INTRODUCTION:

Fatigue is a condition of loss of efficiency and decreased working capacity as well as endurance of the body. Fatigue can also be interpreted as a symptom of a disease that limits physical function thus affecting the quality of life¹. One of the traditional medicinal plants that are empirically and scientifically proven to cure various diseases is Tinospora crispa L. Hook. The part of Tinospora crispa plant that is efficacious as medicine is found in all parts of the plant ranging from roots, stems, to leaves.

Tinospora crispa plants are known to contain compounds of glycosides, alkaloids, flavonoids, saponins, and tannins²^,³. Tinospora crispa plants have pharmacological effects in the form of wound healing⁴, anticancer⁵, antioxidants²^,6, hepatoprotective^7,8, antibacterial⁹^,¹⁰, and sunscreen cream¹¹.

Some studies have proven the tonic activity of extracts containing flavonoid compounds¹². The study states that mulberry leaf extract (Morus alba L.) contains flavonoid compounds and increases swimming time in mice. Flavonoids in ethanol extract of Tartary wheat (Fagopyrum esculentum) were also shown to have tonic activity in mice¹³. Extract ethanol of Tinospora crispa stem has been shown to have a flavanoid content of 32.65 ± 0.20% of routine equivalent¹⁴. Phenolics are the largest group of phytochemicals that account for most of the biological functions in plants or plant products. The secondary metabolites like flavonoids and phenols from plants have been reported to be potent free radical scavengers and immunostimulatory. Such as flavonoid content of Flemingia species¹⁵, Prunus persica L.¹⁶, Sesbania grandiflora¹⁷,and Calotropis gigantea L.¹⁸. Based on these thinking, author is encouraged to conduct research on the tonic effect extract ethanol of Tinospora crispa stem against male mice with the Natatory Exhaustion Method.

MATERIAL AND METHODS:

Extract Preparation:

Tinospora crispa stems are washed and dried in the oven at 40-50^oC. The dried Tinospora crispa stem is preoccupied and extracted using 70% ethanol by the maceration method.

Phytochemical Screening:

Dried Tinospora crispa stem and Tinospora crispa stem extract were performed phytochemical screening against flavonoids and alkaloids.

Experimental:

This research is an experimental study with a posttest matched control group design. The research was conducted after obtaining ethics approval with No. 02/20.05/0449.

Experimental animals:

The tonic effect test was conducted using a swimming test then looked at the time on the endurance of swimming mice. The swimming test method used is Natatory Exhaustion. A total of 25 mice adapted environments in the laboratory for 1 week. Mice were satisfied for 8 hours but were still given a drink. After adapted mice trained swimming in an aquarium with water temperature maintained at 30 ± 10^oC. Swimming exercises were carried out 3 times during the week. Endurance testing of swimming mice before treatment were carried out the day after the last swimming exercise. (Day 0) swimming endurance time (seconds) is calculated from the moment mice is inserted in the aquarium until the mice show fatigue. The sinking of the head and the position of the body was no longer horizontal with the surface of the water until the position of the four legs squeaks slowly moving for no more than 7 seconds which is a marker of fatigue. Mice were divided into 5 different treatment groups, namely:

Group I: Administered 100 mg/ kg body weight of Tinospora crispa stem ethanol extract

Group II: Administered 200 mg/ kg body weight of Tinospora crispa stem ethanol extract

Group III: Administered 400 mg/ kg body weight of Tinospora crispa stem ethanol extract

Group IV: Positive control administered 100 mg/kg body weight of caffeine

Group V: Negative control was given Carboxymethylcellulose sodium 0.5 %

Dosages used based on preliminary tests use the same method with various variations in effective doses and do not cause toxicity. All treatments were administered per oral for 14 days. The day after the last treatment was done testing the survival time of swimming mice. The endurance of swimming time was recorded.

Data Analysis:

Tonic effect test data is the endurance time of swimming mice after the administration of test provision for 14 days tested normality with the Shapiro-Wilk Method. The normality test results were not distributed normally (p<0.05), then the data analysis used is using the Kruskal-Wallis test and followed by the Mann-Whitney test at a 95% confidence level. Data on the endurance time of swimming mice after the administration of Carboxymethylcellulose sodium 0.5%, caffeine and Tinospora crispa stem ethanol extract (100, 200 and 400 mg/kg body weight) were analyzed with differences that meant the time of survival of swimming mice after the root was determined with significant value (p<0.05).

RESULT AND DISCUSSION:

Phytochemical Screening:

In Table 1 above states that dried Tinospora crispa stem and ethanol extract of Tinospora crispa stem contain a group of phytochemical compounds namely flavonoids and alkaloids.

Table 1. Phytochemical screening results of dried Tinospora crispa stem and ethanol extract

No.	Compounds	*Dried Tinospora crispa* stem**	Ethanol Extract
1.	Alkaloids	+	+
2.	Flavonoids	+	+

Swim Endurance Test Results:

The tonic effect test was conducted using a swimming test then looked at the time on the endurance of swimming mice. The swimming test method used is Natatory Exhaustion (Figure 1).

Fig 1. Endurance testing of swimming mice

The normality and homogeneity data tests results of swim endurance test on mice can be seen in Table 2 and Table 3.

Tabel 3. Homogeneity test result

Levene Statistic	Df1	Df2	Sig.
2.865	4	20	0.050

Based on Table 2 it can be seen that the normality test results were obtained abnormal because it had a value (p <0.05). In Table 4 it can be seen that the homogeneity test results indicate that the data obtained were normal due to the value (p =0.05). Therefore used non-parametric analysis of Kruskal-Wallis test. Kruskal-Wallis test results showed that there were significant differences between groups (0.001) in (p<0.05) indicating there were significant differences between the control group and the test group. Subsequent tests. conducted using Mann-Whitney test results showed significant differences between the control group and the test group. Test animal swims endurance data can be found in Table 4.

Table 4. Swimming endurance time

No.	Group	Swimming endurance time (minutes) ± SDM	P
1.	Positive Control	3.31 ±0.113	0.009a
2.	Negative Control	1.21 ± 0.198	0.009b
3.	100 mg/kg body weight of Tinospora crispa stem ethanol extract	2.21 ± 0.154	0.009a 0.009b
4.	200 mg/kg body weight of Tinospora crispa stem ethanol extract	2.60 ± 0.432	0.028a 0.009b
5.	400 mg/kg body weight of Tinospora crispa stem ethanol extract	2.62 ± 0.429	0.009a 0.009b

Description:

a = significant value compared to the Caffeine control group (p < 0.05)

b = significant value compared to Carboxymethylcellulose sodium control group (p <0.05)

The method of tonic effect testing using Natatory Exhaustion method had several advantages when compared to other methods that can know the effects of stimulants that can activate motion coordination. provide an effect of increased working capacity. and minimize fatigue and time used for relatively short observation. and ranking tools used quite simply¹⁹. The working principle applied in this study is to test the effect of tonic on the addition of motor activity that can be seen with the length of time swimming while swimming in the water.

Caffeine is an antagonism of adenosine receptors. Adenosine is a neurotransmitter that works to activate A1 receptors thus inhibiting the lipolysis process. Since caffeine and adenosine compounds have almost the same structure then caffeine has the potential to occupy adenosine receptors so lipolysis takes place. If lipolysis takes place then free fatty acids will increase until there is a delay in the use of glycogen as a source therefore it can improve stamina and delay fatigue ²⁰.

Based on Table 4 it can be seen that there is a noticeable difference in the percentage of tonic effect between the trial animal group was given Na-CMC and the trial animal group given Tinospora crispa stem ethanol extract doses of 100 mg/kg body weight, 200 mg/kg body weight, 400 mg/kg body weight, and caffeine 100 mg/kg body weight. This was shown with significant value (p <0.05). When compared between the group of trial animals given caffeine doses of 100 mg/kg body weight with the trial animal group given 200 mg/kg body weight and 400 mg/kg body weight of Tinospora crispa stem ethanol extract differed noticeably. The doses 200 mg/kg body weight and 400 mg/kg body weight Tinospora crispa stem ethanol extract may increase the tonic effect on mice but not as good as the administration of caffeine doses of 100 mg/kg body weight. Increased swimming time of mice treated between 200 and 400 mg/kg body weight of Tinospora crispa stem ethanol extract did not differ significantly (p>0.05). These results showed that Tinospora crispa stem ethanol extract (200 and 400 mg/kg body weight) had the same ability to increase the survival time of swimming mice. Based on the above results can be concluded that the administration 100 mg/kg body weight, 200 mg/kg body weight, and 400 mg/kg body weight of Tinospora crispa stem ethanol extract have a tonic effect on mice. Administration of 400 mg/kg body weight Tinospora crispa stem ethanol extract produces a tonic effect on mice that differs noticeably with the administration of caffeine dose 100 mg/kg body weight with significant value (p <0.05). Tinospora malabarica which is a family with Tinospora crispa was reported had adaptogenic activity in rats to suppress stress induced changes in biochemical parameters and organ weights in forced swimming and cold induced stress models²¹.

Phytochemical profiles indicated the presence of flavonoids and alkaloids, the tonic effect of ethanol extract Tinospora crispa stem may be due to the presence of alkaloids or flavonoids. Flavonoid have a significant tonic effect with the mechanism that can inhibit ATP to bond on the calcium canal ATPase thus inhibiting the absorption of calcium to enter into the sarcoplasmic reticulum which is the fluid of muscle cells that function where myofibril and myofilament are located. Myofibril is a muscle fiber to be able to contract or relax while models are retractable muscles when contracting that are influenced by actin proteins and elongated when relaxation is influenced by myosin proteins. The absence of these barriers causes calcium levels in the cytosol to then bond with troponin which will cause muscle contractions so that there is no fatigue²². Also flavonoids and alkaloids can activate metabolic by maintaining glycogen content in the liver and muscles. thus helping to reduce the accumulation of lactic acid. ammonia. Creatinine kinase and inhibit lipid peroxidation in liver and muscle tissues thus accelerating recovery²³^,²⁴.

CONCLUSION:

From the study outcome, it can be concluded administration Tinospora crispa stem ethanol extract (100, 200, and 400 mg/kg body weight) has a tonic effect by increasing swimming endurance on male mice. Phytochemical profiles indicated the presence of flavonoids and alkaloids, the tonic effect of ethanol extract Tinospora crispa stem may be due to the presence of alkaloids or flavonoids.

ACKNOWLEDGEMENTS:

The authors would like to acknowledgements a research grant from Ministry of Research, Technology Republic of Indonesia (Deputi Bidang Penguatan Riset dan Pengembangan Kemenristek) for the funding support of the research project (Research program of the 2020 Number: B/87/E3/RA.00/2020).

CONFLICT OF INTEREST:

Author declared that there are no conflicts of interest.

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Received on 11.11.2020 Modified on 05.12.2020

Research J. Pharm. and Tech 2021; 14(10):5439-5442.

DOI: 10.52711/0974-360X.2021.00948

Group	Kolmogorov-Smirnov			Shapiro-Wilk
Group	Statistic	Df	Sig.	Statistic	Df	Sig.
Positive Control	0.338	5	0.063	0.773	5	0.048
Negative Control	0.238	5	0.200	0.914	5	0.495
100 mg/kg body weight of Tinospora crispa stem ethanol extract	0.193	5	0.200	0.953	5	0.760
200 mg/kg body weight of Tinospora crispa stem ethanol extract	0.341	5	0.059	0.798	5	0.078
400 mg/kg body weight of Tinospora crispa stem ethanol extract	0.271	5	0.200	0.888	5	0.347

Fig 1. Endurance testing of swimming mice

Table 2. Normality test results

Tabel 3. Homogeneity test result

Table 4. Swimming endurance time