INTRODUCTION:

Infectious diseases are diseases caused by microorganisms that enter the body and cause organ damage. These microorganisms can be bacteria, viruses, fungi, worms, or protozoa. Tropical infectious diseases are a type of infectious disease that frequently go unnoticed because there is insufficient attention paid to them. The tropical climate and high humidity have allowed several strains of this illness to flourish in Indonesia. Tropical diseases, especially neglected tropical diseases, continue to be a serious public health issue in Indonesia.

This illness has personal and societal repercussions, commonly affecting those in the lower middle class. Twenty diseases caused by bacteria, viruses, protozoa, or helminths are classified as NTDs by the World Health Organization (WHO). Therefore, a plan is required to eradicate the worms and fungi that cause infectious diseases in the tropics^1,2.

Helminthic infections produce helminthiasis, which is widespread, especially in developing nations. More than 60% of Indonesian children may be infected with worms. Over 1.5 billion people, or roughly 24 percent of the world's population, are thought to be infected with soil-transmitted helminths (STH). In the meantime, fungal infections can potentially transform into parasites by acquiring organic substances from living creatures. Fungus' negative impact here is due to the illness it spreads. Among these are the pathogenic fungus Candida albicans and Trichophyton mentagrophytes. Candidiasis is the name given to the sickness brought on by Candida, and 20%–25% of Indonesians have Candidiasis. One fungi that causes ringworm is Trichophyton mentagrophytes, which target the skin of the human feet. Trichophyton mentagrophytes, a dermatophyte fungus with the enzyme keratinase, can infect human and animal skin, hair, and nails^3–5.

Current anthelmintic treatment is a chemical substance that can have undesirable health effects. As one example, albendazole can cause liver damage, eukopenia, and allergic responses. Meanwhile, antifungals are used to treat fungal infections. Although advances have been made in modern medicine, traditional medicine using medicinal plants as an alternative treatment is still in high demand among the people of Indonesia. Medicinal elements having antifungal qualities can be acquired from natural (traditional) and conventional (modern) medicinal substances. Traditional medicine in this region relies heavily on medicinal plants, which have been used for centuries. As a result, there is a pressing need for an effective alternative treatment to combat worms and fungi⁶.

One potential alternative therapeutic approach involves the utilization of traditional medicine. Since traditional medicine is made from all-natural components and has been used for decades, its efficacy, safety, and efficacy have all been well-documented, making it increasingly popular. Dioscorea hispida (Dennts.) is a botanical species indigenous to India and widely distributed over Southeast Asia. Dioscorea hispida (Dennts.) is a plant species that possess numerous advantageous features in nutrition and pharmaceuticals. Dioscorea hispida (Dennts.) possesses diverse secondary metabolites within its tuber, encompassing diosgenin, steroids, saponins, alkaloids, cyanide acid, phenols, and terpenoids. Secondary metabolites such as alkaloids, flavonoids, glycosides, quinolones, saponins, tannins, triterpenoids/steroids, and lignans have been found to have potential anthelmintic and antifungal properties^7,8.

The researcher aims to investigate the anthelmintic and antifungal properties of the ethanol extract and its fractions (n-hexane, ethyl acetate, and water) derived from the tubers of Dioscorea hispida (Dennst.). The objective is to establish a correlation between the physicochemical composition and the observed anthelmintic and antifungal activities. Because of its morphological and physiological similarities to parasitic worms seen in humans, Pheretima posthuma has been widely employed as a pharmacological model organism for the first anthelmintic investigations. The antifungal activity test was conducted with the diffusion method employing paper discs to evaluate the presence of a clear zone for Candida albicans and Trichophyton mentagrophytes.

MATERIALS AND METHODS:

Materials:

The tools used in the research were laboratory quality glassware (Pyrex), autoclave (Fisons), incubator (Fisher Scientific), Air Flow Cabinet (Astec HLF 1200L), analytical balance (Sartorius), oven (Memmert), pipettes micro (Eppendorf), rotary evaporator (Haake D). while the ingredients used are pro analysis from Merck, namely 96% ethanol, ethyl acetate, ketoconazole, n-hexane, Potato Dextrose Agar (PDA), Potato Dextrose Broth (PDB), amyl alcohol, acetic acid anhydride, concentrated hydrochloric acid, concentrated sulfuric acid, iron (III) chloride, isopropanol, chloroform, methanol and pro analysis from smart-lab, namely sodium hydroxide, sodium chloride, anhydrous sodium sulfate, n-hexane, mercury (II) chloride. The test fungi used were Candida albicans and Trichophyton mentagrophytes obtained from the microbiology laboratory of the Faculty of Pharmacy, Universitas Sumatera Utara.

Dioscorea hispida (Dennst.) Tubers were obtained from Deli Serdang, located in North Sumatra, Indonesia. Plant identification was conducted at the Medanese Herbarium (MEDA), located within the Faculty of Mathematics and Natural Sciences at the University of North Sumatra.

Experimental Animal:

The experimental subjects employed in this study were earthworms (Pheretima posthuma). The earthworms were selected for their uniform size (7-10cm). The worms were obtained from soil found in a dump site and subsequently cleansed of impurities through a thorough rinsing with distilled water. Before experimentation, the earthworms were acclimated in a solution containing 0.9% NaCl. The identification of experimental animal specimens was conducted by the Laboratory of Animal Taxonomy, which is affiliated with the Department of Biology at the Faculty of Mathematics and Natural Sciences, University of North Sumatra⁹.

Preparation of Extracts and Fractions, as well as Phytochemical Screening:

The purpose of this research is to investigate the anthelmintic potential of many fractions derived from Dioscorea hispida (Dennst.) tubers, which include ethanolic extracts and fractions (n-hexane, ethyl acetate, and water) against Pheretima posthuma worms. Approximately 4,500 grams of simplicia powder were macerated with 45 liters of 96% ethanol, immersed for the first six hours with intermittent stirring, and allowed to remain for 18 hours. The process of separation involves maceration followed by filtration. The extraction process should be repeated at least once using the same solvent type, with the total solvent volume being reduced to half of the initial screening volume. Gather the macerate and subject it to vaporization using a rotary evaporator until a concentrated extract of substantial viscosity is achieved.

As solvents, n-hexane and ethyl acetate were used to produce the fractions by liquid-liquid extraction. A solution was prepared by dissolving 10g of Dioscorea hispida (Dennst.) tuber ethanol extract in a mixture of ethanol and water in a 1:1 ratio (100ml). This solution was partitioned using a separatory funnel with 100ml of n- to yield two distinct layers. The water solution was subjected to partitioning using ethyl acetate. The n-hexane and ethyl acetate fractions were subsequently subjected to evaporation using a vacuum rotary evaporator. Subsequently, the fractions underwent further evaporation utilizing a water bath.

Secondary metabolites in the simplicia, ethanolic extract and fraction were identified using phytochemical screening.

Anthelmintic Test:

Anthelmintic activity testing was performed on 15 groups, including three control groups comprised of a positive control (1% Albendazole suspension), a negative control (0.9% NaCl solution), and a control (0.9% NaCl solution carrier +1% Tween 80). This study, 12 treatment groups were formed, each consisting of different fractions derived from ethanolic extract, namely the fraction. These fractions were prepared at three distinct concentrations, precisely 10 g/L, 20 g/L, and 30 g/L. Pheretima posthuma were placed into individual petri dishes containing 20 ml of the respective test solution. The researchers then proceeded to observe and document the time at which paralysis occurred and the time of death for each worm. To distinguish worms that do not move between paralyzed or dead, the tail of the worm is dipped into distilled water at a temperature of 50°C; it is declared paralyzed when the worm's tail moves when it is immersed and is declared dead if there is no movement at all ^9,10.

Antifungal Test:

A total of 0.1 ml of fungal inoculum was put into a sterile petri dish, and the test was carried out three times (triple), then 15 ml of media was poured. The petri dish was then shaken on the table's surface (Laminar Air Flow Cabinet) so that the media and fungal suspension were mixed and left for a few minutes until it solidified. The agar diffusion method (Kirby Bauer) assessed the antifungal activity using a paper disc. Place a paper disc that has been soaked with ethanolic extract, a fraction (n-hexane, ethyl acetate, water) of Dioscorea hispida, positive control, and negative control each 10µL on the surface of the solid agar media that has been inoculated with the fungi and left for 15 minutes, then placed in an incubator at a temperature of 25 degrees Celsius for 48 hours. After that, use a caliper to determine the diameter of the resistance encircling the paper disc ¹¹.

Statistic Test:

The research findings are displayed using the mean ± standard deviation format. Statistical analysis was conducted using version 22.0 SPSS and ANOVA. If a significant difference exists, the analysis is continued with the Tukey test. Statistical analysis was performed at a 95% level of confidence.

RESULT:

Phytochemical screening:

Phytochemical screening was performed on simplicia, ethanolic extract, and the three fractions. The outcomes acquired are visible in Table 1.

Test anthelmintic activity:

During the assessment of anthelmintic activity (Figure 1), It was observed that all treatments involving ethanolic extracts and all three fractions showed anthelmintic activity. Notably, the ethyl acetate fraction at a concentration of 30 g/L demonstrated the shortest time to induce mortality compared to the other treatment groups. The experimental group received treatment with Albendazole 1%, while the control group was administered a positive control, as indicated in Table 2.

Table 1: Secondary Metabolite from Dioscorea hispida (Dennst.) tuber

No	Group	Simplicia	Extract	Fractions
No	Group	Simplicia	Extract	n-hexane	Ethyl acetate	Water
1	Alkaloids	+	+	-	+	+
2	Flavonoids	+	+	-	+	+
3	Glycosides	+	+	-	+	+
4	Tannins	+	+	-	+	+
5	Saponins	+	+	-	-	+
6	Triterpenoids/steroids	+	+	+	-	-

Table 2: Result of paralysis time and time of death anthelmintic activity test against Pheretima posthuma

Sample	Concentration	Paralys Time ± SD (Minutes)	Death Time ± SD (Minutes)
Albendazole	1 %	72,63 ± 3,84	108,67 ± 7,02
NaCl	0,9 %	327,33 ± 17,62	410,67 ± 22,03
NaCl 0,9% + Tween 80 1 %		309,67 ± 9,50	350,33 ± 14,50
Ethanolic Extract	10 g/L	43,80 ± 4,95	57,53 ± 8,21
	20 g/L	40,07 ± 5,86	51,07 ± 7,50
	30 g/L	34,15 ± 4,31	43,33 ± 4,80
N-Hexane Fraction	10 g/L	84,40 ± 9,63	109,33 ±8,02
	20 g/L	70,23 ± 2,93	87,67 ± 5,13
	30 g/L	61,43 ± 6,01	82,00 ± 12,49
Ethyl Acetate Fraction	10 g/L	37,17 ± 3,27	47,83 ± 4,15
	20 g/L	35,90 ± 0,79	42,90 ± 5,34
	30 g/L	23,90 ± 2,94	34,20 ± 2,31
Water Fraction	10 g/L	48,87 ± 3,07	71,53 ± 16,00
	20 g/L	44,04 ± 5,81	63,40 ± 6,73
	30 g/L	37,63 ± 2,59	51,17 ± 7,92

Figure 1: Anthelmintic test A (Albendazole 1%, NaCl 0.9%, Tween 1% + NaCl 0.9%), B (ethanol extract 10,20,30 g/L), C (n-Hexane fraction 10,20,30 g/L) D (Ethyl acetate fraction 10,20,30 g/L), E (Water fraction 10,20,30 g/L)

Antifungal Activity Test:

The test solution was made by measuring 5 g of both the ethanol extract and the three fractions. These were then diluted in 10mL of DMSO to achieve a concentration of 500g per L. The extract, n-hexane fraction, and water fraction were diluted to 400, 300, 200, 100, 50, and 25 g/L from an initial concentration of 500g/L. The ethyl acetate fraction was carried out until 50, 25, 20, 15, 10, 5, and 2.5g/L concentrations were obtained. 2% ketoconazole as the positive control was weighed by 0.02g and then dissolved with 1 ml of sterile distilled water.

Table 3: Test Results of Antifungal Activity of Dioscorea hispida

Sample	Concentration (g/L)	Inhibition Zone Diameter (mm)
Sample	Concentration (g/L)	Candida albicans	Trichophyton mentagrophytes
Extract	400	13,97 ± 0,20	12,70 ± 0,43
	300	13,50 ± 0,20	12,13 ± 0,20
	200	12,30 ± 0,26	11,40 ± 0,20
	100	11,27 ± 0,20	10,50 ± 0,30
	50	10,47 ± 0,49	9,17 ± 0,45
	25	6,8 ± 0,10	6,63 ± 0,25
N-hexane Fraction	400	8,97 ± 0,30	8,30 ± 0,26
	300	8,10 ± 0,26	7,80 ± 0,20
	200	7,90 ± 0,26	7,23 ± 0,20
	100	-	-
	50	-	-
	25	-	-
Ethyl Acetate Fraction	50	21,13 ± 0,55	20,23 ± 0,65
	25	16,33 ± 0,25	15,53 ± 0,37
	20	15,10 ± 0,36	13,27 ± 0,15
	15	13,70 ± 0,36	11,60 ± 0,26
	10	12,80 ± 0,10	10,90 ± 0,10
	5	11,00 ± 0,40	9,87 ± 0,32
	2,5	6,60 ± 0,20	6,57 ± 0,15
Water Fraction	400	10,33 ± 0,32	8,76 ± 0,15
	300	11,33 ± 0,45	9,40 ± 0,30
	200	9,83 ± 0,35	8,30 ± 0,40
	100	7,57 ± 0,32	7,33 ± 0,15
	50	-	-
	25	-	-
Positive Control (Ketoconazole 2%)		18,60 ± 0,36	17,30 ± 0,30
Negative Control (DMSO)		-	-

Figure 2. Antifungal Dioscorea hispida Ethanol Extract

Figure 3. Antifungal Dioscorea hispida n-hexane fraction

Figure 4: Antifungal Dioscorea hispida Ethyl Acetate Fraction

Figure 5. Antifungal Dioscorea hispida Water Fraction

DISCUSSION:

The liquid-liquid fraction uses different solvents based on their polarity in hexane solvents, which can dissolve non-polar compounds like steroids and triterpenoids. Ethyl acetate solvent is a semi-polar compound that can attract alkaloids, flavonoids, saponins, tannins, and polyphenols, while water solvents can dissolve glycosides, flavonoids, tannins, and sugars. Based on the results, using semi-polar solvents and polar secondary metabolites is more common than non-polar solvents¹².

The results of statistical testing for the analysis of time of paralysis and time of death of Pheretima phostuma using the SPSS 22 program, showed that the results of the normal distribution test (Kolmogrov-smirnov) produced significant values. (p)>0.05 means that the data tested is normally distributed, so it can be continued with the one-way Anova test. The results of the one-way Anova test obtained a significant value. (p)>0.05, which means there is a significant difference between the average paralysis time value and the average death time value with various concentrations. To see this difference, the post hoc test (Tukey) was continued.

The anthelmintic test uses albendazole as a positive control because it can maintain broad-spectrum activity, excellent tolerability, and good single-dose effectiveness. The mechanism of albendazole is to inhibit glucose uptake by worms so that the production of ATP as an energy source to maintain worm life is reduced, resulting in worm death. From the results obtained, both the extract, the fractions of Dioscorea hispida (Dennst.) tuber extract, and the positive control (Albendazole 1%) have activity as an anthelmintic, and this can be seen from the paralysis time and the time of death of the worms that have been tested where the worms experience a paralytic effect and a death effect in less than 120 minutes. The outcomes above can also be derived from the statistical tests' results, wherein the test solution and the positive control (Albendazole 1%) exhibit distinct mean values for paralysis time and death time compared to the negative control. The Pheretima phostuma worm becomes paralyzed and dies sooner as the tested extract and fractions concentration increases ¹³.

Tannins are one of the primary components responsible for anthelmintic activity. Tannins exert their anthelmintic effects by inducing protein denaturation within the worm's body and disrupting oxidative phosphorylation, thereby leading to metabolic disruptions and disturbances in homeostasis. Tannins can form complexes with unbound proteins within the gastrointestinal tract of worms or glycoproteins present in the worms' cuticle, consequently impeding various physiological processes, including motility, nutrient absorption, and reproductive functions^15,16.

Saponins exert their anthelmintic effect by inhibiting the acetylcholinesterase enzyme, resulting in paralysis and subsequent mortality of the worms. Saponins have been observed to impact the permeability of cell membranes in worms and induce irritation in the gastrointestinal mucous membrane tract of these organisms through interference with food absorption^15,16.

Flavonoids are thought to have anthelmintic activity by denaturing proteins in worm tissue, causing death in worms, and can degenerate neurons in worm bodies, which result in death. Alkaloids exert their mechanism of action by perturbing local homeostasis by inhibiting nitrate, a crucial component in protein synthesis, and suppressing sucrose's transportation to the small intestine. The location at which this active substance exerts its effects is within the central nervous system ^17,18.

Secondary metabolites are organic compounds, including plants, fungi, and bacteria. These compounds are not directly involved. Glycosides and triterpenoids/steroids possess antioxidant properties that can attenuate nitrate production, a crucial component for protein synthesis. Consequently, this leads to retardation in worm development and the suppression transfer by inhibiting the glucose uptake system. As a result, the worms experience energy depletion, ultimately culminating in their demise¹⁹.

The results of negative control measurements consisting of NaCl 0.9% solution obtained paralysis time and death time for Pheretima posthuma worms, respectively 327.33±17.62 minutes and 410.67±22.03 minutes. This time is the longest time obtained from all the test treatments that have been carried out in this study, and this is due to the negative control, which only contains NaCl 0.9% solution, where NaCl 0.9% solution is an isotonic solution. Thus, maintaining the cell membrane of the worm's body to stay alive. 0.9% NaCl contains ions the worm's body needs for its physiological processes.

The criteria for antimicrobial activity on microbial growth are measured from the diameter of the inhibition area, said to be inactive if the inhibition zone is smaller than 6 mm, active if the inhibition zone is 6-8mm, very active if the inhibition zone is greater than 8mm²⁰. Based on the measurement results presented in Table 2, It has been demonstrated that the levels of antifungal activity in ethanol extract, an-hexane fraction, ethyl acetate, and water all differ significantly. Dioscorea hispida tuber fraction in terms of their inhibitory effects on the growth of Candida albicans and Trichophyton mentagrophytes. The ethyl acetate fraction's antifungal activity was superior to the ethanolic extract, n-hexane, and water fraction. This was observed by measuring the inhibition area diameter on the fungal growth. Specifically, a 5 g/L concentration of the ethyl acetate fraction exhibited antifungal activity in the highly active category. In contrast, the ethanolic extract at a concentration of 50 g/L, the water fraction at 200g/L, and the n-hexane fraction at 300g/L did not demonstrate the same level of antifungal activity²⁰.

The antifungal efficacy of the ethanolic extract and fractions derived from Dioscorea hispida is believed to be attributed to the existence of secondary metabolites, including alkaloids, phenols, flavonoids, saponins, and terpenoids, which possess antifungal properties^21,22. The antifungal activity exhibited by the ethanolic extract and ethyl acetate fraction is believed to be attributed to secondary metabolites, specifically flavonoids and saponins. Flavonoids, a prominent class of compounds found in nature, are recognized for their antioxidant properties. These compounds possess phenol groups, contributing to their antibacterial and antifungal effects. Phenolic flavonoids can induce protein coagulation and lower the surface tension of microbial cells²³.

The ethanolic extract of Dioscorea hispida exhibits a higher abundance of secondary metabolites than the ethyl acetate fraction, yet it demonstrates a reduced inhibition diameter. The observed phenomenon is attributed to variations in the concentration of antifungal secondary metabolites attracted by individual solvents ²³. In addition, the ethanolic extract still contains many other metabolites, such as primary metabolites involved during the extraction process and many non-polar compounds that can interfere with the diffusion process.

The antifungal activity of the water fraction is believed to be attributed to its secondary metabolite content, specifically saponins. Saponins exhibit a notable degree of fungicidal toxicity. The antifungal properties of saponins are attributed to their mechanism of action, which involves the interaction between saponins and membrane sterols. Saponin compounds play a role as antifungal agents by effectively lowering the surface tension of the sterol membrane in the fungal cell wall, thereby increasing permeability. Elevated permeability induces the extraction of intracellular fluid with higher concentration, releasing nutrients, metabolic substances, enzymes, and proteins from the cells, ultimately leading to the demise of the fungus²⁴.

The antifungal activity of the water fraction, it was found that the 400g/L water fraction test solution produced a smaller inhibition zone than the 300g/L water fraction test solution. The relationship between the increase in concentration and the diameter of the inhibition zone may not exhibit direct proportionality. This phenomenon may occur due to the high concentration of the extract, which hinders its diffusion into the agar medium^23,24.

Antifungal activity in the n-hexane fraction is thought to be due to its secondary metabolite content, namely terpenoids. Terpenoids can interact with transmembrane proteins located on the outer membrane of microbial cell walls, forming robust polymeric bonds that ultimately cause the degradation of porins. The impairment of the porin, which serves as the primary channel for the transportation of compounds into and out of microbial cells, can lead to a decrease in the permeability of the microbial cell wall. Consequently, the affected microbial cells may experience a deficiency in essential nutrients, resulting in inhibited growth or cell death^23,24.

The antifungal activity of the n-hexane fraction was found to be the least potent when compared to the ethanolic extract, ethyl acetate fraction, and water fraction. This is because the n-hexane solvent attracts non-polar compounds such as oils and fats, which can interfere with antifungal activity. Oils and fats interfere with the diffusion process and protect microbes from antimicrobial compounds, so they cannot inhibit microbial growth^23,24.

The results of statistical tests on the antifungal activity of ethanol extract and gadung tuber fractions against Candida albicans and Trichophyton mentagrophytes using SPSS 22 showed a significance value of P<0.05 in the normality test so it can be concluded that the data is normally distributed. Data testing was continued with the one-way ANOVA test and a significance value was obtained for both mushrooms of 0.000 where sig. P<0.05. So, it can be concluded that there is a significant difference in the diameter of the inhibition zone in the ethanol extract, gadung tuber fractions with various concentrations as well as the positive control and negative control.

CONCLUSION:

This study investigates the anthelmintic and antifungal properties of different fractions of Dioscorea hispida (Dennst.) tubers, specifically the ethanol extract, n-hexane fraction, ethyl acetate, and water. The results indicate that the ethyl acetate fraction exhibits the highest efficacy in anthelmintic and antifungal activities. This can be attributed to alkaloids, flavonoids, glycosides, and tannins within the ethyl acetate fraction. The ethyl acetate fraction exhibited significant anthelminthic activity, as evidenced by a paralysis time of 23.90 ± 2.94 and a time of death of 34.20 ± 2.31, both measured at a concentration of 30 g/L. Additionally, the antifungal activity of the fraction against Candida albicans and Trichophyton mentagrophytes was evaluated at a concentration of 50 g/L, resulting in inhibition zone diameters of 21.13 ± 0.55 and 20.23 ± 0.65, respectively.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

We would like to thank Universitas Sumatera Utara for funding this TALENTA-supported research project.

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Received on 16.09.2023 Modified on 18.12.2023

Research J. Pharm. and Tech 2024; 17(8):3903-3910.

DOI: 10.52711/0974-360X.2024.00606