Abstract
Objective:
This study aimed to explore the phytochemical constituents and anthelmintic activities of four Cassia spp. leaves against Haemonchus contortus.
Materials and Methods:
The extracts were prepared from four species of Cassia spp. (C. siamea, C. fistula, C. surattensis, and C. spectabilis). Phytochemical screening of the extract was done based on the Harborne method. Evaluation of the anthelmintic activities against H. contortus was done in vitro using infective larvae (L3) migration inhibition assay (LMIA). Measurement of larvae migrating was conducted through a nylon filter with a pore size of 20 μm. The doses of Cassia spp. extract implemented were 25, 50, 100, and 200 mg/ml.
Results:
Tannins, alkaloids, phenol hydroquinone, flavonoids, steroids, triterpenoids, and saponins were present in all the extracts, whereas alkaloids were absent in C. fistula. No triterpenoids were found in C. surattensis and C. spectabilis. Movement of H. contortus larvae was significantly inhibited after exposure to Cassia extracts at various dosage levels (p < 0.05). The test results using LMIA on L3 H. contortus showed the lowest inhibition in the negative control. Among the species of Cassia, the C. surattensis (at 200 mg/ml) showed the highest (p < 0.05) inhibition level on the larvae. The latter result corresponded to the effect of albendazole.
Conclusion:
Compared to other Cassia spp., C. surattensis exhibited the highest inhibition against L3 H. contortus. However, the inhibition effect of C. surattensis was still lower as compared to albendazole.
Keywords: Anthelmintic effect, C. siamea, C. fistula, C. surattensis, C. spectabilis, H. contortus
Introduction
The gastrointestinal nematode is one of the animal health risks related to the use of pasture for small as well as large ruminant productions. The most dominant gastrointestinal nematodes in small ruminants in the tropics, including Indonesia, are Haemonchus contortus [1,2]. Control of gastrointestinal nematodes of ruminants mainly relies on the use of synthetic chemical anthelmintics. However, internal parasitic disease control using synthetic chemicals has raised big concern, such as chemical residues in the animal products, especially when the animals on the extensive livestock management system. In this respect, any alternatives substitute to the role of synthetic chemicals is, therefore, of important to be developed. Among the alternatives, tannin-rich plants seemed to be good candidates to control the internal parasitic disease in animals particularly the small ruminants on the pasture rotation system [3].
The research on anthelmintic effect of the plants containing tannin to nematodes has been carried out [4,5]. Cassia spp. is the plants with high condensed tannin content [6]. Kundu et al. [5] showed the broad spectrum anthelmintic effects of C. angustifolia, C. alata, and C. occidentalis crude ethanol extracts against helminth parasite of domestic fowls (Heterakis gallinarum, Catatropis sp., and Raillietina tetragona) in vitro at concentration levels of 10–40 mg/ml. Likewise, Cassia tora leaves extracts showed anthelmintic activity against Pherethima posthuma [7].
In respect, particularly to C. fistula Linn, the methanolic extract from pods, pulp, and seeds from such plant (at the concentration 100 mg/ml) showed an anthelmintic activity against P. posthuma [8,9]. Other Cassia spp., such as C. siamea, C. surattensis, and C. spectabilis, had reported having antibacterial activity, antibiofilm, antifungal, and antioxidant [10–13]. However, anthelmintic activities above mentioned Cassia spp. have never been documented. Anthelmintic activity can be affected by phytochemical compounds. Some phytochemical compounds, such as tannin, anthraquinone glycoside, naphtopyrone glycoside, phenolic compounds, flavonoids, and many others, had isolated from Cassia plants and suggested to the biological activity of the plants [14]. Hence, this study aimed to explore the phytochemical compound and anthelmintic activities against H. contortus of C. siamea, C. fistula, C. surattensis, and C. spectabilis leaves extracts.
Materials and Methods
Ethical approval
All the procedures in the study accordance were done after the approval from the Ethics Committee for Animal Use at the IPB University (approval number 44-2017 IPB).
Plants material
The plants were collected in the area of Diponegoro University, Semarang, Central Java, Indonesia. The collected plants were identified by Herbarium Bogoriense of Indonesian Institute of Sciences as C. siamea, C. fistula, C. surattensis, and C. spectabilis by the reference number 335/IPH.1.01/H.07/II/2016.
Extracts preparation
The leaves of Cassia spp. were separately picked and sorted to remove undesired plant parts. Drying of leaves in the room temperature was done for 7–10 days and then milled to produce homogenous flour particles. The Cassia leaf flours were then turned into extracts using maceration technique with n-hexane and ethanol solvent. Cassia spp. leaf flour (1,000 gm) was initially incubated in n-hexane (5,000 ml) and later in ethanol, 96% (5,000 ml) in the room temperature for 72 h. After incubation, extracting the extract was carried out with Whatman No. 1 filter paper. Filtering results were collected and evaporated using a vacuum rotary evaporator at 50 rpm, 40°C–50°C until the extract was obtained. Storage of the extracts before analysis was used at 4°C analysis.
Phytochemical screening
Phytochemical screening of Cassia spp. extract using the [15] method was done to detect tannins, saponins, alkaloids, flavonoids, steroids, triterpenoids, and hydroquinone phenolics.
Preparation H. contortus infective larvae (L3)
Haemonchus contortus for this study was cultured from stool of a H. contortus purely infected sheep donor. Feces of the donor sheep was collected every morning using an apron. The feces were then mixed with vermiculite to obtain moist and airy medium H. contortus egg hatching. Incubation of the mixture was carried out in the room temperature for 7–8 days so that H. contortus eggs can hatch and develop into infective larvae (L3). The L3 were then harvested by a modification of the Baermann method [16]. In order to remove fecal impurities, the infective larvae harvested are screened. The collected larvae were stored at 4°C before being used for the assay.
Infective larvae assay
An in vitro larval migration inhibition assay (LMIA) was performed to determine anthelmintic effect of the four Cassia spp. Extracts according to Rabel et al. [17] method. The experiment used factorial (4 × 6) design with of Cassia spp. as the first factor and dose levels of the extracts as the second factor [18]. The LMIA started with the mixing of 100 L3 (s) in each Cassia extracts that were prepared into 1.5-ml solution with the dosage levels of 25, 50, 100, and 200 mg/ml diluted in phosphate buffer saline (PBS).
These mixtures were then incubated at the room temperature for 3 h. After incubation, the mixtures were then washed thrice by using PBS, centrifuged at 3,500 rpm for 5 min, before the supernatant was removed. The remaining sediments (the larvae) were then sieved using 20-μm grid sieve mounted on PBS-filled microplate well. Number of the larvae migrated through the sieve to the microplate well was then counted by observing the process under a stereoscopic microscope at 20× magnification. The percentage of larval migration inhibition was then calculated using the formula of [(A − B)/A] × 100 where A is the number of larvae that were prepared (100) and B is the number of larvae that migrated through the sieve at each treatment [17].
Statistical analysis
Results of the phytochemical analysis were presented descriptively, while the results of the LMIA were analyzed based on analysis of variance using SAS v 9.0 [19]. The significant effect of LMIA was further tested with DMRT. The difference in treatment was stated to be significant at p < 0.05.
Results
Percentage yield of extract
The highest percentage of Cassia spp. extract yield was found in C. spectabilis, followed by C. surattensis, C. siamea, and C. fistula. Cassia surattensis extract had the highest tannin content followed by the extracts C. siamea, C. fistula, and C. spectabilis, respectively (Table 1).
Table 1. Percentage yield and tannin content from all four extracts of Cassia spp.
| Extract | Percentage yield (W/W) | Tannin (%) |
|---|---|---|
| C. siamea | 4.49 | 4.92 |
| C. fistula | 2.36 | 3.98 |
| C. surattensis | 5.23 | 7.82 |
| C. spectabilis | 11.37 | 2.67 |
Phytochemical screening of four Cassia spp. extract
Phytochemical compounds identified in the four Cassia spp. extracts are shown in Table 2. Tannins, alkaloids, phenol hydroquinone, flavonoids, steroids, triterpenoids, and saponins were present in all the extracts. Alkaloids were absent in C. fistula, whereas no triterpenoids was found in C. surattensis and C. spectabilis extracts.
Table 2. The phytochemical constituent of extracts of Cassia spp.
| Phytochemical | Extracts | |||
|---|---|---|---|---|
| C. siamea | C. fistula | C. surattensis | C. spectabilis | |
| Alkaloids | + | − | + | + |
| Phenol Hydroquinone | + | + | + | + |
| Flavonoids | + | + | + | + |
| Steroids | + | + | + | + |
| Triterpenoids | + | + | − | − |
| Tannins | + | + | + | + |
| Saponins | + | + | + | + |
+: present; −: absent.
Larval migration assay of extracts Cassia spp.
Table 3 showed that all the Cassia spp. extracts at various doses significantly affected the percentage of inhibited L3 H. contortus (p < 0.05). The H. contortus larval migration inhibition was lowest in the negative control (PBS). Among the various dose and species of Cassia, the C. surattensis at the dose level 200 mg/ml resulted in the highest inhibited of L3 H. contortus (p < 0.05). The later result corresponded to the effect of albendazole.
Table 3. Percentage L3 H. contortus inhibited after exposure to 3 h on the species and dose of extract of Cassia spp. different*.
| Extracts | PBS (negative control) | C-25 | C-50 | C-100 | C-200 | Albendazole (positive control) |
|---|---|---|---|---|---|---|
| C. siamea | 22.11 ± 2.84g | 59.89 ± 1.30f | 63.18 ± 1.09def | 63.34 ± 1.22def | 65.31 ± 1.25cd | 82.71 ± 1.19a |
| C. fistula | 24.89 ± 7.32g | 59.88 ± 1.54bc | 62.30 ± 4.42def | 60.90 ± 3.13f | 63.46 ± 1.49def | 81.82 ± 1.19a |
| C. surattensis | 23.81 ± 0.85g | 63.56 ± 2.41def | 65.90 ± 2.56cd | 67.99 ± 2.06c | 71.62 ± 2.49b | 84.94 ± 0.78a |
| C. spectabilis | 22.56 ± 2.64g | 59.95 ± 1.25f | 61.10 ± 1.71ef | 62.27 ± 1.71def | 64.98 ± 1.17cde | 85.36 ± 0.94a |
Values are mean ± SD of six replicates.
Means within a row and column with different superscripts differ (p < 0.05).
C- means dose of species Cassia extracts (mg/ml).
Discussion
Our data showed that all Cassia spp. used in the present study contained most of the phytochemical compounds, i.e., alkaloids, phenol hydroquinone, flavonoid, steroid, triterpenoid, tannin, and saponin. Unlike C. siamea, C. fistula contained no alkaloids, while C. surattensis and C. spectabilis contained no triterpenoids. Corresponding to our result, Mohammed et al. [20] found that C. siamea contained anthraquinones, alkaloids, tannins, saponins, flavonoids, polyphenols, and glycosides. Likewise, Kamagaté et al. [21] reported that the ethanol extract of C. siamea leaf contained flavonoid (D-pinitol, luteolin), dihydronaphtalen one [(4-trans)-acetyl-3,6,8-trihydroxy-3-methyldihidronapthalenone], triterpenoid (lupeol), and [4-(cis)-acetyl-3,6,8-trihydroxy-3-methyldihydronaphtalenone].
Our finding showed that C. fistula contains phenol hydroquinone, flavonoids, steroids, triterpenoids, tannins, and saponins. However, it contained no alkaloid. In contrast, Panda et al. [22] found alkaloids, flavonoids, tannins and phenolic compound, glycosides, protein, amino acids, saponins, and triterpenoids. In Another study revealed that C. fistula leaves mainly contain oxalic acid, tannin, oxy anthraquinone, and anthraquinone derivative [23]. In general, the composition and concentration of secondary metabolites in particular plant is influenced by several factors, including genetic factors, climate, soil, harvest time, and solar radiation [24].
Compared to PBS group, all Cassia spp. were capable of increasing the percentage inhibition of H. contortus, of the doses, applied. Yet, the values were still lower as compared to the albendazole group. In this respect, C. surattensis had anthelmintic activity, especially against H. contortus. Several studies used else species of the genus Cassia, such as the C. tora, C. auriculata, C. angustifolia, C, ocidentalis, and C. alata which contain active compounds as tannins, flavonoids, saponins, and alkaloids that shown to be anthelmintic [5,7,25]. The anthelmintic activities may be contributed by tannins, alkaloids, flavonoids, steroids, phenol hydroquinone, triterpenoids, and saponins.
Very limited study on the mechanisms of phytochemical compounds of Cassia spp. to destruct H. contortus. However, tannin has been demonstrated to block through uncoupling the oxidative phosphorylation leading to the death of parasites. Another possible mechanism could be through binding of tannins to free proteins in the gastrointestinal tract of the animal, or glycoprotein on the cuticle of the helminth’s body surface causing a paralysis, and thus death [26]. Cuticle and digestive tissue of larvae are significantly damaged due to the presence of tannins. The size of the polymer and the tannin molecule has a relationship in influencing the strength of the anthelmintic effect. In addition, there are effects of the structural units of monomeric tannins because gallocatechin and epigallocatechin monomers have significant anthelmintic activity on the contrary monomer catechin and epicatechin [27]. In addition to tannin, other bioactive compounds in Cassia spp. may contribute the anthelmintic activity against H. contortus. Indeed [28–30], suggested flavonoid, terpenoid, saponin, and alkaloids may exert an anthelmintic effect on H. contortus.
Previous studies in laboratory animals have demonstrated the safety of Cassia spp. as medicinal plants. Thus, no clinical signs of toxicity and mortality were found in mice receiving single oral doses of 2,000–5,000 mg/kg body weight extracts of C. spectabilis [13,31], C. siamea [21], and C. surattensis [32], respectively. However, long-term administration of Cassia spp. extracts may cause reversible hepatotoxicity in rats and mice [21].
Conclusion
Compared to other Cassia spp., C. surattensis exhibited the highest migration inhibition against L3 H. contortus. However, the inhibition effect C. surattensis was still lower as compared to albendazole. Owing to the latter fact, it could be inferred that C. surattensis may be used to substitute the role albendazole as anthelmintic provided that dose is increased. Hence, the future study is necessary to confirm optimal doses of C. surattensis as substitute for albendazole.
Acknowledgments
This research was financed by the Doctoral Dissertation Research Grant of the Ministry of Research, Technology and Higher Education of Republic of Indonesian through Domestic Graduate Scholarship (“BPP-DN”) program. The authors would like to thank Mr. Sulaiman and Department Animal Infectious Diseases, and Veterinary Public Health, Faculty Veterinary Medicine, IPB University, Bogor for providing laboratory facilities during the study.
Conflict of Interests
The authors declare that they have no conflict of interest.
Authors’ contribution
Sri Wahyuni and Fadjar Satrija designed the study, conducted the experiments, analyzed the data, and prepared the article. Sunarso Sunarso and Bambang Waluyo Hadi Eko Prasetiyono corrected the article.
References
- [1].Satrija F. The epidemiology and control of gastrointestinal nematodes of ruminants in Indonesia, with spec I:1 reference of small ruminants in West Java. In: Biological of gastrointestinal nematodes of ruminants using predacious fungi. FAO; Roma, Italia: 1998. Beriajaya; pp. 66–71. [Google Scholar]
- [2].Nurhidayah N, Satrija F, Retnani EBR. Gastrointestinal parasitic infection of swamp buffalo in Sentra Peternakan Rakyat (SPR) of Banten Province Indonesia: prevalence, risk factor and its impact to production performance. Trop Anim Sci J. 2019;41(1):6–12. https://doi.org/10.5398/tasj.2019.42.1.6. [Google Scholar]
- [3].Robertson HA, Niezen JH, Waghorn GC, Charleston WAG, Jinlong M. The effect of six herbages on liveweight gain, wool growth and faecal egg count of parasitised ewe lambs. Proc N Zeal Soc Anim Prod. 1995;55:199–201. [Google Scholar]
- [4].Githiori JB, Athanasiadou S, Thamsborg SM. Use of plants in novel approaches for control of gastrointestinal helminths in livestock with emphasis on small ruminants. Vet Parasitol. 2006;139(4):308–20. doi: 10.1016/j.vetpar.2006.04.021. https://doi.org/10.1016/j.vetpar.2006.04.021. [DOI] [PubMed] [Google Scholar]
- [5].Kundu S, Roy S, Lyndem LM. Broad spectrum anthelmintic potential of Cassia plants. Asian Pac J Trop Biomed. 2014;4(1):5436–41. doi: 10.12980/APJTB.4.2014C1252. https://doi.org/10.12980/APJTB.4.2014C1252. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [6].Alvarez AC, de Ugaz OL. Taninos. Rev Química VI. 1992. pp. 47–63.
- [7].John J, Mehta A, Shukla S, Mehta P. A report on anthelmintic activity of Cassia tora leaves. Songklanakarin J Sci Technol. 2009;31(3):269–71. [Google Scholar]
- [8].Sumi S, Saj OP. Antibacterial, anthelmintic and phytochemical investigations on the pod extracts of Cassia fistula Linn. Int J Med Pharm Sci. 2012;2(1):6–15. [Google Scholar]
- [9].Irshad M, Sing M, Rizvi MA. Assesment of anthelmintic activity of Cassia fistula L. Middle East J Sci Res. 2010;5(5):346–9. [Google Scholar]
- [10].Bhadauria S, Singh H. Bioactive nature of flavonoids from Cassia siamea and Lantana camara. Indian J Fund Appl Life Sci. 2011;1(2):107–10. [Google Scholar]
- [11].Jothy SL, Torey A, Darah I, Choong YS, Saravanan D, Chen Y, et al. Cassia spectabilis (DC) Irwin et Barn: a promising traditional herb in health improvement. Molecules. 2012. pp. 10292–305. https://doi.org/10.3390/molecules170910292. [DOI] [PMC free article] [PubMed]
- [12].Motina BK. Antimicrobial activities of crude extract from Cassia surattensis. NU Sci J. 2013;10(1):10–7. [Google Scholar]
- [13].Suhasini K, Shridhar NB, Sanganal JS, Pattar J, Rao S. Evaluation of acute and subacute toxicity studies of Cassia spectabilis Leaf extract in Wistar Albino Rats. Int J Pharmacol Toxicol Sci. 2013;3(4):24–9. [Google Scholar]
- [14].Singh S, Singh SK, Yadav A. A review on Cassia species: pharmacological, traditional and medicinal aspects in various countries. Am J Phytomed Clin Therap. 2013;3:291–312. [Google Scholar]
- [15].Harborne JB. Phytochemical methods: a guide to modern techniques on plant analysis. 3rd edition. Kluwer Academic Publishers; UK: 1998. pp. 299–312. [Google Scholar]
- [16].Hansen J, Perry B. The epidemiology, diagnosis, and control of helminth parasites of ruminants: a Handbook. ILRAD; Nairobi: 1994. pp. 80–92. [Google Scholar]
- [17].Rabel B, Mcgregor R, Douch PGC. Improved bioassay for estimation of inhibitory effects of ovine gastrointestinal mucus and anthelmintics on nematode larval migration. Int J Parasitol. 1994;24(5):671–6. doi: 10.1016/0020-7519(94)90119-8. https://doi.org/10.1016/0020-7519(94)90119-8. [DOI] [PubMed] [Google Scholar]
- [18].Gomez AA, Gomez KA. Statistical procedures for agricultural research. 1st edition. Vol. 6. John Wiley & Sons, Inc.; Canada: 1984. p. 680. [Google Scholar]
- [19].SAS Institute. The SAS system for windows. Ver 9.0. SAS Institute Inc; Cary, NC: 2002. [Google Scholar]
- [20].Mohammed A, Liman ML, Atiku MK. Chemical composition of the methanolic leaf and stem bark extracts of Senna siamea Lam. J Pharmacogn Phytother. 2013;5(5):98–100. [Google Scholar]
- [21].Kamagaté M, Koffi C, Kouamé NM, Akoubet A, Alain N, Yao R, et al. Ethnobotany, phytochemistry, pharmacology and toxicology profiles of Cassia siamea Lam. J Phytopharm. 2014;3(1):57–76. [Google Scholar]
- [22].Panda SK, Padhi LP, Mohanty G. Antibacterial activities and phytochemical analysis of Cassia fistula (Linn.) leaf. J Adv Pharm Technol Res. 2016;2(1):62–7. doi: 10.4103/2231-4040.79814. https://doi.org/10.4103/2231-4040.79814. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [23].Danish M, Singh P, Mishra G, Srivastava S, Jha KK, Khosa RL. Cassia fistula Linn. ( Amulthus )—an important medicinal plant: a review of its traditional uses, phytochemistry and pharmacological properties. J Nat Prod Plant Resour. 2011;1(1):101–8. https://doi.org/10.1080/01652176.1983.9693876. [Google Scholar]
- [24].Gobbo-Neto L, Lopes NP. Plantas medicinais: fatores de influência no conteúdo de metabólitos secundários. Química Nova. 2007;30(2):374–81. https://doi.org/10.1590/S0100-40422007000200026. [Google Scholar]
- [25].Chaudhary S, Kumar A. Phytochemical analysis and assessment of in vitro anthelmintic activity of Cassia auriculata Linn leaves. Am J Phytomed Clin Therap. 2014;2(2):161–7. [Google Scholar]
- [26].Tiwari P, Kumar B, Kumar M, Kaur M, Debnath J, Sharma P. Comparative anthelmintic activity of aqueous and ethanolic stem extract of Tinospora cardifolia. Int J Drug Dev Res. 2011;3(1):70–83. [Google Scholar]
- [27].Williams AR, Fryganas C, Ramsay A, Mueller-Harvey I, Thamsborg SM. Direct anthelmintic effects of condensed tannins from diverse plant sources against Ascaris suum. PLoS One. 2014;9(5):e97053. doi: 10.1371/journal.pone.0097053. https://doi.org/10.1371/journal.pone.0097053. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [28].Patel J, Kumar GS, Quesha MdS, Jena PK. Anthelmintic activity of ethanolic extract of whole plant of Eupatorium odoratum L. Int J Phytomed. 2010;2:127–32. https://doi.org/10.5138/ijpm.2010.0975.0185.02020. [Google Scholar]
- [29].Botura MB, dos Santos JDG, da Silva GD, de Lima HG, de Oliveira JVA, de Almeida MAO, et al. In vitro ovicidal and larvicidal activity of Agave sisalana Perr. (sisal) on gastrointestinal nematodes of goats. Vet Parasitol. 2013;192:211–217. doi: 10.1016/j.vetpar.2012.10.012. https://doi.org/10.1016/j.vetpar.2012.10.012. [DOI] [PubMed] [Google Scholar]
- [30].Jain P, Singh S, Singh SK, Verma SK, Kharya MD, Solanki S. Anthelmintic potential of herbal drugs. Int J Res Dev Pharm Life Sci. 2013;2(3):412–27. [Google Scholar]
- [31].Sangetha S, Zuraini Z, Sasidharan Suryani S. Fungicidal effect and oral acute toxicity of Cassia spectabilis leaf extract. Jpn J Med Mycol. 2008;49:299–304. doi: 10.3314/jjmm.49.299. https://doi.org/10.3314/jjmm.49.299. [DOI] [PubMed] [Google Scholar]
- [32].Sumathy V, Zuraini Z, Sasidharan S. In vivo toxicity study of Cassia surattensis flower extract. Res J Pharm Biol Chem Sci. 2011;2(3):607–17. [Google Scholar]