Evaluation of anti-hyperlipidemic activity of Lekhana Basti in albino rats.

Lekhana Basti (medicated enema) is a Tikshna formulation which is basically aimed for Apatarpana of the body. The present study (medicated enema) was undertaken to evaluate its anti-hyperlipidemic activity in diet-induced hyperlipidemia animals. Parameters like changes in body weight, organ weight, serum total cholesterol, serum triglyceride, serum HDL cholesterol, and serum (LDL + VLDL) cholesterol were studied to assess the effect of therapy in comparison to the control groups. Lekhana Basti was found to be ineffective in producing anti-hyperlipidemic action potently, but still found to have cytoprotective activity against hyperlipidemia induced organ damage, which was also confirmed by attenuation of cell infiltration and microfatty changes on histopathological examination.

Introduction

Hyperlipidemia is highly prevalent and is closely related to coronary heart disease which is the most common cause of death.[1] Raised cholesterol is estimated to be responsible for 18% of cerebrovascular diseases and 56% of ischemic heart diseases. Overall, these diseases account for about 4.4 million deaths (7.9% of the total).[2] Based upon the etiological factors and symptom complexes, hyperlipidemia can be stated under broad umbrella of “Santarpanjanya Vyadhi,”[3] (diseases caused due to over nutrition and sedentary lifestyle). Apatarpana is the remedy for Santarpanjanya Vyadhi.[4] Taking into consideration the treatment modalities in Ayurveda, “BASTI” seems to be the best treatment in Santarpanjanya Vyadhi as it is the fastest Apatarpana, when prepared with Apatarpaka drugs.[5] In Apatarpana also being more specific “Lekhana” is the treatment which can remove abnormally increased Sneha.[6] Lekhana is nothing but a process of emaciation.[7]

Thus, Basti prepared with Lekhana drugs, seems to be the proper treatment modality for hyperlipidemia, but the experimental data regarding anti-hyperlipidemic potential of Lekhana Basti is lacking. Considering this, the study attempts to evaluate the effect of Lekhana Basti on lipid profile in experimental animals.

Materials and Methods

Test formulation

The raw materials [Table 1] of Lekhana Basti were procured from pharmacy attached to Gujarat Ayurved University, Jamnagar, and authenticated at Pharmacognosy laboratory, Institute for Postgraduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar. Triphaladya Taila[8] was used as Sneha. Putoyavanyadi Kalka[9] was used in formulation as it is a commonly used Kalka (paste of herbs) while preparing Basti.[10] Lekhana Basti was prepared freshly just before administration by classical method.[11]

Table 1

Contents of the Lekhana basti (for preparing 12 Prasruta, i.e. approximately 1000 ml of Niruha)

Animals

Wistar strain albino rats of either sex weighing 190 ± 20 g were obtained from animal house attached to the institute. Animals were exposed to natural day and night cycles with ideal laboratory condition in terms of ambient temperature (22 ± 2°C) and humidity (50-60%). They were fed with Amrut brand rat pellet feed supplied by Pranav Agro Industries and tap water given ad libitum. All procedures and experiments were conducted in day time according to specification of the Indian National Science Academy (INSA). The experiments were carried out after obtaining the permission of Institutional Animal Ethics Committee (Approval number; IAEC/07/2010/09/MD).

Dose fixation

The quantities of ingredients in liquid form were determined in milliliters (ml) by considering the specific gravities of the basic component of the formulation and converting the grams to ml. Thus, the finally prepared 12 Prasruta Basti comes to be of 1000 ml. For present trial, half of the standard dose, i.e. Ardhamatrika Basti was considered as the adult human dose, i.e. 500 ml for Niruha. For Anuvasan Basti, Triphaladya Taila was used and the classical dose is 3 Pala (i.e. 1.5 Prasruta = 160 ml). The dose of Lekhana Basti and Anuvasana Basti were calculated by extrapolating the therapeutic dose to rat dose on the basis of body surface area ratio by referring to the table of Paget and Barnes.[12] Accordingly, the rat dose for Niruha Basti became 45 ml/kg and Anuvasana Basti was 14.4 ml/kg.

Prior to the experiment proper, to rule out any possible practical difficulty while administering this particular dose of Niruha Basti and to know, up to what region of colon this dose of Basti reaches, a pilot study was done in which two rats were used. First rat was administered with above mentioned dose of Niruha Basti and observed for any ill effect. Further, the animal was sacrificed about 5 minutes after the Basti administration with overdose of ether anesthesia and its intestinal tract was observed for presence of Basti material. The Basti contents were found to reach up to cecum. Second animal was first sacrificed with overdose of ether anesthesia and the abdomen was opened by midline incision and the large intestine was carefully exposed. Then, the Niruha Basti was given through rectum. It was confirmed that it reaches up to the ileocecal junction. Thus, the dose decided for the animals was fixed for the present study.

Lekhana Basti is used for Kaphapradhana Vikara. Acharya Charaka has quoted to give Anuvasana on every third or fifth day in “AnativriddhaVata” and “PravriddhaKapha-Pitta Avastha,” (Kapha dominant disease) respectively.[13] As hyperlipidemia is a Kaphapradhana condition, schedule of Basti is fixed as one Anuvasana followed by three Niruha and so on with one Anuvasana on last day for 21 days.

Experimental procedure

The selected animals were grouped randomly irrespective of sex into three groups. To first group, only tap water was administered without hyperlipidemic diet, whereas second and third groups received hyperlipidemic diet. The hyperlipidemic diet included hydrogenated vegetable oil (Vanaspati Ghee - ‘Raag’ brand, Batch No. (AO) VA05, Adani Wilmar Ltd., Gujarat) and cholesterol extra pure powder (Batch No. 5593 Suvidhanath Laboratories, Baroda) made into suspension in 20% coconut oil (Parachute coconut oil, Batch No. PH001 Mumbai). The suspension was administered in the dose of 0.5 ml/100 g body weight of rats, respectively, daily for 21 days (at evening hours between 4 pm-5 pm) to the rats of second and third groups. Concomitantly, second group was treated with water enema (Vehicle) whereas third group was treated with Lekhana Basti. The enema administration was done at morning hours for 21 consecutive days. Lekhana Basti was administered to the animals after doing local Abhyanga (oil massage) at lower abdomen and lower back with lukewarm Triphaladya Taila and Swedana (sudation) with hot water filled in a polythene bag, i.e. the classical enema procedure which is generally adopted clinically, was followed in experimental animals. To administer the test drug, simple rubber catheter (no. 4) attached to 20 cc plastic syringe was used in case of Niruha while the same catheter attached to 3-ml plastic syringe was used in case of Anuvasana Basti. Water enema control group was given tap water enema by using same instrument as of Niruha Basti.

On 22nd day, after overnight fasting, animals were weighed again and anesthetized with diethyl ether. Blood was drawn from supraorbital plexus by puncturing and sent to biochemical laboratory for biochemical estimations. Then, the rats were sacrificed by overdose of anesthesia and important organs like liver, kidney, aorta, and heart were dissected out. Liver, kidney, and heart were weighed and they were (including aorta) transferred to fixing solution (10% formalin) for histopathological examinations. Biochemical parameters like serum total cholesterol,[14] serum triglyceride,[15] serum HDL cholesterol,[16] and serum (LDL + VLDL) cholesterol were studied. Serum (LDL + VLDL) was calculated by subtracting HDL cholesterol value from total cholesterol instead using both values separately, as in rats whose serum cholesterol is <100 mg/dl Friedewald formula overestimates LDL levels.[17] Atherogenic Index (AI) was calculated by using the formula; AI = (Total Cholesterol/HDL-C).[18]

Statistical analysis

The data generated during the study were subjected to student's ‘t’ test for unpaired data to assess the statistical significance. ‘P’; value less than 0.05 was considered as statistically significant.

Results

Data related to effect of Lekhana Basti on body weight of albino rats in different experimental groups have been provided in Table 2. In normal control rats, a marginal decrease in body weight was observed in comparison to their initial values. In contrast, significant increase in body weight was observed in water enema + Cholesterol control rats in comparison to both initial values as well as water control group. In Lekhana Basti-treated group, a marginal increase in body weight was observed in comparison to its initial value; however, in comparison to water enema + cholesterol group significant decrease in body weight was observed.

Table 2

Effect of Lekhana Basti on the body weight in albino rats

The data related to the effect of Lekhana Basti on weight of different organs have been shown in Table 3. Marginal increase of relative weight of liver was found in water enema + cholesterol control group in comparison to water control group which was found to be statistically non-significant. However, increase in Lekhana Basti group was statistically significant when compared to the water enema + cholesterol group.

Table 3

Effect of Lekhana Basti on organ weight in albino rats

Marginal decrease of relative weight of heart was found in water enema + cholesterol control group in comparison to water control group which was found to be statistically non-significant. In Lekhana Basti-treated group, a marginal and statistically non-significant increase in relative weight of the heart was observed in comparison to water enema + cholesterol control group. Significant decrease in relative weight of kidney was observed in water enema + cholesterol control group in comparison to water control group. Administration of Lekhana Basti significantly reversed relative weight of kidney in comparison to water enema + cholesterol control group.

The data related to the effect of Lekhana Basti on serum lipid profile have been shown in Table 4. Statistically non-significant increase in serum cholesterol was seen in water enema + cholesterol control group in comparison to water control group. Lekhana Basti group also have shown marginal increase in serum cholesterol in comparison to water enema + cholesterol control group which was found to be statistically non-significant. Statistically significant increase in serum triglyceride was seen in water enema + cholesterol control group in comparison to water control group. Treatment with Lekhana Basti apparently decreased serum triglyceride level in comparison to water enema + cholesterol; however, the observed change was found to be statistically non-significant.

Table 4

Effect of Lekhana Basti on lipid profile in albino rats

HDL was seen to be decreased in water enema + cholesterol control group when compared to water control group. Treatment with Lekhana Basti apparently increased serum HDL level in comparison to water enema + cholesterol; however, the observed change was found to be statistically non-significant. Administration of cholesterol and treatment with Lekhana Basti did not affect the serum (LDL + VLDL) and AI to significant extent.

Discussion

Administration of hyperlipidemic diet led to significant increase in body weight in albino rats. Lekhana Basti was found to be effective in weight reduction as seen from the significant decrease in body weight in Lekhana Basti-treated group as compared to the water enema + cholesterol control group. This indicates that the test drug has the antagonizing effect against hyperlipidemic diet-induced changes in the body weight. This effect may be attributed to the Shodhana property of the Lekhana Basti that removes excess Kleda from the body. Moreover, the Tikta, Katu, Kashaya Rasa; Ushna Virya, (purificatory) and Katu Vipaka of the Lekhana Basti has an additional action through Rasapanchaka.

Decrease in the organ weight is indicative of degenerative changes or loss of tissue of that particular organ, increase in the weight may be due to hyper functioning of that organ or edematous changes. In present study, administration of hyperlipidemic diet did not affect the weight of heart and liver to significant extent in comparison to normal rats; however, it caused significant decrease in weight of kidney. Furthermore, histopathological studies of the kidney sections from cholesterol control group show fatty degenerative changes, this may be the reason for observed decrease in kidney weight. Administration of Lekhana Basti significantly decreased the weight of kidney and histopathological studies of the kidney sections of this group show almost normal cytoarchitecture. This indicates cytoprotective property of Lekhana Basti.

Lipid profile is the objective measurement of body lipids and AI specifies the cardiac risk. In present experimental trial, serum triglycerides were increased significantly in water enema + cholesterol control group; however, the marginal non-significant decrease was seen in Lekhana Basti group. HDL was found to have non-significant increase as compared to water enema + cholesterol control group which was having non-significant decrease in serum HDL as compared to water control group. But these marginal non-significant changes are not sufficient to prove protective role of Lekhana Basti. This data regarding lipid profile prove Lekhana Basti ineffective in producing anti-hyperlipidemic action more as compared to water enema + cholesterol control group. However, the non-significant increase in serum cholesterol and (LDL + VLDL) complex in water enema + cholesterol control group which was expected to be significant suggests lukewarm water enemas effect on these parameters which can be justified by the colon cleansing leading to protection from autointoxication which is a cause of many metabolic syndromes including lipid disorders.[19] Just a marginal decrease in AI in Lekhana Basti group found as compared to water enema cholesterol control group proves it ineffective in prevention of cardiac risk profile more than the water enema + cholesterol control group.

Histopathological sections from control group show normal cytoarchitecture [Figures 1, 4, and 7]. In contrast, hyperlipidemic diet produced perivascular cell infiltration and microfatty changes in liver, cell infiltration and fatty changes in kidney, and cell infiltration and fatty changes in majority of sections of heart of animals in water enema + cholesterol control group [Figures 2, 5, and 8]. These changes were markedly attenuated by treatment with Lekhana Basti [Figures 3, 6, and 9].

Figure 1

Photomicrograph of representative sections of Liver of albino rat from water control group. (1 × 400 magnification). Hc-Hepatocytes, S: Sinusoid. Note-Normal cytoarchitecture

Figure 4

Photomicrograph of representative sections of Heart of albino rat from water control group. (1 ×400 magnification). Mc: Myocardium. Note-Normal cytoarchitecture

Figure 7

Photomicrograph of representative sections of kidney of albino rat from water control group. (1 ×400 magnification). Cp: Capsule; Ct: Convoluted tubule. Note-Normal cytoarchitecture

Figure 2

Photomicrograph of representative sections of Liver of albino rat from Water Enema + Cholesterol control group. (1 ×400 magnification). Hc: Hepatocytes; CI: Cell infiltration. Note-Diffused microfatty changes and perivascular cell infiltration

Figure 5

Photomicrograph of representative section of Heart of albino rat from Water Enema + Cholesterol control group. (1 ×400 magnification). Fc: Fatty changes; En-Endocardium. Note-Fatty changes

Figure 8

Photomicrograph of representative section of kidney of albino rat from Water Enema + Cholesterol control group. (1 ×400 magnification). CI: Cell infiltration, Fc: Fatty changes. Note-Cell infiltration and fatty changes

Figure 3

Photomicrograph of representative sections of Liver of albino rat from Lekhana Basti group. (1 ×400 magnification). Cv: Central vein. Note-Almost normal cytoarchitecture

Figure 6

Photomicrograph of representative section of Heart of albino rat from Lekhana Basti group (1 ×400 magnification). Mc: Myocardium. Note-Almost normal cytoarchitecture

Figure 9

Photomicrograph of representative section of kidney of albino rat from Lekhana Basti group (1 ×400 magnification). Note-Almost normal cytoarchitecture

Triphala Kashaya is the main ingredient of this formulation. Triphala is proven hypolipidemic combination in experimentally induced hypercholesteremic rats.[20] Moreover, contents of Triphaladya Taila like sesame,[21] Haridra,[22] Guggulu, 23 etc., are also known anti-hyperlipidemic agents. Thus, the moderate anti-hyperlipidemic changes and protection from the organ damage due to hyperlipidemia can be attributed to the combined effect of all the components of Lekhana Basti. Also, it is hypothesized that Basti has action on enteric nervous system (ENS). It is recognized that the ENS has a unique ability to mediate reflex activity independently of input from the brain or spinal cord.[24] The ENS contains sensory receptors, primary afferent neurons, interneuron's, and motor neurons. The events that are controlled, at least in part, by the ENS are multiple and include motor activity, secretion, absorption, blood flow, and interaction with other organs such as the gall bladder or pancreas.[25] Basti has the prime function of colon cleansing. The colon cleansing has the effect on whole body that can be compared to the Srotoshodhana. The rationale for colon cleansing is the concept of “auto-intoxication,” the idea that food enters the intestine and rots.[26] Thus, cytoprotective activity of the Lekhana Basti can also be attributed to the colon cleansing and ENS stimulation action of Bastikarma.

Conclusion

On the basis of this study, it can be concluded that Lekhana Basti is ineffective in producing anti-hyperlipidemic action significantly, but still it has cytoprotective activity which prevented hyperlipidemia-induced organ damages. Also, significantly lower the values of body weight as compared to water enema + cholesterol control group which suggests its anti-obesity activity in comparison to ant-hyperlipidemic activity.