The anti-leech potential of the solvent extract of Bornean neem leaves and ultra-high performance liquid chromatography-high-resolution mass spectrometry profiling

1 Introduction

Aquaculture is an important activity in Malaysia and a vital tool for the increment of export revenue, food production and employment (Othman et al., 2017). In 2018 the fisheries department of Sabah reported 159,773.11 tonnes of fishery products from aquaculture in Sabah, Malaysia (Department of Fisheries Sabah, 2020). Aquaculture is an important source of protein for over 30 million population of Malaysia. Unfortunately, the spread of diseases and parasitic infestation is impeding aquaculture developments (Venmathi Maran et al., 2009; Venmathi Maran et al., 2012).

In Malaysia, aquaculture farmers culture various types of marine fishes such as snappers, groupers and sea bass. The highly prevalent species of the groupers are hybrid grouper Epinephelus fuscoguttatus × E. lanceolatus, tiger grouper E. fuscoguttatus, orange-spotted grouper E. coiodes and Malabar grouper E. malabricus. The highly prevalent snappers are the yellow-streak snapper Lutjanus lemniscatus, mangrove snapper L. argentimaculatus, John’s snapper L. johnii and red snapper L. erythropterus (Othman et al., 2017). The culture of groupers and snappers impeded greatly due to the infestation of some parasites (Venmathi Maran et al., 2009) in addition to marine parasitic leech Zeylanicobdella arugamensis de Silva, 1963 (Hirudinea, Piscicolidae) (Cruz-Lacierda et al., 2000; Ravi and Shariman Yahaya, 2017). They attached to the gills, eyes, and fins of the host, feed the blood by sucking and cause lesions and wounds on the body surface which leads to bacterial infection and ultimately result in host mortality within 3–4 days (Cruz-Lacierda et al., 2000). Farmers use formalin, pesticides and other chemicals for the disinfestation of the parasites (Shariff et al., 2000). Such chemicals are highly toxic to fish (Pitten et al., 2000). Thus, it is very important to develop a natural control agent to reduce the consumption of harmful chemicals. Plant-derived extracts can act as a biocontrol agent against leeches due to the presence of different metabolites (Bahmani and Rafieian-Kopaei, 2014).

The neem plant (Azadirachta indica) (Meliaceae) has been reported with insecticidal activity (Schmutterer, 1990), antiparasitic activity (Venmathi Maran et al., 2021) and antivenom activity against black-necked spitting cobra (Naja nigricollis) (Sani et al., 2020). The antihelminthic efficacy of the neem extract has been reported against the gastrointestinal parasite of animals (Jamra et al., 2015). Antibacterial potential of the aqueous extract of neem leaves has been reported against Citrobacter freundii gram-negative bacteria isolated from diseased fingerlings of Oreochromis mossambicus (Tilapia) (Thanigaivel et al., 2015). Malaysian neem plant leaves have been reported with antifungal activity against the fungi Aspergillus niger and Candida albicans (Arumugam et al., 2015). However, no research has been reported on the evaluation of the anti-leech potential of the methanol extract of the plant against Z. arugamensis. The study aimed to provide a natural control agent against the parasitic leech Z. arugamensis by the evaluation of the antiparasitic potential and phytochemical investigation of the methanol extract of the neem leaves.

2 Materials and methods

2.3

2.3 Antiparasitic bioassay

The parasitic leeches were collected from the infested hybrid groupers (Epinephelus fuscoguttatus × E. lanceolatus) (Fig. 1) from Universiti Malaysia Sabah. The selected adult parasitic leeches (0.8–1.2 cm long) were categorized into 5 groups (6 leeches per group) negative control (group 1), positive control (group 2, treated with 0.25% formalin solution), extract treatment (groups 3, 4 and 5 administered with 25, 50 and 100 mg/ml of the methanol extract of neem leaves). The selected concentrations were based on optimization. The behaviour of parasitic leeches was noticed keenly during the exposure (Fig. 2). Parasites inactivity and mortality were noted up to 120 mins. At the end of the experiment, the leeches were further observed for 720 mins (after every 60 min interval) (Shah et al., 2020c).

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Fig. 1

Infestation of Zeylanicobdella arugamensis on hybrid grouper fish (Epinephelus fuscoguttatus × E. lanceolatus).

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Fig. 2

Control and plant treated leeches. a = control, normal leech, treated with seawater only, b = leech treated with 0.25 % of formalin solution, c = leech treated with methanol extract of neem leaves.

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3 Results

3.1

3.1 Phytochemical profile of neem leaves methanol extract

The name of the metabolites noticed in the methanol extract of neem leaves via UHPLC-HRMS along with the class, retention time and formula are shown in Table 1 and the structures are indicated in Fig. 3. The data showed the presence of flavonoids (isorhamnetin, myricetin, myricetin-3-O-galactoside, trifolin, quercetin), aromatics (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, 2,4-quinolinediol, indole-3-acrylic acid), phenolics (2-(3,4-dihydroxyphenyl) acetamide, apocynin, phloretin, p-coumaric acid, scopoletin), and terpenoids (carvone, pulegone).

Table 1 List of the 41 metabolites in the methanol extract of neem leaves identified using UHPLC-HRMS.

No.	Metabolites	Class	Retention Time	Formula	m/z	Adduct	Mass Error, ppm
[1]	Histidine	Amino acid	0.69	C6H9N3O2	156.07664	[M + H]+	1.35
[2]	N3,N4-Dimethylarginine	Amino acid	0.85	C8H18N4O2	203.15031	[M + H]+	0.45
[3]	Arginine	Amino acid	0.69	C6H14N4O2	175.11884	[M + H]+	0.92
[4]	Valine	Amino acid	0.75	C5H11NO2	118.08642	[M + H]+	0.26
[5]	Leucine	Amino acid	0.90	C6H13NO2	132.09036	[M + H]+	1.07
[6]	1-Aminocyclohexanecarboxylic acid	Amino acid	1.20	C7H13NO2	144.10190	[M + H]+	0.35
[7]	Proline	Amino acid	0.94	C5H9NO2	116.07070	[M + H]+	1.04
[8]	Jasmone	Cyclic ketone	7.04	C11H16O	165.12721	[M + H]+	0.97
[9]	2-Methylcyclohexan-1,3-dione	Cyclic ketone	1.50	C7H10O2	127.07545	[M + H]+	0.00
[10]	Decanamide	Fatty Acyl	8.09	C10H21NO	172.16940	[M + H]+	0.88
[11]	4-oxododecanedioic acid	Fatty Acyl	5.17	C12H20O5	245.13652	[M + H]+	0.66
[12]	3-Hexenoic acid	Fatty Acyl	1.87	C6H10O2	115.07545	[M + H]+	1.23
[13]	Isorhamnetin	Flavonoid	5.54	C16H12O7	317.06509	[M + H]+	1.52
[14]	Myricetin	Flavonoid	4.91	C15H10O8	319.04434	[M + H]+	1.10
[15]	Myricetin 3-galactoside	Flavonoid	4.72	C21H20O13	503.07938	[M + Na]+	0.44
[16]	Trifolin	Flavonoid	5.41	C21H20O11	471.08945	[M + Na]+	1.16
[17]	Quercetin	Flavonoid	5.03	C15H10O7	303.04953	[M + H]+	1.79
[18]	6-Methyl-2-pyridinemethanol	Heterocyclic	1.08	C7H9NO	124.07578	[M + H]+	0.73
[19]	Cytidine	Heterocyclic	0.71	C9H13N3O5	282.04807	[M + K]+	1.07
[20]	Guvacoline	Heterocyclic	1.07	C7H11NO2	142.08617	[M + H]+	0.14
[21]	Glucosamine	Heterocyclic	0.61	C6H13NO5	180.08640	[M + H]+	0.78
[22]	Pyroglutamic Acid	Heterocyclic	0.83	C5H7NO3	130.04984	[M + H]+	0.46
[23]	β,β-Dimethyl-γ-methylene-γ-butyrolactone	Heterocyclic	1.61	C7H10O2	127.07500	[M + H]+	0.08
[24]	Pipecolic acid	Heterocyclic	1.05	C6H11NO2	130.08623	[M + H]+	0.85
[25]	1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid	Heterocyclic aromatic	1.34	C10H11NO2	178.08582	[M + H]+	0.45
[26]	2,4-Quinolinediol	Heterocyclic aromatic	3.24	C9H7NO2	162.05475	[M + H]+	1.37
[27]	Indole-3-acrylic acid	Heterocyclic aromatic	1.94	C11H9NO2	188.07060	[M + H]+	0.59
[28]	Prolylleucine	Peptide	1.53	C11H20N2O3	229.15443	[M + H]+	0.53
[29]	2-(3,4-dihydroxyphenyl)acetamide	Phenolid	0.93	C8H9NO3	168.06551	[M + H]+	0.54
[30]	Apocynin	Phenolic	2.93	C9H10O3	167.06998	[M + H]+	0.60
[31]	Phloretin	Phenolic	5.40	C15H14O5	275.09100	[M + H]+	1.50
[32]	p-Coumaric acid	Phenolic	3.21	C9H8O3	165.05435	[M + H]+	0.61
[33]	Scopoletin	Phenolic	4.35	C10H8O4	193.04947	[M + H]+	0.52
[34]	1-Methyladenine	Purine	0.83	C6H7N5	150.07732	[M + H]+	0.47
[35]	Guanine	Purine	0.79	C5H5N5O	152.05652	[M + H]+	1.26
[36]	2′-O-Methyladenosine	Purine	1.61	C11H15N5O4	282.11963	[M + H]+	1.35
[37]	Adenine	Purine	0.80	C5H5N5	136.06166	[M + H]+	0.67
[38]	Carvone	Terpenoid	5.04	C10H14O	151.11153	[M + H]+	0.87
[39]	Pulegone	Terpenoid	3.61	C10H16O	153.01779	[M + H]+	0.72
[40]	Nicotinamide	Vitamin B3	0.89	C6H6N2O	123.05534	[M + H]+	0.66
[41]	Nicotinic acid	Vitamin B3	0.84	C6H5NO2	124.03929	[M + H]+	0.49

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Fig. 3

Structure of the 41 identified metabolites from the methanol extract of neem leaves.

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3.2

3.2 Antileech activity of neem leaves methanol extract

The administration of neem leaves extract resulted in the killing of leeches at lower (25 mg/ml), medium (50 mg/ml) and higher (100 mg/ml) concentration in an average period of 35.81 ± 5.40, 14.18 ± 0.99 and 9.21 ± 1.51 min. While an average time of 3.77 ± 0.25 min was taken by the formalin solution to kill the leeches. No mortality was noticed in the negative control group treated with seawater. Significance differences were noticed by multiple comparisons among the negative and positive control and extract-treated groups. The differences are highlighted by various symbols (Fig. 4). The mortality percentages of leeches are shown in Table 2. Total mortality (100 %) was noticed in leeches treated with neem leaves extract and formalin solution as compared to normal control group (no mortality).

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Fig. 4

Mortality time of parasitic leeches at different concentrations of methanol extract of neem leaves.

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Table 2 Mortality percentage of parasitic leeches at different concentrations of methanol extract of neem leaves.

No	Group	Mortality of parasites in percentage (%)
1	Normal control	0
2	Positive control (Formalin 0.25%) (v/v)	100
3	Neem (25 mg/ml)	100
4	Neem (50 mg/ml	100
5	Neem (100 mg/ml)	100

4 Discussion

The parasitic leech Z. arugamensis is well distributed throughout the Indian Ocean, affects a wide range of marine fish species (Cruz-Lacierda et al., 2000; Kua et al., 2014; Ravi and Shariman Yahaya, 2017). The leech resulted in the morality of the host in a very short period of time (Kua et al., 2014). Toxic and non-degradable drugs and chemicals are applied for the control of parasitic infestation (Forwood et al., 2013; Pitten et al., 2000). However, the chemicals could be hazardous to living organisms and the environment (Jayaraj et al., 2016; Pitten et al., 2000). Hence, to control the leech infestation, plants with potential bioactive compounds can be applied as a natural remedy (Kayser et al., 2003). Thus, the main reason for this study was to develop a natural and biodegradable control agent against parasitic leech by the application of the plant. Our study indicated that the methanol extract of neem leaves resulted in a total mortality of leeches at a concentration of 25 to 100 mg/ml in an average time period of 35.81 ± 5.40 to 9.21 ± 1.51 min, respectively. Some other plants with antiparasitic properties have also been reported recently. The methanol extract of fern Nephrolepis biserrata (Nephrolepidaceae) (100 mg/ml) resulted in a total mortality of Z. arugamensis in 4.8 min (Shah et al., 2020b), however the chromatographic fraction (2.50 mg/ml) took 1.9 mins (Shah et al., 2021). The methanol extract of neem leaves (100 mg/ml) took more time (9.21 min) for the total mortality of leeches compared to the methanol extract and fraction of N. biserrata. This might be due to the presence of various antiparasitic compounds of different nature in N. biserrata extract and fractions including ivalin, isovelleral, kaempferol 7,4′-dimethyl ether, piscidic acid, chlorogenic acid, phytosphingosine, pyrethrosin, warburganal, isodomedin and pheophorbide a (Shah et al., 2020b; Shah et al., 2021), however the neem leaves extract indicated completely different antiparasitic compounds (Table 1), discussed below in details. Similarly, the methanol extract of shrubby Dillenia suffruticosa (Dilleniaceae) (100 mg/ml) resulted in a total mortality of the parasitic leech in 14 min. (Shah et al., 2020c) and the chromatographic fractions (20 mg/ml) took 31 min. (Shah et al., 2020a). The methanol extract of the neem leaves indicated high antiparasitic efficacy in less time compared to the methanol extract of D. suffruticosa. All plants are available naturally in Sabah without any cost. Baikal Skullcap Scutellaria baicalensis (Lamiaceae) was tested against the marine parasitic leech Piscicola geometra (Hirudinea) infesting Cobia fish (Rachycentron canadum) (Rizky et al., 2018). The leeches were exposed to different serial dilutions (ranging from 1/50x to 1/1000x) of the extract for 96 h and mortality (100%) was recorded in an average time period of 8 h, 40 h, 48 h, 72 h, and 96 h (Rizky et al., 2018). The various serial dilutions (ranging from 1/50x to 1/1000x) of the extract of Noni Morinda citrifolia (Rubiaceae) was applied against Piscicola geometra for 96 h and 80% of leech mortality was reported (Rizky et al., 2018). The methanol extract of the ginger Zingiber officinale (Zingiberaceae) at a concentration of 32 × 10⁴ ppm killed the freshwater leech Limnatis nilotica in 24 min (Forouzan et al., 2012). The above-mentioned reported plants evidently proved that the medicinal plants have anti-leech potential. In addition, compared to other plants the methanol extract of neem plant has shown an effective anti-leech activity in less than 10 min at higher concentration (see: Fig. 4) except N. biserrata.

The UHPLC-HRMS analysis of the methanol extract indicated the presence of compounds such as quercetin, carvone, phloretin, trifolin, jasmone and nicotinamide. The anti-leech activity of these compounds have not yet been reported against Z. arugamensis but they showed a various degree of potential effects against mammalian and plant parasites (Abdel-Rahman et al., 2013; Frank et al., 2002; Juan et al., 2013; Li et al., 2005; Mamani-matsuda et al., 2004; Mead and McNair, 2006; Sereno et al., 2005; Wuyts et al., 2006). In an animal study, a significant reduction in lengths of the gastrointestinal nematodes Haemonchus contortus has been reported in carvone treated sheep (Katiki et al., 2019). The application of carvone does not limit to mammalian parasites but plants as well. Aside from being effective in suppressing potato sprout, carvone has also been reported with antifungal properties against several plant parasitic fungi such as Fusarium, Phoma, and Helminthosporium (Hartmans et al., 1995; Oosterhaven et al., 1995). On the other hand, nicotinamide is also a well-documented antiparasitic compound against Plasmodium falciparum (Prusty et al., 2008), Trypanosoma cruzi (Soares et al., 2012), and Leishmania sp. (Sereno et al., 2005). Nicotinamide significantly deregulated the growth of T. cruzi by changing the conformation of sirtuin TcSir2, a NAD-dependent deacetylase which involved in the parasite cell cycle (Soares et al., 2012). Thus, we believe that the above-mentioned compounds in the methanol extract of neem leaves might contribute to the mortality of the parasitic leech. In the current study, the Bornean neem plant was easily accessible from nature without any cost. As mentioned above the crude extract of the plant possessed a mixture of vital antiparasitic chemical components. The cost of this naturally obtained chemical components mixture is very less as compared to the synthetically prepared chemical components.

5 Conclusion

Our data indicated that the methanol extract of neem leaves showed a significant anti-leech activity against the parasitic leech Z. arugamensis with a total mortality at various concentrations ranging from 25 to 100 mg/ml in average time period from 35.81 ± 5.40 to 9.21 ± 1.51 min, respectively, compared to untreated control. The ultra-high performance liquid chromatography–high–resolution mass spectrometry system revealed the presence of different bioactive compounds such as flavonoids, aromatics, phenolics, and terpenoids. Thus, it is indicated that the methanol extract of neem leaves act as an effective anti-leech agent against Z. arugamensis of cultured groupers due to the presence of vital bioactive compounds. Besides, our current sample indicated 100% mortality of Z. arugamensis in less time as compared to our previous sample D. suffruticosa. This research will be highly beneficial to fish farmers in Sabah, Malaysia to control the leeches in natural manners in aquaculture. However, further isolation, purification and characterization of the pure bioactive compounds with antiparasitic potential is required.