Rasayana effect of Guduchi Churna on the life span of Drosophila melanogaster.

INTRODUCTION: The Drosophila melanogaster is one of the most extensively studied animal models for understanding the process of aging and longevity. This fly is being used for genetic studies since almost a century. Guduchi (Tinospora cordifolia [Wild.] Miers.) is an Ayurvedic drug used as Rasayana (rejuvenation) due to its antiaging effect. AIM: To explore the potential of Guduchi Churna (powder of T. cordifolia) on the life span of D. melanogaster using adult feeding method.
MATERIALS AND METHODS: For experimental setups, the regular food media were mixed with Guduchi Churna in different concentrations (0.25 g/100 ml, 0.50 g/100 ml, and 0.70 g/100 ml). Flies were exposed to these concentrations for 30 days.
RESULTS: Increase in the lifespan was observed in both parent and F-1 generation. Applying of ANOVA revealed significant difference between control and drug groups. The longevity effect, in the drug group was seen even on F-1 generation.
CONCLUSION: Guduchi Churna enhances the life span of D. melanogaster in both parent and F-1 generation.

Introduction

Rasayana (rejuvenation therapy), one of the most essential branches of Ayurveda,[1] practiced effectively and extensively since ages, is a group of medicinal preparations which are immunostimulants and capable of preventing the causation of many ailments such as untimely aging. The term Rasayana refers to nourishment or nutrition. Rasayana drugs act essentially on nutrition dynamics and rejuvenate the body and psyche. Rasayana drugs also promotes intellect and strength, prolongation of life and helps in the prevention of diseases.[2]

Aging is a process of unfavorable progressive changes associated with decline in vigor resulting in death. In humans, aging represents the accumulation of changes in a human being over time.[3] Aging is one among the largest known risk factors for most human diseases. However, every individual wants to stay healthy, look younger, and live longer. In recent years, scientists are interested to discover the scientific clues to the aging process and to determine if the process of aging is genetically or environmentally controlled, or by both. To this respect, different food components have been shown to increase life span of many organisms; however, no conclusive evidence in favor of any particular food component has yet been established.

The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics.[4]

Guduchi (Tinospora cordifolia [Wild.] Miers.) is an Ayurvedic drug used for Rasayana Chikitsa.[5] The biological effect of this drug on life span has not been tested till now, but only being prescribed to human beings. Therefore, to revalidate the concept of Rasayana Chikitsa and in particular to assess the longevity effect of Guduchi Churna, the present study was carried out using D. melanogaster as the test system.

Materials and methods

Preparation of food media

Wheat cream agar medium was used as a food medium for D. melanogaster. This medium was prepared by boiling 1000 ml of distilled water along with 100 g of jaggery. After complete dissolution of jaggery, 100 g of wheat flour (Suji or Rava) was added to the medium and then 10g of agar and 7.5 ml of propionic acid (antifungal) were added gently. The medium was distributed to glass vials of 8 cm × 2.5 cm size. The mouth of the vials was kept closed with sterilized cotton.[6] One day later, one or two drops of yeast solution were added to the food media. This medium was used after 24 h. At every step, heat vials were used for preparing medium, to prevent outbreak of pests and diseases. This culture was used as control culture. For experimental culture, different dose were calculated for T. cordifolia. There was no dietary restriction for the flies in both control and experimental cultures.

Preparation of cultures

To study the impact of T. cordifolia on longevity of D. melanogaster (Mysore strain), flies were obtained from Drosophila stock center, Department of Zoology, Mysore. The pure culture of these flies was maintained under standard food medium.[7] The effect of T. cordifolia was studied by adult feeding method. For this purpose, stocks of the flies were built up for five to six generation from isofemale line. The virgin females and bachelor males emerged from the normal media were isolated under ether anesthesia within 3 h of eclosion. Four groups were made and each group contain equal number of male (10) and female (10) flies and was marked as control, V group, S group, and T group. Test group flies were fed with 0.25g/100 ml, 0.50g/100 ml and 0.70g/100 ml of test drug with food media respectively. These flies were transferred to 8 cm × 2.5 cm autoclaved glass culture vials (to avoid contamination) containing equal quantity of normal food media used for Drosophila culture.

Experimental design

The stems of Guduchi was procured from its natural habitat, authenticated at Pharmacognostical Laboratory of IPGT and RA, Jamnagar, and made into fine powder (mesh size #72). For experimental setups, the regular food media were mixed with Guduchi Churna in different concentrations (0.25 g/100 ml, 0.50 g/100 ml, and 0.70 g/100 ml). For longevity (life span) study, twenty virgin females and bachelor males from all the four cultures were used. Daily, these flies were transferred to the fresh vials of all the three cultures and a controlled experiment (without any drug content) was also run simultaneously. Flies were exposed to experimental cultures of respective concentrations for 30 days. Later on, each vial was minutely monitored and the number of dead flies were recorded. For each experiment, life span of each fly was noted by simply noting the survivability of flies. All these experiments were conducted inside a BOD (Bio-Oxygen Demand) incubator with constant temperature (25°C) and other environmental conditions. The results were calculated in terms of number of days.

Statistical analysis

The data were statistically analyzed by one-way ANOVA test.

Results

Results revealed significant effect of Guduchi Churna on lifespan of D. melanogaster. Results clearly indicates that there was 85.91% increase in life span of T group and the mean life span was 53.08 ± 0.14 [Table 1 and Figure 1].

Table 1

Effect of Guduchi Churna in different concentration on life span of male and female Drosophila flies (n=100)

Figure 1

Comparison between percentage survivability among all the groups

The linear plot [Figure 1] shows that in control group, there was no survivability of flies in 30–40 days. In V group, the rate of survivability was 62% for 80–90 days. In S group, the rate of survivability was 47% for 70–80 days. In T group, the rate of survivability was 39% for 60–70 days. Slight differences in the rate of survivability of D. melanogaster flies were found in both the sexes [Table 2]. The life span of female flies was slightly higher when compared to male flies in all the groups [Figure 2].

Table 2

Comparative effect of Guduchi Churna in different concentration on life span of male and female Drosophila flies

Figure 2

Comparative effect of Guduchi Churrna in different concentration on life span of male and female flies

The difference in mean life span of control group and V group flies was 55.50 days which is statistically significant (P > 0.001). The difference of mean between control group and S group was 44.40 days, which again reflecting toward the positive effect of drug on the longevity of flies (P > 0.01). However, comparison between control and T group shows statistically insignificant result (P < 0.05) though the difference of mean was 23.80 days [Table 3].

Table 3

Mean difference of longevity in different groups, multiple comparisons versus control group (Holm-Sidak method)

In F-1 (first generation) of control group, the life span of male and female flies was 24 days. The highest life span of flies (F-1 generation) was observed in 0.25 g/100 ml concentration, i.e., 46 days. In 0.50 g/100 ml concentration (F-1 generation), the life span of flies was 33 days. In the highest concentration, i.e., 0.70 g/100 ml (F-1 generation), the rate of survivability is less compared to other drug groups as the dose of Guduchi Churna increased, the life of flies was decreasing [Table 4].

Table 4

Comparative effect of Guduchi Churna in different concentration on life span of Drosophila flies in F-1 generation

The differences in longevity period between F-1 generation of V group and control group was 22 days which is statistically significant (P < 0.001). The difference of mean between control group and S group was only 9 days which is statistically insignificant (P > 0.05). However, comparison between control and T group shows statistically insignificant result (P > 0.05) though the difference of mean was only 5.00 [Table 5].

Table 5

Multiple comparisons versus control group (Holm-Sidak method) in F-1 generation

Discussion

The fruit fly D. melanogaster has been used as a model in biological research on aging for exploring the longevity phenotypes, artificial and natural selection responses.[8] T. cordifolia whole plant extract had shown neuroprotective property in vivo in Drosophila.[9] In view of the health-promoting potential of Guduchi Churna, impact on longevity in Drosophila was planned.

In control group, the life span of male and female flies was 29 and 28 days, respectively. The highest life span of male and female flies was observed in 0.25 g/100 ml concentration, i.e., 83.2 and 84.8 days, respectively. In 0.50 g/100 ml concentration, the life span of both male and female flies was 33 days. This is suggestive of Guduchi Churna exhibits Rasayana effects irrespective of sex. It was also observed that increase in the concentration of drug led to decrease in the survivability rate. At lower concentration of Guduchi Churna there was a significant increase in lifespan of the flies. Higher concentrations progressively reduced the life span of flies significantly. Increasing the concentrations further did not show more increase in lifespan. This indicates that there is a particular concentration of Guduchi Churna which causes maximum lifespan extension (around 0.25 g/100 ml of food). Concentrations beyond that limit do not further increase the lifespan, which may be due to the absorption threshold of Guduchi Churna. The increase in lifespan of the flies due to Guduchi Churna can be attributed due its high antioxidant properties. Researches suggest that, the alcoholic and aqueous extracts of T. cordifolia are reported to have beneficial effects on the immune system[10] and have been tested successfully for their immunomodulatory activity.[11121314] Although the exact mechanisms of the action of Guduchi Churna by which this plant's products are able to enhance the survivability of D. melanogaster could not be ascertained from this study, but has validated the concept of Vayasthapana and Rasayana effect of Guduchi Churna.

Conclusion

Guduchi as Rasayana, enhances the life span of D. melanogaster which validates the concept of Rasayana. Increase in the life span of F-1 generation of Drosophila is suggestive of Rasayana effect of Guduchi Churna even prevails to next generation.

Financial support and sponsorship

IPGT and RA, Gujarat Ayurved University, Jamnagar, Gujarat.

Conflicts of interest

There are no conflicts of interest.