Hepatoprotective potential of kumaryasava and its concentrate against CCl4-induced hepatic toxicity in Wistar rats.

OBJECTIVE: Kumaryasava (KS) is a marketed Ayurvedic formulation containing Aloe vera as the main ingredient. It has been used widely for the treatment of liver disorders; however, there is a lack of modern scientific data on hepatoprotection. The recommended dose of KS is high and up to 60 mL/day. The present study describes the preparation of new KS concentrate and evaluation of comparative hepatoprotective activity of KS and prepared KS concentrate at one-third of KS dose against CCl4-induced hepatic toxicity.
MATERIALS AND METHODS: Animals were divided into different groups (n = 6). The first group received normal saline (control) 1.0 mL/Kg/day p.o. for 10 days. The second group (toxicant) was given normal saline 1.0 mL/Kg/day p.o. for 10 days with CCl4 in olive oil (1:1 v/v) at 1.0 mL/Kg/day p.o. Third, fourth, and fifth groups received KS, KS concentrate and a marketed formulation as standard) at doses of 5.0 mL/Kg/day p.o., 1.6 mL/Kg/day p.o., and 100 mL/Kg/day p.o. (tablet suspended in water using 0.1% carboxymethyl cellulose) respectively for 10 days along with CCl4 as given to the toxicant group. On the 11(th) day, blood was withdrawn from retro-orbital plexus and serum was separated for biochemical estimation of serum glutamic-oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), alkaline phosphatase (ALP), and albumin levels. Later, animals were sacrificed under high dose of anesthesia to remove liver tissue, which were removed and washed with ice cold saline for the estimation of lipid peroxidation. Liver tissue from each group was also fixed in 10% formalin for histopathological analysis.
RESULTS: Results demonstrated that both KS and KS concentrate showed the protection against CCl4-induced hepatic toxicity. This was evident from the reduction in serum SGOT, SGPT, ALP levels, and elevation in serum albumin levels observed post treatment of CCl4 treated rats with KS and KS concentrate, which were supported by histopathological data.
CONCLUSION: KS concentrate can be a useful hepatoprotective formulation which may help in reducing the high dose of KS to approximately one-third of the recommended dose.

Ayurveda is the plant-based indigenous system of medicine practiced in India since ancient times.[1] However, there is a growing need for scientific evidence toward the efficacy of the Ayurvedic formulations.[2] Asavas (fermented infusions) are considered as unique and valuable therapeutics in Ayurveda.[3] These are the medicinal preparations made by soaking the drugs (powder or mixing decoction) in a solution of jaggery for a specified period of time. It undergoes the fermentation process by generating alcohol, which facilitates the extraction of active principles of drugs. The alcohol generated during the process also serves as a preservative.[4] Kumaryasava (KS) is a marketed Ayurvedic herbal formulation mentioned in the Ayurvedic formulary of India. It is a self-fermented galenical containing about 40–50 crude drugs and Aloe vera as the main ingredient.[5] It is one of the most widely used over the counter products and used for the treatment of liver disorders.[6] However, there are lack of modern scientific data for the use of KS in the treatment of liver disorders. Another issue in the use of KS is the high dose of the product which goes up to 60 mL of the daily dose.

The present manuscript describes the preparation of new KS concentrate and comparative evaluation of hepatoprotective activity of KS and KS concentrate against CCl4 induced toxicity in Wistar rats as per the method described by Bhoopat et al.[7]

Materials and Methods

KS and KS concentrate were obtained as a gift sample from Dabur India Ltd. The KS concentrate was prepared by transferring 450 mL of kumaryasva in 1000 mL round bottom flask and evaporated to approximate 50–60% of the original sample below 80°C temperature in the rotary evaporator. Further, concentrated sample was transferred into a 1000 mL beaker and kept on water bath (maintained at 90–100°C) for 2–3 h, so that, 30% w/w of its original moisture remained.

Hepatoprotective activity

The hepatoprotective activity of KS and KS concentrate were carried out on Wistar albino rats as per the protocol described by Bhoopat et al.[7] Thirty animals were obtained from the central animal house facility of Hamdard University. The study was approved by the institutional animal ethic committee and carried out strictly as per the guidelines.

The first group received normal saline (control) 1.0 mL/Kg/day p.o., for 10 days. The second group (toxicant) was given normal saline 1.0 mL/Kg/day p.o., for 10 days with CCl4 in olive oil in 1:1 ration given at 1.0 mL/Kg/day p.o., Third, fourth, and fifth groups received KS, KS concentrate and new Livfit (standard) at doses of 5.0 mL/Kg/day p.o., 1.6 mL/Kg/day p.o., and 100 mL/Kg/day p.o., (tablet suspended in water using 0.1% CMC), respectively for 10 days along with CCl4 as given to the toxicant group.

On the 11th day, blood was withdrawn from the retro-orbital plexus, and serum was separated for biochemical estimation. Later, animals were sacrificed under high dose of anesthesia to remove the liver tissue which were removed and washed with ice cold saline for biochemical estimation. Liver tissue from each group was also fixed in 10% formalin for histopathological analysis. In the serum glutamic-oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), alkaline phosphatase (ALP), and albumin estimation were carried out using commercially available kits. Lipid peroxidation was estimated in the liver tissue using the method described by Okhawa et al.[8]

Statistical analysis

The values were expressed as mean ± standard deviation. The statistical analysis was carried out by one-way analysis of variance followed by Tukey's test. P < 0.05 were considered as significant.

Results and Discussion

Toxicant group showed significantly elevated (P < 0.001 vs. control) levels of SGOT, SGPT, serum ALP, and malondialdehyde (MDA), whereas significantly lower (P < 0.001 vs. control) level of serum albumin was compared to control group. Significantly lower (P < 0.001 vs. control) level of serum albumin was observed in the toxicant group compared to control.

Treatment with KS concentrate showed significant reversal (P < 0.001 vs. toxicant) in SGOT, SGPT, ALP, and MDA levels as well as in serum albumin level. Similar results were obtained in KS and standard treated groups. Statistical analysis revealed that for different biochemical parameters showed that there was the insignificant difference between KS and KS concentrate as well as in standard (new Livfit) treated groups.

The histopathological evaluation of liver tissue of different treatment groups was carried out, which showed the normal architecture of liver tissue [Figure 1a] in the control group. CCl4 treated toxicant [Figure 1b] showed a high degree of cellular necrosis and vacuolization. Histopathology after treatment with KS [Figure 1c] revealed the reduced hepatic damage with little vacuolization. KS concentrate and standard groups showed the normal histological features [Figure 1d and e].

Figure 1

Histological study of liver tissue in different treatment groups showing (a) Control group with normal hepatic tissue architecture, (b) toxicant group hepatocellular necrosis with high vacuolization, (c) kumaryasava treated group showing reduced hepatic damage with little vacuolization, (d) kumaryasava concentrate treated group showing normal histology of liver tissue, (e) liver tissue from standard group treated with new Livfit showing normal histological architecture

The present study is the first study undertaken to prepare and compare the efficacy of KS concentrate with its traditional formulation with the purpose to reduce its dose to one-third of its administration (currently given in 60 mL/day). A significant increase in the level of SGOT, SGPT, and ALP indicated the hepatic damage caused by CCl4 [Table 1].[9] Additionally, the hypoalbuminemia was also observed in CCl4 treated rats which may indicates the liver cirrhosis. The increased levels of MDA indicated the lipid peroxidation and membrane damage due to oxidative stress.[10]

Table 1

Effect of different treatment groups on SGOT, SGPT, serum albumin, and serum ALP level

KS as well as KS concentrate lowered the increased level of these enzymes indicating a reversal of CCl4 induced liver damage. Similarly, a reversal of hypoalbuminemia by KS and KS concentrate reinforces their protective effect. KS and KS concentrate restored MDA level toward normal indicating interference with oxidative damage and their anti-lipid peroxidative property. Further, KS and KS concentrate may stabilize the plasma membrane leading to the reduced extent of lipid peroxidation.[11] Results of biochemical estimations were supported by similar results of the histopathological examination.

The KS concentrate (and KS) contains Aloe barbadensis and honey as its major constituents. Previously, A. barbadensis has been reported to possess hepatoprotective activity due to its antioxidant activity.[12] Similarly, in a comparative study, honey has been reported to be more hepatoprotective than black seed. These agents can have contribution toward the hepatoprotective effect of the formulation.

Comparative analysis during the study showed that there was statistically insignificant difference between the efficacy of KS and KS concentrate. For optimum outcome, approximately 60 mL of the KS is to be taken by oral route per day. However, the equivalent efficacy was observed with kumaryasava concentrate (20 mL) in the animal study indicates that it may help in reducing the high dose of KS to approximately one-third of the traditional dose.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.