INTRODUCTION:

Nematodes infectionsstill occur in huge numbers affecting, directly and indirectly, humans, livestockalso the environment^1,2.Nematode infection especially Haemonchus contortuscauses major economic loss in livestock^3–5. Adult Haemonchus contortus is attached and feeds the blood on the abomasumcausingblood loss oedema, diarrhoea, depression,and even death⁶. The application of synthetic anthelmintic is the most common method to eliminate Haemonchosis. The potential useof the medicinal plant as a natural anthelmintic has been documented⁷^–12. Moreover, researchers¹²^,¹³ try to combine several medicinal plants as a novel polyherbal formulation and its anthelmintic activity and the result is promising.

The disadvantages of the medicinal plant are such as invariability of effect, and theexactness of dosage depends on a lot of factors.

Nanotechnology becomes the breakthrough solution to make the materials at the nano proportions, in which this nano size can be utilized at various applicationsas including drug discovery and delivery¹⁴^–¹⁵, and also as diagnostic tool¹⁶. The mechanism to deliver drugs is by improving the biodistribution of encapsulated compounds more effectively and more selectively to the pathological site. According to several researcher¹⁷^-¹⁸ nanoparticles improve the stability and solubility of drugs inside the body by protecting the drug molecule inside systemic circulation. Because of the nanosize, they can penetrate throughout the body, and also can interact with or penetrate microorganisms^17-¹⁹.The frequent methods to synthesis nanoparticles are physical and chemical²⁰^-²⁴. Implementation of the physical method needs a lot of external energy to decrease the sizeof the particle²¹. The implementation of toxic compounds for the synthesis of silver nanoparticles using chemical methods is an obstacle in its applications. Researcher developing new green methods to synthesize nanoparticles. Biological methods provide a wide range of environmentally acceptable methodologies and their applications in various fields.²²^-²³

Phytochemicals content such as alkaloids, phenolic compounds, terpenoids, as well assteroidsare huge in extracts of medicinal plantsincluding fungi, bacteria such as Actinomycetes those bioactive can decrease silver salts. ²⁴^–³¹ Those phytochemicalscompounds can be used to synthesize silver nanoparticles because they are simple, effective, economical/low cost and environmentally friendly, inexpensive, safe, and do not require any sophisticated instrumentation. Any part of the tree such as the leaf, root, stem, peel, or fruit, can be utilizedto synthesis silver nanoparticles.³²^,^28,32,33

The use of plants for the synthesis of nanoparticles is a rapid, low-cost, eco-friendly, adaptable to underdeveloped countries and single-step method that can be directly used for drugs¹⁵³⁴³¹³⁵. Melastoma malabathricum has belonged to the Melastomataceae family, this plantisof medicinal plants is commonly found in Indonesia, Thailand, andMalaysia. Melastoma malabathricum is used in traditional medicine for various diseases such as bleeding, diarrhoea, swelling, anti-spasmodic, gynaecological tumours,antiviral and cytotoxic activity. Our findings showed that Melastoma malabathricum have capability as wormicidal can inhibit egg hatch and larva development of Haemonchus contortus in vitroas well as in vivo on local goats. Our research was conducted to determinethe anthelmintic impact of the Ag-Extract Melastoma malabathricum combination on the adult motility test of Haemonchus contortus.

MATERIAL AND METHODS:

Preparation of extract of Melastoma malabathricum:

Fresh leaves Melastoma malabathricum were collected around the University of Bengkulu by handpicking manually. The plant leaves were dried in the shade for about 6 days and pulverized into a coarse powder.200gram Melastoma malabathricum powder was macerated in1000 ml 96% ethanol for 24 hours then filtered. The filtered crude ethanol extract of Melastoma malabathricum was distilled for 4 hours at 85⁰C and then stored in the fridge.

Qualitative Phytochemical Test:

Qualitative phytochemical analysis was done to carry out the metabolic seconder of Ethanolic extracts of Melastoma malabathricum.

Liebermann- Burchard Test for Terpenoids and Steroids:

Ethanolic extracts of Melastoma malabathricum dissolved into chloroform then acetic anhydride is added followed by sulphuric acid fromthe sideofthetube. The green colour demonstrate the existence of steroids and pink colour terpenoids

QualitativeTest for flavonoids: Sodium hydroxide (NaOH) test:

Four (4) ml of 10 % Na O H were put on 2ml of Melastoma malabathricum extracts the formation of a yellow precipitate indicates the presence of flavonoids in the extract.

Test for tannin: Ferric chloride (FeCl) test:

The test extract was mixed with a few drops of 5% FeCl, bluish-green colour indicated the presence of hydrolyzable tannins

Preparation of 1^-2M silver nitrate solution:

Sterile distilled water was used for preparation of solution,briefly 0.1699g of AgNO3 (100% purity) wasdissolve in 100ml of sterile distilled water, then stirred well and stored at 4°C for further studies²⁶.

Green synthesis of AgNP:

The green synthesissolution was done according to the method of²⁶ with modification, briefly, 10ml ethanolic extract of Melastoma malabathricum as describe previously was mixed with 90ml of 1^-2M Silver Nitrate (Merck, Germany) This solution was stirredconstantlyforonehour using a magnetic stirrer and incubated at room temperature. AUV-Vis spectrophotometer (Nano-Photometer P 360-Implant-Germany) was employed for the spectrometric analysis. The reduction of silvernan oparticles wasmeasured in wave lengths ranging from 200 to 700nm. The colour change of the solution from brown to reddish-brown was indicated the depletion of Ag+ to Ag− nanoparticles.

In vitro assay:

The adult worms Haemonchus contortus gathered from the abomasum of goat to be slaughtered in the local abattoir, the abomasum was opened in 0.9% saline at 37 C and washed away. The active female worms were collected for the assay. In this assay 10 worms which still actively moving put in Petri dishes containing various test concentrations (AgNPs- Extract 0.2mg/ml, AgNPs- Extract 0.1mg/ml, AgNPs- Extract 0.05mg/ml, M malabathricum extract 200mg/ml, M malabathricum extract 100mg/ml, Albendazole 40mg/ml and Normal Saline), four replicates for each concentration were used. The total number of motile and immotiledead Haemonchus contortus were calculated individually under a dissecting microscopeevery ten minutes after exposure and mortality were recorded for each concentration. Inaddition, the onset/time of worms paralyze and death was noted.

Data Analysis:

The mean percentage of adult motility assay was summarized as means ± SEM, while the different concentrations between means were compared by one-way ANOVA and Duncan Multiple Range Test. The time need of each treatment required to kill 50% of adult worms’ motility (LT₅₀) and LT 99 and their 95% confidence intervals (CI) were generated using probit analysis using SPSS.

RESULTS AND DISCUSSION:

Qualitative Phytochemical Test.

Melastoma malabathricum in ethanolic leaf extract was tested for the presence of terpenoid, steroid, tannin, flavonoid, and alkaloid. Sign ‘+’ indicated relative abundance of secondary metabolite contents in Melastoma malabathricumleaves extract, while ‘-’ sign the non-existence of secondary compound in this extract (Ferdous, 2018). Qualitative assessment (Table 1) indicates the appearance of terpenoids, tannin, flavonoids, but an absence of steroids and alkaloids. Danladi reported the presence of tannin, alkaloids, flavonoids, phenols, terpenoids, but no glycosides and saponins were demonstrated consistency on phytochemical screening of ethanolicextract of Melastoma malabathricum. However, Danladi also found invariably content of both phenolic and flavonoidmethanol extract Melastoma malabathricum in a different location.

Table 2: Adult motility assay

Treatments	Worms Motility (%).
	Time (minutes)
	0	10	20	30	40	50	60	70	80
AgNPs- Extract (0.02 mg/ml)	100	37.5	0	0	0	0	0	0	0
AgNPs- Extract (0.01 mg/ml)	100	66.7	25	0	0	0	0	0	0
AgNPs- Extract (0.005 mg/ml)	100	100	79.17	58.33	29.17	0	0	0	0
M. malabathricum extract 200 mg/ml	100	66.7	41.67	0	0	0	0	0	0
M. malabathricum extract 100 mg/ml	100	100	75.00	50.00	12.50	0	0	0	0
Albendazole 40 mg/ml	100	66.7	16.67	0	0	0	0	0	0
Normal Saline	100	100	100	100	100	100	100	100	100

The twitching motion or body stiff is used as an indicator of non-motile worms. The changes in the worm's motility are observed every 10minutes and the percentage of motility is represented in Table 2. The highest immobilization was found in synthesized AgNPs -Extract Melastoma malabathricum 0.02mg/ml against mature female Haemonchus contortus. While the efficacy of AgNPs-Extract Melastoma malabathricum 0.01mg/ml similar to control positive (Albendazole 40 mg/ml), and Melastoma malabathricum extract 200 mg/mlit means that AgNPs-Extract Melastoma malabathricum 0.01mg/ml was found as effective as Albendazole. Results showed the immobility of the worms as evidence of the efficacy of extract of Melastoma malabathricum as a natural anthelmintic. As tannin is main phytochemical compound in Melastoma malabathricum ethanolic extract, this compound could be responsible immobilize the adult worm. According to ⁴⁰ presence of tannins disturb the larval ensheathment. Acevedo-Ramirez et al confirmed that tannins from chestnut wood showed toxic activity on the L₃ of the parasite⁴¹. Based on the scanning electron microscope reported ⁴²that condensed tannins caused significant damage to the larval cuticle and gastrointestinal tissue of the worms.

The minimum time required for paralysis and death of Haemonchus contortuswas 9.29±0.68and 27.22±1.59 minutes for green synthetic nanoparticles AgNPs-Melastoma malabathricum extract, followed by AgNPs +extract (0.1mg/ml) 14.98+1.38 minutes and 48.90.0± 2.23 minutes. Our findings showed paralysis and death time of adult Haemonchus contortus AgNPs- Extract 0.01mg/ml comparable (AgNPs- Extract (0.01mg/ml) tothestandardanthelmintic used (Albendazole 40 mg/ml). Several researchers reported that plants rich in tannins as a potential alternative for the decrease in the motility and migration of H. contortus larvae.

The paralyzes and death time for extract Melastoma malabathricum 200mg/ml occurred within 16.58±1.57min and 43.97±1.40. It seems that the highest concentration is in line with the faster paralysis and death time. Similarly, the result was also reported by⁴³ who working with Ophioxylon album L. Ag-NPs- Extract Momordica charantia possessed high anthelmintic activity against Pheretima posthuma⁴⁴. Our result also showed that the distilled water did not affect even after 120 minutes of exposure. The metabolic seconder in the extract could interrupt the neuromuscular transmission causes paralysis of musculaturewormsleading to their death⁴⁵. According to Sen (2014),⁴⁶ phytochemicals on plan extract can attach glycoprotein to the cuticle of the parasite causing deaths⁴¹. The effect of In addition, Khade et al.¹³ said that the phytochemical compound such as tannins in medicinal plants could bind the free proteins in the gastrointestinal tract of the animal causing the death of the parasites.

The wormcidal test of M.charantia and AgNPs was studied by⁴⁴ using Pheretima posthuma. It was reported that the most effective is the combination of M. charantia and AgNPs (50mg/ml compared to the extract alone.

Capturing Agnanoparticles on the extract could be the reason the combination is more effective due to These two components acting as the synergistic effect of eachother. Asaresult, a strong wormicidal effect was found. The wormicidal activity of Ag-Extractnanoparticles against earth worms suggests that they are also effective against parasitic infections of humans.

Table 3: Paralysis and death time of adult worms

Treatments	Paralysis time + SEM			Death time + SEM
Treatments	Minutes
AgNPs- Extract (0.02 mg/ml)	9.29	+	0.68^a	27.22	+	1.59^a
AgNPs- Extract (0.01 mg/ml)	14.98	+	1.26^b	50.39	+	2.00^bc
AgNP AgNPs- Extract (0.005 mg/ml)	30.82	+	2.01^c	55.25	+	2.01^c
M malabathricum extract 200 mg/ml	16.58	+	1.57^b	43.97	+	1.40^bc
M malabathricum extract 100 mg/ml	29.71	+	1.89^c	50.00	+	1.72^bc
Albendazole 40 mg/ml	14.98	+	1.21^b	39.42	+	1.32^b
Normal Saline	> 120			>120

Table 4: Lethal Time (LT) 50 and Lethal Time(LT) 99 as well as Confident Interval (CI)

Treatments	LT 50	95% CI	LT 99	99% CI
AgNPs- Extract 0.02 mg/ml	24.57	11.30 - 29.65	42.69	37.84 - 58.10
AgNPs- Extract 0.01 mg/ml	49.14	41.07 –53.66	69.14	61.66 - 93.32
AgNPs- Extract 0.005 mg/l	53.25	41.95 - 53.12	82.78	73.07 - 111.36
M malabathricum extract 200 mg/ml	42.91	38.49 –45.88	55.54	51.26 - 67.08
M malabathricum extract 100 mg/ml	45.89	39.93 - 49.21	67.63	61.41 - 85.03
Albendazole 40 mg/ml	37.73	32.47 - 40.51	55.10	49.56 - 73.85

Table 5: Lethal Concentration (LC) AgNPs- ExtractMelastoma malabathricumfor 30- and 60-minutes examination

Lethal Concentration	AgNPs- Extract M malabathricum (mg/ml)
Lethal Concentration	30 minutes	60 minutes
LC-50	0.0187	0.0046
LC-60	0.0197	0.0058
LC-75	0.0215	0.0079
LC-99	0.0301	0.0292

Overall the anthelmintic activity revealed the concentration-dependent. The data clearly show even with a low concentration of AgNPs- Extract (0.02 mg/ml) exerts greater anthelmintic effects than Albendazole and ethanolic extract of Melastoma malabathricum (Table 4). Probit analysis revealed lethal concentration (LC₅₀) of AgNPs+ Extract Melastoma malabathricum against adult Haemonchus contortus 0.0187mg/ml in 30 and 0.0046 in 60 minutes). The study implemented of silver nanoparticles (AgNPs) synthesized using the aqueous extract of Azadirachta indica against adult Haemonchus contortus showed the lethal concentration (LC₅₀) 0,5885mg/ml⁴⁷. It seems tannin in extractMelastoma malabathricum have ability to inhibit adult Haemomchus contortus. Our finding indicated that the lethal consentration 50% of Melastoma malabathricumis smaller than that of Azadirachta indica. Several authors reported that tannin could physically damage around the mouth and reproductive organs and external cuticle of the parasite, also inhibit nutrient availability and metabolism of parasite, separate the sheath of the cervical and caudal regions of the larva⁴⁰. Table 4 showed that combination AgNPs- Extract 0.02mg/ml have the shortest time to kill 50% of the worms. As the same result also reported⁴⁴ that M. charantia and AgNPs combination of was more effective than their individual. Perhaps when the extract was capped on Ag nanoparticles, these two components act as the synergistic effect of each other therefore strong inhibition effect was found.

CONCLUSION:

Extract Melastoma malabathricum is capable of producing silver nanoparticle, and have the capability effect as anthelmintic again adult Haemonchus contortus. Further experiment still needed to examine the active ingredients that may lethal to the worm during in vitro test and also the effect of this in vivo on goats.

ACKNOWLEDGEMENT:

The authors are grateful for the financial support of the Ministry of Education-Indonesia, through competitive grand Strategic National Research contract number 679/UN30.15/AM/LT/2021. Also thankful to Animal Science Laboratory, Faculty of the Agriculture University of Bengkulu for providing us with the necessary facilities to carry out the present work.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

AUTHORS’ CONTRIBUTIONS:

Tatik Suteky concepted the study, collected materials, and did experimental work, wrote the manuscript. Dr Dwatmadji supervised data analysis and wrote the manuscript to ensure publication.

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Received on 16.11.2021 Modified on 26.02.2022

Research J. Pharm. and Tech 2023; 16(4):1913-1918.

DOI: 10.52711/0974-360X.2023.00314

Secondary Metabolites	Methods	Observation	Result
Terpenoids	Liebermann- Burchard Test	The pink colour indicates the presence of terpenoids	+
Steroid	Liebermann- Burchard Test	The green colour indicates the presence of steroids	-
Tannin	Ferric Chloride Test	The formation of bluish-green colour indicated the presence of hydrolyzable tannins.	++
Flavonoid	Sodium hydroxide test	The formation of intense yellow colour indicated the presence of flavonoids which turns colourless with the addition of dilute acid.	+
Alkaloid	Mayer’s test	A creamy white precipitate for alkaloids.	-

The Synthesis AgNPs of Leaves Melastoma malabathricum Extract reduce Adult Haemonchus contortus Motility Assay