INTRODUCTION:

Diarrhea is a disease that often occurs in society. Poor nutrition, community attitudes, socio-economic conditions, and the environment are some triggering factors for diarrhea.¹ The most common causes of diarrhea are bacterial infections (Salmonella, Escherichia coli, Shigella, Vibrio cholerae, Campylobacter, and Clostridium difficile²) or viral infections of the digestive system.³ Diarrhea is still a significant public health problem in developing countries,⁴ especially Indonesia, both in terms of morbidity⁵and mortality.⁶ Until now there are frequent outbreaks of diarrhea in various areas accompanied by several deaths.

The main treatment for diarrhea is rehydration therapy.⁷ This therapy is carried out to restore lost body fluids due to being wasted with feces in diarrheal conditions.⁸ If the dehydrated condition has been treated, diarrhea treatment can be combined with anti-diarrheal drugs. However, currently, there are still some people in Indonesia, especially in rural areas, who have not been reached by formal health services, so they often use natural ingredients to treat diarrhea.⁹ One of these natural ingredients is kejek tea leaves (Camellia sinensis L.).

Kejek tea is a traditional green tea originating from Garut, West Java, Indonesia. This tea is different from other teas because the manufacturing process is still traditional, namely by being “kejek” (trampled). This process makes kejek tea has distinctive flavour compared to other teas. Kejek tea contains secondary metabolites include as tannins, alkaloids, and flavonoids.¹⁰ The tannin content shows the activity as an astringent which can reduce intestinal peristalsis, so it is thought to be effective as an anti-diarrheal.¹¹ Furthermore, it contains flavonoids, which can inhibit acetylcholine release and intestinal contractions, as well as alkaloids, which act as inhibitors of the growth of microorganisms in the intestine that cause diarrhea.¹² Based on this background, a study was conducted on the antidiarrheal activity of kejek tea infusion (C. sinensis L.) in Swiss Webster male mice. This study is expected to provide information to the community, particularly Garut residents, regarding the use of kejek tea (C. sinensis L.) which has anti-diarrheal properties. In addition, it can be used as a reference material for further research and can be used as an alternative treatment for diarrhea.

MATERIALS AND METHODS:

Plant Material:

Kejek tea leaf simplicia (C. sinensis L.) was obtained from the tea plantation of PTPN VIII Dayeuhmanggung, Cilawu District, Garut Regency, West Java. Plant determination was carried out at the Bandungense Herbarium, School of Life Sciences and Technology, Bandung Institute of Technology.

Infusion Manufacturing:

The infusion method was chosen because the simplicia of tea leaves has active metabolites that dissolve in water and is also adapted to the technique used in the community in the form of boiling green tea leaves and this method is relatively easy to make in a short time. Kejek tea leaf infusion as a test preparation was made in various concentrations, namely 3%, 4%, and 5% w/v. To make a stock solution with a concentration of 5%, 5 grams of kejek tea leaf simplicia is needed, then put it in an infusion pot and add 100mL of water. The pot is heated over a water bath with a temperature of 90⁰C for 15 minutes while stirring occasionally, then the resulting filtrate is filtered with filter paper or fine cloth. The volume of the filtrate was measured, then hot distilled water was added to an infusion volume of 100 mL. To make an infusion with a concentration of 3% and 4%, a 5% infusion solution is diluted.

Phytochemical Screening:

The Kejek tea infusion obtained was then subjected to qualitative tests to identify the content of secondary metabolites such as flavonoids, alkaloids, steroids, tannins, saponins, quinones, and phenols. This is done using simple and standard qualitative methods.¹³

Animal Experiment:

Male mice (Swiss Webster strain) weighing about 20-30 g were obtained from the School of Life Technology, Bandung Institute of Technology. The mice were first adapted to the conditions of the cage for 1 week. During the adaptation period, mice were given food and water ad libitum. The research was conducted at the Pharmacology Laboratory of Garut University and received ethical approval from the Research Ethics Committee of Padjadjaran University number (1030/UN6.KEP/EC/2022).

Antidiarrheal Activity:

a) Method of Protection against Induction of Oleum Ricini:

Thirty male swiss webster mice fasted for 1 hour. Before being given treatment, all mice were weighed first, then their body weight was recorded and grouped into 6 groups, each consisting of 5 mice. All groups of mice were given oleum ricini as much as 0.75ml/20 gBW as an inducer of diarrhea except for group I (negative control) which was only given 1% tragacanth suspension. After giving oleum ricini induction, it was left for 60 minutes and then each group of test animals was given treatment. Group I, namely the healthy condition group (negative control), was only given 1% tragacanth suspension, group II, namely the sick condition group (positive control), was given 1% tragacanth suspension, groups III, IV, and V, which were the test groups, were given infusion with a concentration of 3%, 4%, and 5% as much as 0.2mL/20 gBW orally and group VI, namely the control group, was given loperamide HCl as much as 0.01mg/20 gBW orally. Mice were stored in a place based on filter paper which had been weighed then the response of each mouse was observed at intervals of 30 minutes for 360 minutes or 6 hours. The parameters observed in this method include the frequency of defecation, the consistency and weight of the feces, the time diarrhea appears, and the duration of diarrhea.¹⁴

b) Intestinal Transit Method:

A total of 25 male swiss webster white mice fasted for 18 hours while still being given a drink. Before being given treatment all mice were weighed first then their body weight was recorded and grouped into 5 groups, each consisting of 5 mice. All test animals were given 0.75ml/20 gBW of oleum ricini and then allowed to stand for 60 minutes. After 60 minutes of induction, all groups of test animals were given treatment. Group I, the negative control group, was only given 1% tragacanth suspension, group II, the positive group, was given Loperamide HCL as much as 0.01mg/20 gBW orally, groups III, IV and V, which were the test groups, were given infusion with a concentration of 3%, 4%, and 5% w/v as much as 0.2mL/20 grBW orally.

Forty-five minutes later after being given the treatment, the test animals were given 0.2mL/20 gBW of 5% norit suspension orally. After sixty-five minutes of administration of 5% norit suspension, all test animals were sacrificed by dislocation of the neck bones, then their intestines were carefully removed. The intestine is stretched without being stretched, scissors are used to break the connective tissue in the intestine, and the intestine is stretched on paper. Measuring the length of the intestine that is passed by the marker (norit) from the pylorus to the end (black color) and the length of the entire intestine, from the pylorus to the colon using a ruler. Then for each test animal, the percent ratio of the passages traversed by the marker to the total intestinal length was calculated.¹⁵

The ratio of paths traversed by norit is measured using the formula:¹⁵

The length of the intestine through which the norit passes

The ratio of paths traversed by norit = ---------------------------------x100

Overall intestinal length

Statistic analysis:

The data obtained was then subjected to one-way analysis of variance (ANOVA) followed by the LSD test. Values with p<0.05 are considered significant.

RESULT AND DISCUSSION:

This plant was determined taxonomically at Herbarium Bandungense, Sekolah Ilmu dan Teknologi Hayati, Institut Teknologi Bandung as Camellia sinensis L. from the Theaceae family, collection number 5331/ITI.C11.2/TA.00/2022.

Phytochemical screening result:

The results of the phytochemical screening test on Simplicia and the kejek tea infusion on plant secondary metabolites are summarized in Table 1.

Table 1. Results of phytochemical screening of simplicia and infusion of kejek tea leaves (C. sinensis L.)

Phytochemical screening	Simplicia	infusion
Flavonoids	+	+
Alkaloids	+	+
Steroids	+	-
Tannins	+	+
Saponins	+	+
Quinones	+	+
Phenol	+	+

Description (+) : detected ; (-) : not detected

The group of phytochemical compounds contained in that simplicia and kejek leaf infusion (C. sinensis L.) contains flavonoids, alkaloids, steroids, tannins, saponins, quinones, and phenols. Kejek tea leaf infusion (C. sinensis L.) contains the most important phytochemical compounds, namely tannins, alkaloids, and flavonoids. These three groups of compounds have an important role as antidiarrheals.

Castor oil-induced diarrhea was used to test the Kejek tea leaf infusion (C. sinensis L.) anti-diarrheal effectiveness.¹⁶ Because autocoids and prostaglandins are involved and have been linked to the cause of diarrhea in humans, the use of the castor oil-induced diarrhoea model in our research is well warranted.¹⁷ Castor oil is also said to cause diarrhea by enlarging the intestinal cavity and preventing water from being reabsorbed. The intestinal mucosa becomes irritated and inflamed as a result of the production of ricinoleic acid from castor oil,¹⁸ leading to release of prostaglandins,¹⁹ which stimulate motility and secretion.²⁰ (85–95%) of the ricinoleic acid in castor oil has laxative properties.²¹ In the lumen of the intestine, the ricinoleic acid thus released easily transforms into ricinoleate salts with sodium and potassium. In the mucosal surface, the ricinoleate salt that results from this process acts like soap or a surfactant.²²

Andiarrheal acitivity evaluations:

Method of protection against induction of oleum ricini:

The first parameter observed in the diarrhea protection method induced with oleum ricini is the time of onset of diarrhea and duration of diarrhea which can be seen in Table 2. The onset of diarrhea is the time parameter calculated after the mice are given the treatment/test preparation. The faster the time for diarrhea to appear, the weaker the anti-diarrheal ability of the test preparation, conversely the longer the time for diarrhea to appear, the stronger the anti-diarrheal ability of the test preparation. Meanwhile, the duration of diarrhea was calculated when the mice had diarrhea for the first time which was characterized by the consistency of watery stools and ending with the consistency of stools that returned to normal/dense. The results showed that the positive control group had a greater onset (40.60 minutes) and duration (297.80 minutes) than the other groups. This shows that the induction of diarrhea using Oleum ricini is successful. The 4% Kejek Tea Infusion (ITK) test group showed the longest time for diarrhea to appear, namely at 152.40 minutes, meaning that the 4% ITK group had the best antidiarrheal activity to prevent diarrhea due to oleum ricini induction. The 5% ITK test group showed the shortest duration of diarrhea, 170.80 minutes, meaning that the 5% ITK test preparation had the best antidiarrheal ability to stop diarrhea induced by oleum ricini.

Table 2. Observations on the onset and duration of Kejek tea leaves (C. sinensis L.)

Group	Onset (minutes)	Duration (minutes)
Positive Control	40,60 ± 15,53	297,80 ± 22,35
ITK 3%	88,60 ± 59,16	234,40 ± 82,30
ITK 4%	152,40 ± 60,99^*	207,60 ± 60,99^*
ITK 5%	150,20 ± 140,51^*	170,80 ± 94,36^*
Loperamide	141,80 ± 30,59^*	180,40 ± 48,50^*

* Significantly different compared to the positive control group (P <0.05)

The second parameter observed in this test was the frequency of diarrhea which can be seen in Table 3. The frequency of diarrhea was monitored by counting the number of times the mice defecated at 30-minute intervals during the 6 hours of observation. The results showed that in the 3% ITK test group there was a decrease in the frequency of diarrhea and were significantly different compared to the positive control group (P<0.05) at the 210th, 240th, 270th, 300th, and 330th minute of observation. In the 4% ITK test group there was a decrease in the frequency of diarrhea and was significantly different compared to the positive control group (P<0.05) at the 180th, 210th, 270th, and 330th minutes of observation. In comparison, in the 5% ITK test group there was a decrease in the frequency of diarrhea and was significantly different compared to the positive control group (P<0.05) at the 120th, 210th, 240th, 270th, and 330th minute of observation.

Table 3. Observation Results of the Average Frequency of Defecation

Group

The average frequency of diarrhea in minutes to

0-30

31-60

61-90

91-120

121-150

151-180

181-210

211-240

241-270

271-300

301-330

331-360

Negative Control

1.40 ± 1.34

0.20 ± 0.44

0.00 ± 0.00

0.20 ± 0.44*

0.00± 0.00*

0.00 ± 0.00*

0.20 ± 0.44*

0.00 ± 0.00*

0.20 ± 0.44*

0.00 ± 0.00*

0.00± 0.00

Positive Control

1.60 ± 1.51

0.20 ± 0.83

2.00 ± 1.22

1.80±

1.30

2.00±

1.00

1.60±

0.54

2.20 ±

0.83

1.60 ±

0.89

1.80 ±

0.83

1.00 ±

0.00

1.00±

0.00

0.00± 0.00

ITK 3%

1.80 ± 2.68

2.20 ± 2.49

2.00 ± 1.22

1.40 ±

0.89

1.40 ±

2.07

0.80±

0.83

0.20± 0.44*

0.20 ± 0.44*

0.60 ±

0.54

0.40 ± 0.54*

0.00± 0.00

ITK 4%

1.20 ± 1.09

1.20 ± 2.16

0.80 ± 1.09

1.00 ±

1.00

0.80 ±

1.30

0.60 ± 0.89*

0.40 ± 0.54*

1.00 ±

1.22

0.40 ± 0.54*

1.00 ±

0.70

0.00 ± 0.00*

0.00± 0.00

ITK 5%

1.60 ± 3.57

1.00 ± 1.41

0.80 ± 1.78

0.40 ± 0.54*

1.00 ±

1.00

0.80 ±

0.83

0.40 ± 0.54*

0.20 ± 0.44*

0.40 ±

0.54

0.00 ± 0.00*

0.00± 0.00

Loperamide

2.40 ± 1.51

1.00 ± 1.00

0.60 ± 0.89

0.60± 0.89*

1.20 ±

0.83

0.40 ± 0.89*

0.40 ± 0.54*

0.00 ± 0.00*

0.40 ±

0.54

0.00 ± 0.00*

0.00± 0.00

* significantly different compared to the positive control group (P <0.05)

Table 5. Observation results of stool consistency

Group	T30	T60	T90	T120	T150	T180	T210	T240	T270	T300	T330	T360
Negative Control	0	0*	0*	0*	0*	0*	1	0*	0*	0*	0*	0
	1	1*	0*	0*	0*	0*	0	0*	0*	0*	0*	0
	0	0*	0*	1*	0*	0*	0	0*	0*	0*	0*	0
	1	0*	0*	0*	0*	0*	0	0*	0*	0*	0*	0
	1	0*	0*	0*	0*	0*	0	0*	0*	0*	0*	0
Positive Control	0	3	3	3	3	3	0	3	3	3	3	1
	1	3	3	3	3	3	3	3	3	3	3	0
	0	3	3	3	3	3	0	3	3	0	3	1
	3	3	0	3	3	0	3	3	3	3	0	0
	0	3	3	0	3	3	0	3	0	3	3	0
ITK 3%	0	0	2	2	0	3	0	0*	0	3	3	0
	0	0	0	1	3	0	3	3*	3	0	3	0
	0	2	3	3	3	3	0	0*	0	3	0	0
	3	3	2	0	1	0	0	0*	0	0	0	0
	2	3	3	3	0	2	0	0*	0	0	0	0
ITK 4%	1	1*	2*	3	3*	0	3	3	0	3	0*	0
	0	0*	0*	3	0*	2	2	3	0	3	0*	0
	0	0*	0*	0	0*	3	0	3	3	3	0*	0
	1	2*	2*	0	3*	0	0	0	0	0	0*	0
	2	0*	0*	2	0*	0	0	0	3	3	0*	0
ITK 5%	0	0*	0	0*	1*	2	0	0*	0	2	0	0
	3	3*	0	2*	0*	2	0	0*	0	0	0	0
	0	2*	3	0*	3*	0	0	3*	3	0	0	0
	0	0*	0	0*	0*	0	0	0*	0	3	0	0
	0	0*	0	2*	2*	2	0	0*	0	0	3	1
Loperamide	1	1*	0	0	3	3	3	3*	0*	3	0	0
	2	2*	3	0	1	0	0	0*	0*	0	0	0
	2	0*	3	0	3	0	0	0*	0*	0	3	0
	1	2*	0	3	0	0	3	0*	0*	0	0	0
	0	0*	0	3	3	0	0	3*	0*	3	0	0

*significantly different compared to the positive control group (P <0.05)

Intestinal Transit Method:

The next parameter observed was the intestinal transit method which can be seen in table 6. The results showed that the comparison group and the test group, namely 5% ITK, 4% ITK, and 3% ITK, were significantly different from the positive control group (P <0.05), This means that the Kejek tea infusion test preparations with various concentration variations have anti-diarrheal activity by slowing the movement of intestinal peristalsis and prolonging the emptying of the stomach contents of the test animals. The smaller the value of the norit passage ratio, the stronger the antidiarrheal activity produced by the test preparation. The 5% kejek tea infusion test preparation has the smallest average norit passage ratio among the other test preparations so it has a better effect in suppressing intestinal peristalsis.

Based on the results of the study, it was found that the infusion of Kejek tea with various concentrations, namely 3%, 4%, and 5% had anti-diarrheal activity by reducing the weight of the stool, reducing the frequency of diarrhea, reducing the stool consistency score, shortening the duration of diarrhea, slowing the appearance of diarrhea and reducing the ratio value. norit pathway in Swiss Webster strain male mice induced by oleum ricini. The best antidiarrheal activity was shown by the Kejek tea infusion test preparation with a concentration of 5%. The antidiarrheal activity is thought to be due to the content of secondary metabolites found in kejek tea such as tannins, alkaloids, and flavonoids. The tannin content in green tea shows the activity as an astringent. Tannins have chelating and spasmolytic effects that cause a reduction in intestinal peristalsis.²³ This spasmolytic effect, however, may also cause the bacterial cell wall or cell membrane to contract, impairing the cell's permeability. Cells cannot function normally when permeability is disrupted, which prevents them from growing or even causes them to die.²⁴ As tannins are thought to have a similar effect to phenolic chemicals, they may also have anti-bacterial capabilities through precipitating proteins.²⁵ Tannins have antibacterial properties that include interactions with cell membranes, enzyme inhibition, and the destruction or inactivation of bacterial genetic material.²⁶ In addition, a flavonoid derivative is quercetin. Numerous neurotransmitters that cause spasms can be blocked by quercetin. One of the neurotransmitters that might cause intestinal spasms is acetylcholine, which can rise as a result of intestinal bacterial inflammation.²⁷ Reduced intestinal contractions brought on by cholinergic inhibition will reduce diarrhea.²⁸ Alkaloids which work as inhibitors of the growth of microorganisms in the intestine that cause diarrhea.²⁹

CONCLUSION:

Based on the study, it can be concluded that Kejek tea infusion at a concentration of 5% shows the most effective anti-diarrheal effect because it has anti-diarrheal activity by reducing the weight of the stool, reducing the frequency of diarrhea, reducing the stool consistency score, shortening the duration of diarrhea, slowing the appearance of diarrhea and reducing the value of norit pass ratio compared to kejek tea infusion 3% and 4%. Further studies are needed to support the source of new potential agents as anti-diarrhea derived from natural ingredients.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The author would like to thank to FMIPA UNIGA research funding program No. 005/FMIPA.UPPM/I/2022.

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Received on 11.02.2023 Revised on 15.12.2023

Accepted on 04.06.2024 Published on 24.12.2024

Available online from December 27, 2024

Research J. Pharmacy and Technology. 2024;17(12):6091-6097.

DOI: 10.52711/0974-360X.2024.00923

Group	Stool weight (g)
Negative Control	0.05 ± 0.34*
Positive Control	1.06 ± 0,79
ITK 3%	0.52 ± 0,31
ITK 4%	0.47 ± 0.37*
ITK 5%	0.44 ± 0.27*
Loperamide	0.40 ± 0.45*