In Vitro Anthelmintic Activity of the Extract of Coffee Husk Fermented with Pleurotus Ostreatus for Ascaridia Galli

The objective of this research was to evaluate the anthelmintic efficacy of the ethanolic and aqueous extract of coffee husk fermented by Pleurotus ostreatus against Ascaridia galli. In vitro study of anthelmintic activity was conducted by counting the number of paralyzed worm body of Ascaridia galli during 24 hours in petri dish containing different levels of ethanolic and aqueous extract of fermented coffee husk, namely 0%, 1%, 2%, 3% and 4% (w/v) and compared to piperazine 0,5% (w/v) and NaCl 0,9%. The powdered samples of fermented coffee husk were macerated in the 30% ethanol solution with the ratio samples to solution = 1:3. In aqueous extract, samples to solution ratio was 1 : 7. The extract of fermented coffee husk possess phytochemical compunds which responsible for anthelmintic activity. The anthelmintic activity of ethanolic extract was better than aqueous extract. Fermented coffee husk ethanolic extract exhibited mortality of A.galli at concentration 2% while showed highest mortality at concentration 4%. Fermented coffee husk ethanolic extract have the potential to aid in the control of gastro intestinal parasites in animal


Introduction
Gastro intestinal parasites (GIP) have direct effects on and remain one of the main problems to productivity of the livestock. Ascaridia galli, an intestinal nematode that affects production of hens and other domestic wild birds, causes economic losses in avian production (Marcos-axtutegi et al., 2009). The common control mode of GIP based on the use of anthelmintics is now strongly questioned because of the increasing development of resistance and residual effect to animal product (Wolstenholme et al., 2004;Sissouma et al., 2011). Thus, alternative GIP control strategies are necessary.
Alternative environment friendly sustainable strategies are required, which could reduce the exclusive reliance on anthelmintic treatment. Mushrooms have been used from ancient times to cure diseases of man and animals and as food supplement for medicinal properties.
One of the mushrooms is Pleurotus ostreatus (P. ostreatus) containing a number of active compounds that help as health-enhancers, potential against diseases and as biological response modifiers (Wong et al., 2011;Patel et al., 2012). Ganeshpurkar et al. (2012) reported that there was anthelmintic potential of Pleurotus florida to Ascaridia galli. Haque et al. (2105) concluded that the extract of Pleurotushighking have potent anthelmintic activity when compared with the conventionally used drug. The content of bioactive compounds from mushroom may be responsible for anthelmintic action.
P. ostreatus was the same genus with Pleurotus florida, so it is expected to have anthelmintic activity.
In several studies, the use of plant as an alternative gastro intestinal control strategies may be attributed to the presence of active ingredients responsible for the activity. In few cases, the phytochemical examination of concerned plants has also been made and the active principle has been isolated and tested for anthelmintic activity. In all these studies, }u‰ OE]•}v• }( ‰o vš•[ ((] Ç Z • v u with one or other synthetic anthelmintic drug such as albendazole, piperazine citrate, etc. (Ekeanyanwu and Etienajirhevwe, 2012) Pleurotus ostreatus can be cultivated on a wide range of lignoselulosic substrates because produce ligninolytic extracellular enzymes, such as laccases, lignin peroxidases and Mn peroxidases (Widiastuti and Panji, 2008;Alemawor, 2009).
The ability of P. ostreatus to degrade a wide variety of lignoselulosic substrates plays an important role in managing organic wastes with problematic disposal. Coffee husk is one of the principal by products produced during the operation of coffee cherry to get coffee grain by sun drying (Murthy and Mannomani, 2008;Fan and Soccol, 2005). Indonesia is one of coffee-producing countries but the coffee husk is barely utilized.
Present study intends to perform the preliminary phytochemical screening of ethanol and water extract of coffee husk fermented by P. ostreatus and to evaluate the anthelmintic activity to A.galli in vitro examination.

Coffee Husk Cultivation with Pleurotus ostreatus
Coffee husks were sun dried until its moisture content ranged 10%-15%. The cultivation method and the composition of solid substrate were conducted according to Herliyana et al. (2008) with slight modification. The substrate consisted of 82.5% coffee husks, 15% rice bran, 1.5% gips and 1.0% CaCO3. The clean water was added into the substrate as much as 65-70% (v/w). All the components were composted for 24 h, weighed 400 g to store into polypropylene bags. The bag logs were sterilized on 121 o C for 30 min. After cooling, each bag was inoculated aseptically with P. ostreatus grain spawn and incubated in an incubating room at 23-24 o C and approximately 80% relative humidity. Each spawned bag was closed with a small sterile cotton plug inserted in the middle of its opening. All bags were placed in incubating room and after 60d the substrates were fully colonized and the primordial started to appear. The fully colonized substrates were sun dried and ready to use.

Preparation, Extraction and Phytochemical Properties of the Extracts
The samples were dried and pulverized into fine powder. The water and ethanol extracts were prepared using the maceration. The powdered samples were macerated in the 30% ethanol solution with the ratio samples to solution = 1:3.
The suspensions were stirred and precipitated overnight then be refined. The supernatants were retained and the residue was discarded. The supernatants were dried in vacuum rotavapor at 45 o C to obtain the concentrate extracts. The obtained extracts were examined for the presence of alkaloids, flavonoids, phenol hidroquinon, steroids, triterpenoids, tannin, and saponin as described by Harborne (1989).

In vitro Anthelmintic Assay
Ascaridiagalli were collected from freshly slaughtered native chicken intestines in the local abattoir. The worms were washed repeatedly using NaCl 0,9%. As much as 20 ml NaCl 0,9% were placed into petri dish plate and the extract of fermented coffee husk were added into the petri dish with the final concentration 1, 2, 3 and 4% (w/v). The piperazin 0.5% (w/v) and NaCl 0.9% were used as positive and negative control. Five adult worms without any sexing, with the same weight and body length were put into the plates. There were four replicates for each extract concentration and control. The plates were further incubated for 24 h in temperature room. Paralysis was said to occur when the worms were not able to move even in lukewarm normal saline.The research was arranged in Completely Randomized Design with ten treatments and four replications. The data were analyzed for statistical significance using anova. For detailed test used Duncan Range Test. Difference at P<0.05 was considered as significant.

Phytochemical Screening
The results of phytochemical evaluation of secondary phytoconstituents are listed in Table 1. The phytochemical evaluation confirmed the presence of saponin, glycosides, flavonoids, tannins and phenolic compounds.
Ethanolic extractionyield of fermented coffee husk was 2.105% and water extract was 5.75%. The ethanol extract contained alkaloids, tannins, saponins, flavonoids and triterpenes. On water extract contained saponin and flavonoid. The solubility of secondary substances in ethanol solvent were much more than water. Gamse (2002) stated that the important considerations to select solvent were the selectivity, the characteristic of the solvent, the abililty to extract, no harmful, easy to evaporate and not expensive. Coffee husk subtrate fermented by P. ostreatus showed a great potential in phytochemical contents. Coffee husks were rich in phenolic compound (tannin, saponin, flavonoid) and alkaloid (caffeine) (Esquivel and Jimenez, 2012).

In vitro Anthelmintic Assay
The anthelmintic activity of ethanol extract was better than that of water extract ( Table  2). The difference of anthelmintic activity between ethanol and water extract was regarded to the their phytochemical profile ( Table 1).
The ethanol extract of fermented coffee husk on the concentration 3% showed a significant increase in the mortality. The use of 4% was not significantly different from the 3%. Adult mortality assay of A. galli soaked in ethanol extract solution on the concentration of 1, 2, 3 and 4% were 0 worm (0.0%), 3 worms (15.0%), 9 worms (45.0%) and 12 worms (60.0%), respectively (Table 2). It indicated that the higher concentration of the ethanol extract, the higher anthelmintic activity.
Piperazin had the strongest anthelmintic activity, while on NaCl 0.9% solution no mortality of worms showed. NaCl 0.9% named physiological NaCl was the solution the same with the condition of worm body solution.
The mechanism of fermented coffee husk as anthelmintic may be related to the presence of bioactive molecules. Pathak et al. (2013) reported that the leaf from forage containing condesed tannin could be used as anthelmintic. Gupta et al. (2012) reported that Pleurotus florida revealed the presence of different secondary metabolites contain carbohydrates, alkaloids, glycosides, flavonoids, tannins, saponins and steroids.
Another study reported the anthelmintic activity of Jatropha curcass Linn (Suharti et al., 2010), a plant rich in phytochemicals that may contribute to the anthelmintic activity. The possible anthelmintic effect of tannins is that they can bind to free proteins in the gastrointestinal tract of host animal or glycoprotein on the cuticle of the parasite, and may cause death (Min and Hart, 2003).  The main possible actions of saponins are changes in membrane permeability and pore formation, similar with two conventional anthelmintic drugs such as praziquantel and toltrazuril. The anthelmintic drug affects the permeability of the cell membrane of worm, causing vacuolisation and disintegration of the teguments (Wang, 2010). Alkaloids may have acted on the central nervous system of the earth worms causing paralysis (Roy, 2010). The effect could be due to presence of the steroidal alkaloids oligosaccharides which have been reported to suppress the transfer of sucrose from the stomach to the small intestine which could diminish the availability of glucose to helminths together with its antioxidant effect which is capable of reducing the nitrate generation.

Conclusion
The ethanol extract of fermented coffee husk can be used as natural anthelmintic. The concentration of ethanolic extract of fermented coffee husk as much as 4% were effective as anthelmintic in vitro.