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Ethanol extraction of Sambiloto leaves (Andrographis paniculata Nees) and evaluation of its immunomodulatory activity towards gastrointestinal nematode infestation in Ettawah crossbred goats

Rositawati Indrati, Nur Ihsan, Osfar Sofjan and Suyadi Suyadi

Faculty of Animal Science, University of Brawijaya, Malang 65145, Indonesia


Sambiloto (Andrographis paniculata Nees) leaves contain various immunomodulatory and anthelmintic compounds such as saponin, tannin, and andrographolide. This study aims to analyze and evaluate the andrographolide from Sambiloto leaves through ethanol extraction, as well as the immunomodulatory activity of Sambiloto leaves extract (SLE) in Ettawah crossbred goats observed from the blood profile and gastrointestinal nematodes (GIN) infestation. This study was conducted as experimental research with a completely randomized design. The data were analyzed with one-way ANOVA, and followed with LSD test to determine the differences. The study used 5 treatments, consisted of positive control (PC) treatment (supplemented with 500 mg CurminVitPlus, a commercial immunomodulatory drug) and negative control (NC) treatment (without any immunomodulatory drugs supplementation) as the control group; 750 mg SLE supplementation (SLE750); 1125 mg SLE supplementation (SLE1125); and 1500 mg SLE supplementation (SLE1500). The results of the study showed that ethanol extraction would yield SLE as much as 33.6% of the dry weight with 1.12 ± 0.04% andrographolide concentration. Moreover, the immunomodulatory observation showed that SLE supplementation improved the blood profile quality of the Ettawah Crossbred goats compared to the control group. The best SLE supplementation was found at 1125 mg dosage, which suppressed the GIN eggs infestation up to 96.8%. The research concludes that ethanol-extracted Sambiloto leaves showed an immunomodulatory, immunosuppressant, and anthelmintic potential towards GIN infestation in Ettawah Crossbred goats.

Keywords: alternative drugs, extract supplementation, immunosuppressant, medicinal herbal


Immunomodulators are compounds that have the ability to enhance body defense mechanisms while also stimulate the immune system whether through specific immune responses and non-specific immune responses (Schulz 2004; Mun'im and Hanani 2011). The immune system plays an important role to protect the body from pathogens and destroy antigens in the body. The utilization of herbal medicine, or widely known as phytopharmaca, has been extensively studied on its efficacy as well as its immunomodulatory effect. Immunomodulatory compounds are found in various plants. One of the widely used and studied phytopharmaca is Sambiloto (Andrographis paniculata Ness) leaves.

Sambiloto leaves contain various immunomodulatory compounds, such as lactone in the form of deoxy-andrographolide, andrographolide, neoandrographolide, 14-deoxy-11,12-didehydroandrographolide, and homoandrographolide. Andrographolide is an active chemical compound with immunomodulator activity and acts as immunostimulant by increasing leukocyte production that attack bacteria and other antigens (Naik and Hule 2009). In addition, other compounds in Sambiloto leaves, such as flavonoids (an anti-inflammatory compound), and tannins (an anthelmintic) would coagulate proteins of nematodes, then intervene in their metabolism and homeostasis condition, thus killing the nematodes as the end result (Williams et al 2014). Moreover, another important compound found in Sambiloto leaves is saponin. Saponin is a glycoside which has anthelmintic ability by lowering surface tension of the membrane wall, thus inhibits acetylcholinesterase activity which leads to muscle paralysis and death of the nematodes (Ali et al 2011).

The common internal parasites found in goats is gastrointestinal nematode (GIN) endoparasites, which are mostly found in goats under extensive farming. The infection is known to cause weight loss up to 38%, moderate to severe anemia, stunting, decreased endurance, and up to 28% chance to cause death (Partoutomo 2004; Suhardono et al 2002). One of the efforts to prevent nematode infection is by giving anthelmintic drugs to the goat. However, the drug supplementation would cost the farming finance, not to mention with the occurrence of drug resistance as well. Various researches (Waller 1993; Waller et al 1996; Baltrušis et al 2018; Ridwan et al 2000) showed that the drug resistance genes would have passed to the offspring of the nematodes as well.

An approach to increase the immune system against GIN infection in goats is by using natural herbal ingredients such as Sambiloto leaves (Andrographis paniculata Nees). The immunomodulatory compounds in Sambiloto leaves like andrographolide, would kill the nematodes as well as increase the antibody production (Prapanza and Marianto 2003; Naik and Hule 2009). The andrographolide supplementation by feeding the Sambiloto leaves extract (SLE) is expected to suppress the chemical drug usage and simultaneously reduce losses due to disease . Bastiawan et al (2001) showed that the examination of GIN infection could be done through haematological examination. In this research, we evaluated the SLE production through ethanol extraction and analyzed its anthelmintic properties through GIN infestation observation, as well as its immunomodulatory effectivity through haematological observation.

Materials and methods

Sambiloto leaves extraction and capsule production

Five kilograms of Sambiloto leaves from UPT Materia Medica (Indonesia) were dried and then ground into powder (at the size of 60 mesh) and macerated by using 96% ethanol for three days. The result of three-day maceration was then extracted by using a rotary evaporator and the Sambiloto leaves extract (SLE) is then obtained. As much as 1680 g SLE was taken for phytochemical tests, which include flavonoids, alkaloids, tannins, terpenoids/steroids, saponins, and quinones (Harborne 1987). The phytochemical analysis to measure the andrographolide concentration was carried out through Thin Layer Chromatography Method in the UPT Materia Medica analytical laboratory, Batu, East Java, Indonesia.

The SLE capsule was produced by adding Sacharum lactis and Manihot amylum mucilage to the SLE before made into capsule. The SLE capsules in this research were arranged to have 750 mg, 1125 mg and 1500 mg, and were upplemented orally to the Ettawah Crossbred (PE) goats to observe the immunomodulatory efficiency.

SLE capsule supplementation

The goats in this research were male Ettawah crossbreds aged 3 to 5 months with the body weight of 13 to 19 kg. The goats were then divided into 5 treatment groups based on the immunomodulatory supplementation, with each group consisted of 5 goats. The detail of each treatment group is as follow:

Negative control = without any immunomodulatory drug supplementation

Positive control  = supplemented with 500 mg Curmin VitPlus;

SLE 750 = supplemented with SLE capsule (750 mg);

SLE1125 = Supplemented with SLE capsule (1125 mg); and

SLE1500 = Supplemented with SLE capsule (1500 mg)

The goats were fed 3 times a day, with concentrates at 8:00 a.m., and King grass (Pennisetum purpurhoides) at 10:00 a.m. and 16:00 p.m for 10 months. Drinking water was given adlibitum.

Haematology and nematode eggs analysis

The blood and feces samples were collected every two weeks for 10 months. The blood samples were taken through the jugular vein and kept in 5 ml EDTA tube for haematology observation (total erythrocytes, haemoglobin, leukocytes, eosinophils, hematocrit levels and immunoglobulin E/IgE). The haematology profile was examined as follow: Haemoglobin concentration was measured by conventional Sahli method (Wintrobe 1975) with the help of hemoglobinometer; the erythrocyte, leukocyte, and eosinophils cells were measured with Neubauer Haemocytometer (Bull et al 2000); the hematocrit test was performed by using hemotocritic standard tubes (Daice and Lewis 1984); while the Immunoglobulin E (IgE) level was measured by using the enzyme-linked immunosorbent assay (ELISA/ Shanghai Crystal Day Biotech Co.Ltd, China) method with goat IgE ELISA Kit Cat No.E0045Go BT-LAB. The feces samples were collected to measure the total nematode eggs infestation by following McMaster floatation method (Hansen and Perry 1994). All of the haematology profile and nematode eggs analysis were conducted in the Laboratory of Epidemiology and Laboratory of Physiology, University of Brawijaya.

Data analysis

This study was conducted in a completely randomized design (CRD) with 5 replications. Analysis of the helminthiasis incidence was carried out by using descriptive data from the total samples with positive endoparasites infestation and the eggs per gram (EPG) of feces. Data from this study were analyzed by using analysis of variance and followed with least significant difference test (Mattjik and Sumertajaya 1999) to determine the difference between  treatments.

Results and discussion

Phytochemical Analysis of Sambiloto Leaves Extract

The ethanol extraction of 5000 g Sambiloto leaves yielded 1680 g of Sambiloto leaves extract (SLE) or equal to 33.6%. The result is higher than three-days maceration extraction by using pro methanol analysis which yielded 8% SLE (Cahyaningsih et al 2007). Moreover, the SLE in this research had andrographolide levels at 1.12 ± 0.04%, which is relatively similar to the finding by Raina et al (2007) which proposed that the HPTLC analysis of Andrographis paniculata Nees showed andrographolide levels at 1.14-2.50%. Research by Pandey et al (2010) showed that the andrographolide compounds in Sambiloto leaves grown in central India were ranging from 1.04-2.24%. The different andrographolide levels were caused by various factors, such as the plant age, the cultivation environment, and also the extraction method. The longer extraction time would result in higher andrographolide levels in the Sambiloto leaves, and at certain extent would be decreased.

Immunomudulator activity of SLE in Ettawah Crossbred

The haematology profile observation showed that the SLE supplementation improved (p<0.05) total erythrocytes, haemoglobins, hematocrit, leukocytes, eosinophils, and immunoglobulin E (IgE) of the Ettawah Crossbred goats (Table 1). Moreover, the results also showed that higher dosage of SLE supplementation resulted in better haematology profile. The amount of erythrocytes, hemoglobins, and hematocrit indicates the blood ability to distribute oxygen to the tissues and excrete carbon dioxide from the body. Wardhana et al (2001) stated that the result of blood profile observation indicates an in-depth health status of the animals, as each blood profile factor functions and correlates between each other.

Table 1. The haematology profile of Ettawah Crossbred goats supplemented with SLE at different dosages

Haematolog y profile







Erythrocytes (x106µl)






Hb (g/dl)






Hematocrit (%)






Leukocytes (x10³cell/µl)






Eosinophils (cell/µL)












Different superscripts on the same line indicate differences (p<0.05). NC = Negative Control (without any drugs supplementation), PC = Positive Control (supplemented with Curmin VitPlus: 500mg), SEL750 = ESL 750 mg, SEL1125 = ESL 1125 mg, SEL1500 = ESL 1500 mg

The curcuminoid and volatile oil compounds in CurcuminVitPlus (PC treatment) are the main substances which give immunomodulatory effect by increasing the circulated leukocytes and erythrocytes in blood. In this research, it is shown that CurcuminVitPlus and 750 mg SLE supplementation had a similar effect to the total erythrocytes, haemoglobins and hematocrit. The higher erythrocyte cells in blood will increase the blood capacity to carry O2 and CO2 (Alberts et al 2002) which is essential for nutrient metabolism (Oburoglu et al 2016; Frandson et al 2009).

The research on other herbal immunomodulatory supplement (Raguati and Rahmatang 2012)  showed that  2.5% ginger in USMB-Plus supplementation increased the total erythrocytes (7.07-13.63 million/mm 3) and haemoglobins (8.77-10.41 gr/100 ml) in Ettawah Crossbred goats aged 5-6 months. Sugiharto (2004) added that 20% curcumin infusion could protect the erythrocytes and haemoglobins against oxidation while also enhance the detoxifying enzymes synthesis in the liver.

The increased SLE dosage also showed a positive correlation to the total erythrocytes, haemoglobins and hematocrit, with the best immunomodulatory activity obtained in SEL1500. The similar result in other immunomodulatory supplementation substances was shown in the finding by Dhama et al (2011) that immunomodulatory dosage supplementation would increase the blood cells production. Suhirman and Winarti (2010) stated that the andrographolide supplementation would induce the immunosuppressants and immunostimulants. Meyer and Harvey (2004) and Lukistyowati (2012) explained that the total of erythrocytes, hemoglobin and the hematocrit percentage can be used to determine the impact of immunostimulant use, and is used as an indication of livestock health condition.

The results also showed that increased dosage would affect total leukocytes, eosinophils and IgE. Research by Suhirman and Winarti (2010) showed that SLE could be used as immunosuppressant and immunostimulant. The immunostimulant compounds are divided into two groups, the first group stimulate immunity to specific antigens and characterized by an increase in the total lymphocyte cells in the blood circulation, and the second group is non-specific immunity stimulation characterized by an increase in the number of neutrophil, eosinophil and basophil cells. Moreover, the different white blood cells production would affect the immune system differently, such as in antigen recognition, and inflammation process (Corwin 2000). According to Widhyari et al (2017), the number of leukocyte cells is regulated physiologically within a certain threshold to respond to  any inflammation or infection. Research by Dhama et al (2011) indicated that the administration of immunomodulatory substances could increase the leukocyte cells activity to improve body immunity, while also suppress the development of endoparasitic gastrointestinal.

The total of eosinophil cells in Ettawah Crossbred goats were different between each SLE dosage (750 mg, 1125 mg, and 1500 mg). The SEL1125 and SEL1500 showed higher total eosinophil cells compared to other treatments. The result thus indicates that higher SLE dosage had a certain role in increasing the total eosinophils, a blood cell which prevent pathogenic infections from outside the body, in Ettawah Crossbred goats. The normal eosinophils in goats according to Weiss et al (2010) ranges from 50-650 (cells/µL). The observation of eosinophil cells is important to determine the body immunity, as the cell is able to destroy pathogenic agents and produce enzymes that can neutralize inflammatory factors, inflammation and allergies (Lokapirnasari and Yulianto 2014). According to Roitt and Delves (2001), the eosinophil cells formation is determined by the T-cells stimulation against antigens as a defensive response. The process is initiated by antigen attachment to the Th2 cell receptor by the antigen presenting cell (APPC), which then results in cytokine (IL-5) production by the Th2 cell. Eosinophil cells contain various chemical mediators such as hydrolytic enzymes and cytotoxic substances that would destroy large extracellular antigens. In nematodes infections, the eosinophil cells would migrate towards the infection site and release destructive enzymes which kill the nematodes.

The IgE observation showed that the IgE levels in Ettawah Crossbred goats began to increase after supplemented with SLE at 1125 mg, with the highest level found at the dosage of 1500 mg or in SEL1500 treatment. The results thus showed that SLE supplementation would begin to show immunomodulatory effect at 1125 mg. Moreover, the results also showed that the IgE level has a positive correlation to the SLE dosage. According to Kumar et al (2011) Sambiloto leaves contained andrographolide, 14-deoxyandro-andrographoside and neoandrographolide, thus will increase proliferation and induction of blood peripheral lymphocyte Interleukin 2 (IL-2). The IL-2 would induce TNF-α production and increase lymphocyte cell proliferation, which is vital for the immune system. In addition, the process also stimulates CD4+ in Th2 cells subset to release IL-4 which will produce IgE to bind the surface area of nematodes and activate eosinophils.

Effect of SLE supplementation on GIN infestations

The results of SLE supplementation on the average observed nematodes egg per gram (EPG) in each treatment are as follow: 155EPG (NC); 134 EPG (PG); 103 EPG (SEL1125); 104EPG (SEL1125); and 97 EPG (SEL1500). Moreover, the statistical analysis showed that there was no difference in different SLE dosage supplementation to the total of nematode eggs found in feces. In this research, commercial herbal supplement (Curmin VitPlus, Patent ID P000040583) is used as a positive control (PC) to compare the anthelmintic and immunomodulation of SLE in different dosages. In this research, the nematode eggs found in all treatments ranged from 1 to 499 EPG. The degree of nematode eggs infestation in this research thus classified as light according to standard by Thienpont et al (1986).

All the  Etawah Crossbreed Goats faecal samples in all treatments (NC, PC, SEL750, SEL1125, and SEL1500) showed GIN infestation with mixed endoparasites species. Subronto and Tjahajati (2001), and Suhardono et al (2002) reported that the presence of GIN infestation can be identified through nematode eggs examination in feces, while factors affecting GIN infestation in livestock include environmental factors, rearing management and the supplementation of anthelmintics.

The fecal sample examination in this study showed that there were 7 GIN species found in the sample, which were Trichuris sp.,Oesophagustomum sp., Strongylus sp.,Strongyloides sp., Trychostongylus sp., Haemonchus sp., and Bunostomum spp. These GIN species are widely known to infest ruminant livestock (Pfukenyi and Mukaratirwa 2013; Purwaningsih et al 2017). In Figure 1, is present ed the total of nematode eggs in all observation times.

Figure 1. Total GIN eggs infestation (T1=750, T2 1125 and T3 1500 mg SEL)

The overall total of nematode eggs in all treatments decreased during the 10-weeks observation. After 10 weeks, the GIN infestations were reduced as much as 65.49% (NC); 89.94% (PC); 94.45% (SEL750); 96.8% (SEL1125); and 92% (SEL1500). This showed that the highest immunosuppressant/anthelmintic activity was found in 1125 mg SLE supplementation (SEL1125). According to Ali et al (2011); Williams et al (2014); and Naik and Hule (2009), the immunosuppressant and anthelmintic activity are due to the saponin, tannin, and andrographolide compounds in Sambiloto leaves which would kill the nematodes through various ways. Saponin would act as acetylcholinesterase inhibitor which will disrupt the neurotransmitter and paralyse the nematodes, while tannin would coagulate the proteins in nematodes’ cell wall, which then disrupt its homeostatic condition and inhibit the larvae migratory process.

Research by Partoutomo (2004) and Haryuningtyas (2008) reported that synthetic anthelmintic drugs supplementation could lead to the growth of resistant nematodes, aside of its negative effect to the environment. Therefore, controlled grazing management added with medicinal herbal supplementation is recommended for sustainable farming practice (Coles et al 2006). In this study, the supplementation of SLE showed the potential to be used as immunosuppressant and anthelmintic alternative for Ettawah Crossbred goats, thus could replace the synthetic anthelmintic drugs and provide more sustainable farming practice.



The authors would like to thank the Directorate General of Education Ministry of Research and Technology and Higher Education, Republic of Indonesia, for providing research fund through the Domestic Graduate Education Scholarships program.


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Received 30 October 2019; Accepted 7 November 2019; Published 2 December 2019

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