Evaluation of Ratnaprash for its effect on strength, stamina and fatigue using swim endurance test and biochemical estimation in swiss albino mice.

CONTEXT: Traditional medicines have been considered as important resources for postponing fatigue, accelerating elimination of fatigue related metabolites and improving physical ability. Rasāyanās or rejuvenative therapies are mentioned as one of the eight clinical specialties in Ayurveda for attaining longevity, healthy life and regulation of bodily balance. Eventhough more detailed studies are needed to confirm the claims of benefits in the light of evidence based research, Ratnaprash, a herbo-mineral rasāyana formulation, is proposed here to be an antifatigue supplement that is good in promoting strength and stamina.
MATERIALS AND METHODS: In the present study, anti fatigue, strength and stamina enhancing properties of Ratnaprash were examined based on swim endurance capacity and the change in biochemical parameters in Swiss Albino mice. Treatment groups were orally administered Ratnaprash at various test doses (500, 1000, 2000 mg/Kg per day), while the control group received distilled water at similar dose volumes. Effect of therapy was evaluated after 28 days of treatment.
RESULTS: At the end of study period, the swimming times to exhaustion were longer in the treated groups than in the control group. Plasma lactate levels of treated groups were lower than those of the control group (P < 0.05) while tissue ATP levels were higher. These effects were dose dependent and the strongest effect was seen in groups treated at 1000 mg/Kg.
CONCLUSION: Ratnaprash enhanced the forced swimming capacity of mice and exhibited elevated anti-fatigue activity, reduced blood lactate levels and increased tissue ATP levels in preclinical models in comparison to vehicle control, exhibiting possible role in increasing strength and stamina and contributing anti-fatigue activity.

INTRODUCTION

Strength and stamina are integral components of a healthy human being. Limited stamina restricts a person's ability to exert in terms of limit in the length of time one can apply energy to an activity (endurance) or a limit in being able to exert the effort needed for a particular task (strength). Limited strength and stamina may lead to fatigue, a complex phenomenon that can be described as 'time dependent - exercise induced reduction in the maximal force generation capacity of a muscle’.[1] Fatigue is characterized as physical and/or mental weariness resulting in negative impacts on work performance and exercise intensity, family life and social relationships. Hard work or intense exercise can lead to the production and accumulation of excess reactive free radicals, which results in oxidation stress injury to the body. Exhaustion theory suggests that energy source depletion and excess metabolite accumulation lead to fatigue. Long term physical and mental fatigue leads to health damage and chronic fatigue.[23456]

Recent studies suggest that the supplementation of exogenous dietary antioxidants can decrease the contribution of exercise-induced oxidative stress and improve an animal's physiological condition.[78] The quest for safe and effective anti fatigue methods has drawn the attention of researchers towards traditional and alternative medicine.[9] Rasāyanās or rejuvenative therapies form one of the eight clinical specialties in Ayurveda. These therapies help attain longevity, healthy life and help regulate the body balance. Many of the medicines used as rasāyanās are rejuvenators, nutritional supplements and possess strong antioxidant activity. They also have antagonistic actions on the oxidative stressors, which give rise to the formation of different free radicals.[1011] Presently, research on specific nutrients from traditional medicine and rasāyana herbal supplements are needed to find agents that reduce metabolite production and/or improve energy utilization.[12] Even though more detailed studies are needed to confirm the claims and its traditional benefits in the light of evidence based research, Ratnaprash, one such herbo-mineral rasāyana formulation, is proposed here to be an antifatigue supplement that is good in promoting strength and stamina. Previously, Ratnaprash was found to be safe in Repeated Dose 28-day Oral Toxicity Studies.[13] The aim of the present study is to evaluate/validate the strength, stamina and fatigue related properties of Ratnaprash utilizing an animal model.

MATERIALS AND METHODS

Study product

Ratnaprash is a herbo-mineral Ayurvedic Lehya (linctus) formulation. It comprises over forty herbs in appropriate proportions. The ingredients of Ratnaprash act in synergy and have individual roles in providing key health benefits. Ingredients of Ratnaprash such as Brāhmī, Mūsalī, Keśara, Abhraka, Muktā piṣṭi, Ātmaguptā, Āmlakī, Aśvagandhā and Śatāvarī have been documented to possess rasāyana properties and to be good for increasing strength, stamina, vigour and overall health.[14151617] The composition of Ratnaprash and its proximate analysis are given in Tables 1 and 2.

Table 1

Contents of Ratnaprash* (with part used)†

Table 2

Proximate analysis of Ratnaprash

Experimental animals

The study was carried out in the Pharmacology Division of Althea Lifesciences Ltd, New Delhi - 110029, India. Thirty two (32) 8–14 weeks old male Swiss albino mice[18] (Mus musculus), weighing 32–48 g, procured from Althea Lifesciences Ltd were used for present study with prior approval from institutional animals ethics committee (IAEC approval no. IAEC/22/209, Date of approval - 6/SEP/2012). They were given free access to RO drinking water and conventional feed (Golden feeds, New Delhi); and were acclimatized to laboratory conditions (21.8 to 24.9°C temperature, 51 to 62% humidity, 12 h light and dark cycle) for 15 days before study initiation.

Experimental design

After acclimatization, Animals were randomized based on Swim Endurance (SE) time in four groups on day 0 (baseline) comprising eight animals each as:

Group 1: Control group with mice given distilled water for 28 days (G1)

Group 2: Mice treated with Ratnaprash (500 mg/Kg) for 28 days (G2)

Group 3: Mice treated with Ratnaprash (1000 mg/Kg) for 28 days (G3)

Group 4: Mice treated with Ratnaprash (2000 mg/Kg) for 28 days (G4).

Test substance was prepared in distilled water and fed orally using gavage from Day 1 - 28 consecutively at dose volume of 10 ml/Kg, q.i.d. Group (G1) served as control receiving only distilled water at similar dose volume. Ratnaprash is an Ayurvedic lehya formulation. Human dose of lehya was used as the basis to calculate the animal dose.[19] Different dose levels were chosen to observe any dose response effect.

The swimming exercise of mice was measured with a plastic round tub (63 × 49 × 35cm) filled with water maintained at 34 ± 1°C.[20] All the animals were allowed to swim till exhaustion. End point of SE test was considered when the mouse drowned more than thrice.

Determination of swim endurance (SE) time and antifatigue activity

Swim Endurance (SE) test was performed on Day 0, 14 and 28; Percent antifatigue activity was calculated using the formula:

% Anti fatigue Activity = (Test SE Time – Control SE Time)/Control SE Time ×100

Biochemical analysis

Estimation of blood lactate levels

Following SE test on Day 28, blood samples were collected from all the animals of groups G1-G4, and the serum was subjected to lactate estimation using Kit Method (Kit: BioVision Inc. Catalog #K627-100; Lactate Assay Kit II, Method: Serum samples were diluted with lactate assay buffer; reaction mix was added to each well containing the lactate standard and test samples and incubated in the reaction mix for 30 min at room temperature, Optical Density (OD) reading was measured at 450 nm in a micro-plate reader). Blood lactate levels were calculated for each animal and the mean value was tabulated for each group using a standard error mean (SEM). The % change was calculated with respect to vehicle treatment group.

Estimation of tissue adenosine triphosphate (ATP) levels

Following SE test on Day 28, thigh muscle of each mouse was dissected and tissue samples were collected from all the animals in the groups G1-G4 and subjected to tissue ATP estimation using Kit Method (Kit: BioVision Inc. Catalog #K354-100; ATP colorimetric Assay, Method: Tissue samples were homogenized and centrifuged ice cold at 15,000×G for 2 minutes and supernatant was collected, reaction mix was added to each well containing the ATP standard and test samples and incubated at room temperature for 30 minutes, protected from light. OD reading was measured at 570 nm in a micro-plate reader.) Tissue ATP levels were calculated for each animal and mean value was tabulated for each group with SEM. The % change was calculated with respect to vehicle treatment group. Group wise mean SE time was calculated and represented in tabular and graphical forms.

All data were expressed in Mean ± SEM and analyzed by one-way ANOVA for statistical significance.

OBSERVATIONS AND RESULTS

Effect on swim endurance test

The results were analyzed in two parts - (i) effect on SE Time and (ii) effect on anti-fatigue activity. At the time of baseline randomization, no statistical difference in SE time was obtained among the study groups. In Ratnaprash treated groups, animals treated at test doses of 500 and 1000 mg/Kg exhibited an increase in mean SE time on day 14 and 28 as compared to baseline [Table 3 and Figure 1]. Anti-fatigue activity of Ratnaprash on Day 14 and 28 was found to be elevated to 9.9 and 50.0 % and 31.0 and 51.4 %, respectively at doses of 500 and 1000 mg/Kg in comparison to vehicle control [Table 4 and Figure 2]. Results indicated that treatment with Ratnaprash at the above doses lengthened SE time in comparison to vehicle control to exhibit possible role in contributing anti-fatigue activity.

Table 3

Mean SE time (min) elevation

Figure 1

Elevation in Mean Swim Endurance (SE) Time after Treatment with Ratnaprash

Table 4

Percent elevation in anti.fatigue activity

Figure 2

Percent elevation in anti-fatigue activity in treated groups

Effect on biochemical parameters

Observations on swimming induced biochemical changes revealed a significant reduction in the ratio of blood lactate levels in treated animals at all the three test doses in comparison to control group where the ratio remained unchanged. Maximum reduction was seen at doses of 1000 mg/Kg followed by 500 mg/Kg. Results indicated that after swimming on day 28, the concentration of blood lactate in animals treated with Ratnaprash were significantly lower than that of control group [Table 5 and Figure 3].

Table 5

Percent decrease in blood lactate levels

Figure 3

Percent decrease in blood lactate levels in treated groups

Estimation of tissue ATP levels in treated animals after swimming on day 28 showed an increase at all the tested dose levels of Ratnaprash in G2-G4 in comparison to vehicle control. The effect was dose dependent and maximum increase was observed at doses of 1000 mg/Kg followed by 500 mg/Kg [Table 6 and Figure 4]. At higher test doses, the decrease in blood lactate and increase in muscle ATP levels was observed to be lesser.

Table 6

Percentage increase in muscle ATP levels

Figure 4

Percent increase in muscle ATP levels in treated groups

DISCUSSION

The present study investigates the anti fatigue activity and strength and stamina related properties of Ratnaprash in comparison to vehicle control in male Swiss albino mice. The swimming exercise was employed to evaluate anti-athletic fatigue activity of Ratnaprash in mice as it is commonly accepted that swimming is an experimental exercise model.[2122]

At the end of study period, it was observed that the average swimming time of animals treated with Ratnaprāśa at 500 and 1000 mg/Kg increased with elevation of anti fatigue activity on day 14 and 28, in comparison to vehicle control, indicating Ratnaprash could lengthen the swim endurance time. Effect was more pronounced at dosage of 1000 mg/Kg. Cumulative higher doses could have contributed in other extra pyramidal response which is independent of anti fatigue activity.

Swimming exercise is known to induce biochemical changes. Swimming exercise induced biochemical changes were measured in order to determine and understand anti-fatigue mechanism.[23] In the present study, blood lactate levels in the Ratnaprash treated groups were found to be lesser than the control group indicating that Ratnaprash could effectively increase the swim endurance time and postpone the appearance of fatigue at the dosage of 500 and 1000 mg/Kg. Tissue ATP level was found to be increased in both the treated groups in comparison to vehicle control at the above doses.

Fatigue is associated with many physiological factors including reduced neural input (central and peripheral) and disruptive metabolic changes in skeletal muscle such as lactic acidosis and the production of oxidative free radicals.[4] Glycolysis of carbohydrates under anaerobic conditions is the main energy source for intense exercise in a short period of time and produces blood lactate as end product.[24] Therefore, blood lactate level is one of the important indicators for determining fatigue. Decrease in blood lactates after swimming can be used as an indicator for the degree of fatigue. In the current experiment, reduced blood lactate level in the Ratnaprash treated groups reflect the energy expenditure indicating prolonged swimming activity and is one of the important indicators for judging the degree of fatigue. ATP level in muscles is another important biochemical parameter related to muscle fatigue. The effect was found to be dose-dependent, strongest effect being with 1000 mg/Kg dose.

Aerobic metabolism is more efficient than anaerobic glycolysis in producing ATP which indicates antioxidants assist in the undisrupted oxygen supply that creates the aerobic environment. Besides inhibiting cell damage, antioxidants may enhance the overall physical performance under the stress conditions.[25] In the present experiment, the ATP level in muscle was found to be increased in 500 and 1000 mg/Kg treated groups in comparison to vehicle control suggesting that Ratnaprash possibly has antioxidant potential and could have resulted in anti-fatigue effects on mice. The above effect was found to be dose-dependent, strongest effect being with 1000 mg/Kg dose. Thus, contribution of anti-fatigue activity for Ratnaprash was established in low and intermediate doses.

CONCLUSION

Ratnaprash supplementation exhibited prolongation of swim endurance time against forced swim, reduced accumulated blood lactates and enhanced tissue ATP levels in preclinical models. The effect was dose-dependent, and strongest effects were observed at doses of 1000 mg/Kg. It can be concluded that Ratnaprash, at the treated doses could appreciably lengthen the swim endurance time to exhibit possible role of treatment in contributing anti-fatigue activity through increasing the ATP level in the muscle and utilization of blood lactate against swim.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.