, Musta, Vidanga, Chavya, Hapushpa, Ativisha, Pata, for treatment of  hypercholesterolemia. Earlier studies have confirmed that the polyherbal formulation, Lipovedic, increases serum HDL level and significantly decreases in serum cholesterol, serum LDL, serum VLDL levels and hence has anti-obesity activity. It is hypothesized that, as a result of these findings drug can be used as antihypercholesterolemic.⁵ Since Lipovedic is a drug intended for lifelong use, it is necessary to evaluate genotoxicity of the drug.  In the present study, an attempt is made evaluate genotoxicity of the polyherbal formulation, Lipovedic, by moderating the genetic damage induced by cyclophosphamide.

 

MATERIALS AND METHODS:

Animals: Ten to twelve weeks old Swiss albino mice, of either sex, with an average weight of 27±2.5 g, obtained from the Central Animal Research Facility, NIMHANS, Bangalore, India were used in this study. Animals were housed in groups (6 animals/ group) and maintained under standard conditions of temperature (25±2°C), relative humidity 55 ± 10%, and natural light and dark cycle and fed with standard pellet diet and water ad libitum. The experimental protocol, which is in accordance with the OECD (Organization of Environmental Carcinogen Detection) guidelines No.470 and WHO guidelines for mutagenicity studies in animals⁶ was approved by the Institutional Animal Ethics Committee before starting the experiments.

 

Drugs and chemicals: Cyclophosphamide, Colchicine, bovine serum albumin, May-Grunwald’s stain, Giemsa stain, Potassium chloride, Methanol, Phosphate buffer, Lipovedic.

 

Experimental design²: The animals were divided into eight groups consisting of six animals each. Group one served as normal control (Distilled water 10ml/kg), group two, three, four was treated with  Cyclophosphamide 100 mg/kg, i.p. and bone marrow was collected after 24, 48 and 72 h of clastogen administration, respectively. Group five was treated with Lipovedic (260 mg/kg, p.o.)⁵ for seven days. Groups six, seven, eight were treated with Lipovedic for seven days followed by Cyclophosphamide challenge. Bone marrow was collected at 24, 48 and 72 h after clastogenic challenge.

 

Bone marrow micronucleus assay:⁷

The animals were sacrificed and the bone marrow was aspirated from femur and tibia into one ml of 5% bovine albumin in phosphate buffered saline (pH 7.2) at 24, 48 and 72h after the clastogenic challenge. The cell suspension was centrifuged (1000 rpm for 5 min) and the smears were prepared from the pellet on chemically cleaned glass slides and stained with May-Grunwald’s and followed by Giemsa stain. The smears were analyzed under oil immersion using Labomed- Model Digi 2 microscope (90 - 260 V) for the presence of Micronuclei (MN) in polychromatic erythrocytes(PCE) and normochromatic erythrocytes (NCE). P/N (Polychromatic erythrocyte/Normochromatic erythrocyte) ratio was determined by counting a total of about 500 erythrocytes per animal and 2000 erythrocytes were examined for the presence of micronuclei.⁸

 

Chromosomal aberrations test: ^{9, 10}

On seventh day, each animal was injected with 0.04% colchicine in a dose of four mg/kg i.p, 90 min prior to sacrifice, for mitotic arrest. The bone marrow was aspirated from femur and tibia into suspending medium 0.075 M KCl, centrifuged and supernatant was discarded. The pellet was mixed with fixative (3:1, methanol: acetic acid) and then centrifuged. The preparation was given two changes of fixative and smears were prepared. The slides were flame-dried and stained with 10% Giemsa at pH 6.8 for 15 - 20 min. Smears were screened for different types of chromosomal abnormalities-rings, breaks, exchanges and minute.

 

Statistical analysis:

The results were expressed as Mean ± SEM and analysis was carried out by One way ANOVA followed by Tukey’s multiple comparison test to estimate the significance of difference between various individual groups. P < 0.001 was considered significant.

 

RESULTS:

There was no significant decrease in the micronucleus formation (Table 1, Figure 1a) and chromosomal aberrations (Figure 1b, Figure 1c) in Lipovedic treated mice against clastogenic challenge. In Lipovedic alone treated mice of group five, there was no significant change in the P/N ratio (Table 1), total no. of aberrations and mitotic index (Figure 1b) as compared to the cyclophosphamide control and the effect is comparable with normal control.

 

Figure 1a: Micronuclei in PCE and NCE

Figure 1b: Effect of LIPOVEDIC on chromosomal aberration after 24hr, 48hr & 72hrs of Cyclophosphamide challenge.

 

n=6, Values are expressed as Mean ± SEM, Oneway ANOVA followed by Tukeys multiple comparison test. ^***p< 0.0001 vs. normal control.

[Note: CPA-Cyclophosphamide; LIPOV-lipovedic; LCPA-lipovedic+Cyclophosphamide

CPA24, CPA48, CPA 72: Bone marrow extraction after 24, 48 and 72hrs of cyclophosphamide administration respectively.

LCPA 24, LCPA 48, and LCPA 72: Bone marrow extraction after 24, 48 and 72hrs of cyclophosphamide challenge in the mice pretreated with Lipovedic respectively]

 

 

Table 1: Effect of LIPOVEDIC on micronucleus formation after 24, 48 and 72hrs of cyclophosphamide challenge.

Group	Treatment	%MN PCE	%MN NCE	P/N
1	Normal control	0.508±0.068	0.233±0.025	0.971±0.006
2	Cyclophosphamide(100mg/kg)p.o., (24 h)	2.233±0.095***	0.817±0.112***	0.828±0.033***
3	Cyclophosphamide(100mg/kg) p.o., (48 h)	2.825±0.122***	1.167±0.123***	0.751±0.025***
4	Cyclophosphamide(100mg/kg) p.o., (72 h)	3.158±0.16***	1.533±0.133***	0.692±0.018***
5	Lipovedic (260mg/kg) p.o.	0.467±0.079	0.217±0.044	0.97±0.005
6	Lipovedic+ Cyclophosphamide(100mg/kg) p.o., (24 h)	2.142±0.189	0.792±0.109	0.843±0.016
7	Lipovedic+ Cyclophosphamide(100mg/kg) p.o., (48 h)	2.767±0.299	1.083±0.084	0.785±0.019
8	Lipovedic+ Cyclophosphamide(100mg/kg) p.o., (72 h)	3.075±0.203	1.433±0.199	0.721±0.025

n=6, Values are expressed as Mean ± SEM, Oneway ANOVA followed by Tukeys multiple comparison test. ^***p< 0.0001 vs.Normal control.

24h, 48h and 72h: Bone marrow extraction after 24, 48 and 72hrs of cyclophosphamide challenge in the mice pretreated with Lipovedic respectively.

 

Figure 1c: Chromosomal aberrations

 

 

DISCUSSION:

In the present study, an attempt is made to evaluate the genotoxicity of Lipovedic by mouse bone marrow micronucleus test and chromosomal aberration test. Other than bone marrow micronucleus test and chromosomal aberration test a few other tests like Comet assay, Ames test, Cell array, Peripheral blood micronucleus test, Sperm morphology test have been used for genotoxicity testing. Cyclophosphamide yields clearly positive results in the micronucleus test and therefore is used as a clastogenic control.² Cyclophosphamide gets metabolized to phosphoramide mustard and acrolein before it can act as a mutagenic agent to promote chromosomal aberrations. Chromosomal aberrations are due to lesions in DNA caused by phosphoramide mustard which lead to discontinuities of the DNA helix.¹¹

A time dependant significant (p<0.0001) rise in %MNPCE and % MNNCE was observed upon administration of Cyclophosphamide, which indicates Cyclophosphamide induced chromosomal damage in mouse bone marrow cells. These fragmented chromosomes were condensed to form micronuclei which are not included in the main nucleus. When the Clastogenic control (group 2, 3 and 4) was compared against normal control, there is a significant decrease in P/N ratio, mitotic index, increase  in %MNPCE,  %MNNCE, total number of aberrations which may be due to cyclophosphamide induced chromosomal damage.¹¹  The decrease in mitotic index could be due the affected cell division in the bone marrow by cyclophosphamide.⁹ Whereas when Lipovedic was administered alone in group five there was no significant decrease in  P/N ratio, increase in %MNPCE,  %MNNCE, total number of aberrations, which indicates Lipovedic to be free of genotoxicity.

 

When the results of group 6, 7 and 8 mice  are compared against the results of group 2, 3 and 4 mice respectively, it is found that, there is no significant decrease in cyclophosphamide induced clastogenicity in mice  of group 6, 7 and 8.  This may be due to the inability of Lipovedic to interfere with the cyclophosphamide induced aberrations and inability to interfere with affected cell division. Hence, Lipovedic is not antimutagenic.

 

CONCLUSION:

Lipovedic does not provide protection against mutagenicity, but it is free of genotoxicity.

 

List of abbreviations:

MNT - Micronucleus test

CAT - Chromosomal Aberration test

CPA - Cyclophosphamide

PCE - Polychromatic erythrocytes

NCE - Normochromatic erythrocytes

MN - Micronuclei

MNPCE - Polychromatic erythrocytes

MNNCE - Normochromatic erythrocytes

 

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Received on 10.02.2011       Modified on 01.03.2011

Accepted on 24.03.2011      © RJPT All right reserved