Evaluation of in vitro Anthelmintic activity of Lannea coromandelica (Houtt.) Merr. against Pheretima posthuma and Ascardia galli

 

Rajesh. R*, Selvakumar. K

Department of Pharmaceutical Analysis, Acharya & BM Reddy College of Pharmacy, Soldevanahalli, Hessarghatta Main Road, Bengaluru - 560107.

*Corresponding Author E-mail: rajeshr@acharya.ac.in

 

ABSTRACT:

Helminthiasis is a major public health concern and in developing nations, this leads to the prevalence of pneumonia, malnutrition, eosinophilia and anaemia. In current research, the development of resistance to anthelmintics has led to the evaluation of the alternative source of medicinal plants as an anthelmintic drug. Methanol and aqueous extracts of Lannea coromandelica bark were evaluated at various concentration levels of 10, 25 and 50mg/ml for in-vitro anthelmintic activity against Pheretima posthuma and Ascardia galli obtained from soil. Results were reported in paralysis time and worm death time in minutes. As a reference standard, piperazine citrate was used (15mg/ml). In both extracts, activity based on dose was observed. The result revealed that the extract of methanol (MELC) is more active than the aqueous extract (AELC) and can therefore be useful as anthelmintics. From these observations, it was concluded that the anthelmintic activity observed due to the presence of secondary metabolites such as flavanoids, saponins, tannins, alkaloids and terpenoids. Our results report for the first time that Lannea coromandelica bark extracts have therapeutic values with prominent anthelmintic properties.

 

KEYWORDS: Anthelmintic, piperazine citrate, Lannea coromandelica, Pheretima posthuma, Ascardia galli.

 

 


INTRODUCTION:

It is estimated by the World Health Organization (WHO) that 80 percent of the population of developed nations depends on traditional medicines to meet their key needs for medical treatment, predominantly plant medicines. As a consequence of the increased toxicity of synthetic drugs and their allergic symptoms, the use of medicinal plants is increasing globally. Helminths are large multi-cellular parasitic microorganisms. It is claimed that about a one third human population of the world is contaminated with helminth parasites. This causes serious public health problems, particularly in regions with poor sanitation or poor access to safe drinking water. Many of these helminth parasites are spread from the soil and cause gastrointestinal infections after pasture or water infected with their eggs is ingested1.

 

The majority of these worm infections are usually limited predominantly in the tropical regions and the incidence was increased due to poverty and unhygienic lifestyle, which also contributes to symptoms such as anaemia, eosinophilia and pneumonia2. For effective therapeutic activities, secondary metabolites are essential plant constituents. The presence of this particular group of compounds has been reported to have specific therapeutic actions. Often traditionally reported, but little scientific validation is available. Among these activities, the activities of antioxidant and anthelmintics are quite important. Furthermore, parasitic worm infections are serious human issues, creating numerous diseases worldwide, and helminth is one of them. Helminths are common human infectious agents that cause substantial mortality and morbidity in developing countries. Helminths consist of nematodes (roundworms-intestinal and filarial worms) and platyhelminths (flatworms- flukes and schistosomes)3. More than two billion people worldwide are infected with parasitic worms, according to the World Health Organization report4.

 

 

The Lannea coromandelica (Houtt.) Merr. (Anacardiaceae) is a widely distributed in Bangladesh as tropical deciduous tree, India and other tropical nations. It has wide scope around the world5. In cuts, wounds, ulcers, ophthalmia, gout, ulcerative stomatitis, odontalgia, sprains, cardiac disease, diarrhoea, and dysentery, the bark is effective. For impetiginous eruptions and serious ulcerations, the astringent bark is used as a lotion6. Traditionally, various plant parts have been used as therapeutic agents for various diseases. By synthetic drug treatment, several harmful effects are caused and helminthes are resistant. There are also requirements for secondary metabolites of natural plants in treating and mitigating this chronic problem. The bark of Lannea coromandelica (Houtt.) Merr is traditionally used to treat skin and worm infections in Mayurbhanj district, Odisha, India7. To relieve ulcers and pain, leaf juice was taken orally, while fruit sap was used to treat cold and cough8-9. The bark of L. coromandelica was used in the treatment of gout, dyspepsia, dysentery, skin eruption, ulcers and toothache10-11. L.coromandelica aqueous extract antagonises glucagon receptor and the capacity to decrease glucagon mediated liver glucose production and antinociceptive behaviour12-13. The presence of secondary metabolites in the plant that were first assessed from methanol and aqueous bark extracts, the current study was performed to determine anthelmintic activity.

 

MATERIALS AND METHODS:

Collection and identification of barks:

The bark of Lannea Coromandelica has been obtained from the southern region of Tamil Nadu, India. Plants were identified and certified with the help of Dr. V. Chelladurai, Research Officer, Central Council for Research in Ayurvedic Sciences, Government of India, Tirunelveli, Tamil Nadu. A specimen voucher is kept for future reference in the department.

 

Preparation of extracts:

For several days, the fresh bark of Lannea coromandelica was shade-dried and ascertained for fungal infections in between. The barks were dried and made to coarse powder and 250g of the above powder was subjected to 4 hours of light extraction with petroleum ether in a Soxhlet extractor and defatted the materials. With four solvents, the barks were extracted successively with chloroform, methanol (80%) and maceration with aqueous solvent for 7 days. The reflux method was used 7-8 hours separately for all extracts (Prior to extraction with the next solvent, the marc was collected and dried in the air) preparation and finally, after the solvents were removed, the yield was calculated by rotational evaporation (at 45°C). For further investigation, the dried extract that was kept in a refrigerator at 4-5°C.14

 

Phytochemical screening:

Preliminary phytochemical study of bark extracts was carried out in order to find out the presence of flavonoids, steroids, cardiac glycosides, alkaloids, anthraquinones, tannins, glycosides and saponins using the standard protocol described by Khandelwal and Kokate15-16.

 

Drug and chemicals:

Piperazine citrate was acquired from Glaxo Smithkline Pharmaceutical Ltd. and other chemicals were analytical grade.

 

Anthelmintic activity:

Anthelmintic activity was performed with minor modifications in according to the method stated by Ajaiyeoba et al.17. The preliminary assay has been carried out on adult earthworms, Pheretima posthuma belongs to the Oligochaeta class and Ascardia galli belongs to the Secementea class. For preliminary anthelmintic activity they have been widely used due to its easy availability and their anatomy and physiological similarity to human intestinal round worm parasites18-20.  Due to easy availability, they have been mostly used for the initial in vitro evaluation of anthelmintic molecules21-22.

 

The anthelmintic study used Indian adult earthworms (Pheretima posthuma) was obtained from moist soil and washed to clear all faecal matter with normal saline. For the experimental protocols, the earthworms of size 3-5 cm in length and 0.1-0.2cm in width were used. The experiment was performed in seven groups of six worms in each group. Three different concentrations of fifty millilitres of formulation, each containing MELC and AELC bark extracts (25, 50, 100mg/ml in normal saline). The experiment was carried out in duplicate in all the extracts as well as standard. The mean time of paralysis in minutes was calculated on the basis of no movement of any kind, when the worm was shaken vigorously. Time for death in minutes was calculated after confirming that there is no movement of the earth worm while shaken vigorously or dipped in hot water maintained the temperature of 50șC. Death was confirmed, as the worms lost their motility, their body colours faded away. Piperazine citrate (15 mg / ml) was used as a reference standard. For Ascardia galli worms, the same experiment was performed, and in normal saline solutions the solutions were prepared.


 

 

 

 

 

Table No 1. In vitro anthelmintic activity of bark extracts of MELC and AELC

Treatment

Concentration

Pheretima posthuma

Ascardia galli

Paralysis

Death

Paralysis

Death

Piperazine citrate

15 mg/ ml

14.65 ± 0.15

25.96 ± 0.35

21.28 ± 0.37

32.18 ± 0.29

MELC

25 mg/ml

26.8 ± 0.41

35.13 ± 0.29

32.26 ± 0.1

43.23 ± 0.12

MELC

50 mg/ml

22.17 ± 0.32

26.54 ± 0.18

25.54 ± 0.16

29.19 ± 0.03

MELC

100 mg/ml

11.23 ± 0.22

19.14 ± 0.17

17.51 ± 0.15

23.85 ± 0.04

AELC

25 mg/ml

32.98 ± 0.32

41.89 ± 0.25

39.93 ± 0.12

47.41 ± 0.09

AELC

50 mg/ml

26.23 ± 0.31

31.75 ± 0.18

32.17 ± 0.03

37.35 ± 0.11

AELC

100 mg/ml

15.91 ± 0.28

21.97 ± 0.13

20.24 ± 0.06

26.35 ± 0.13

 

 

Fig. 1. In vitro Anthelmintic activity of bark extracts of MELC and AELC against Pheretima posthuma

Values are expressed as mean ± SEM.(n=6) ** P<0.01 significant as compared to the corresponding value of the standard.

 

 

Fig. 2. In vitro Anthelmintic activity of bark extracts of MELC and AELC against Ascardia galli

Values are expressed as mean ± SEM.(n=6) ** P<0.01 significant as compared to the corresponding value of the standard.

 


RESULTS AND DISCUSSION:

Preliminary phytochemical studies on Lannea coromandelica have reported the existence of steroids, carbohydrates, alkaloids, tannins, proteins and flavanoid glycosides. It was proposed from these results that the anthelmintic activity was observed due to the presence of these secondary metabolites in MELC and AELC. Each 25, 50 and 100mg/ml of MELC and AELC produced dose-dependent anthelmintic activity. Mean time of paralysis (P) and death (D) for the shortest period at a concentration of 100mg/ml was observed. MELC produced paralysis in 11.23 min and death in 19.14 min at a concentration of 100mg/ml, while AELC produced paralysis in 15.91 min and death in 21.97 min against Pheritima postuma. MELC produced paralysis at a concentration of 100mg/ml at 17.51 min and death at 23.85 min, while AELC produced paralysis at 20.24 min and death at 26.35 min for Ascardia galli.

At concentration of 15mg/ml, the standard drug piperazine citrate produced the paralysis and death at 14.65 min and 25.96 min respectively in Pheritima postuma. The standard drug induced paralysis of Ascardia galli in 21.28 minutes and death in 32.18 minutes.

 

A flaccid paralysis caused by the peristalsis expulsion of the worm is the dominant effect of piperazine citrate on the worm. Piperazine citrate causes hyper polarisation and decreased excitability by increasing conductance of chloride ion of the worm muscle membrane, resulting in muscle relaxation and flaccid paralysis23. Tannins are polyphenolic molecules that have been shown to produce anthelmintic activity24. Synthetic phenolic anthelmintics like bithionol, niclosamide and oxyclozanide have been shown to interfere with helminth parasite energy generation by uncoupling oxidative phosphorylation25. Similar effects may have been caused by tannins found in Lannea coromandelica extracts. Tannins have an anthelmintic effect since they may bind to free proteins in the gastrointestinal tract or glycoprotein on the cuticle of the host animal on the cuticle of the parasite and cause death.26-27

 

In conclusion, as the extracts exhibited activity against Pheritima postuma, the traditional claim of the bark as an anthelmintic of Lannea coromandelica has been established. There is a need for further study to isolate and reveal the active compound present in MELC and AELC responsible for the anthelmintic activity and to determine the mechanism of action.

 

ACKNOWLEDGEMENT:

The author expresses his gratitude to the principal and management of Acharya & BM Reddy College of Pharmacy, Bengaluru for providing support.

 

REFERENCES:

1.      Neil Mabott A. The influence of parasite infections on host immunity to co-infection with other pathogens. Front Immunol. 2018; 9:2579. doi.org/10.3389/fimmu.2018.02579

2.      Swathi H. Shekshavalli T. Invitro evaluation of anthelmintic activity of Rhus mysorensis leaves. Res. J. pharmacology and pharmacodymanics. 2016; 8(3):115-117. doi.org/10.5958/2321-5836.2016.00021.5

3.      Singh AK. Amit kumar S. Madan S. Vijay KY. Nidhi S. In vitro anthelmitic activity of stem bark extracts of Saraca indica Roxb. Against Pheretima posthuma. Asian J. Research Chem. 2014; 7(2):141-143. https://ajrconline.org/AbstractView.aspx?PID=2014-7-2-6

4.      Mulla WA. Thorat VS. Patil RV. Burade KB. Anthelmintic activity of leaves of Alocasia indica Linn. International Journal of Pharm.Tech. Research. 2010; 2(1):26. https://www.cabdirect.org/cabdirect/abstract/20103302236

5.      Tripathi AK. Verma RK. Gupta AK. Gupta MM. Khanuja SP. Quantitative determination of phyllanthin and hypophyllanthinin Phyllanthus species by high-performance thin layer chromatography, Phytochem Anal. 2006; 17(6):394-397. doi.org/10.1002/pca.936

6.      Nadkarnis KM. Indian Materia Medica. Vol I. Popular Prakashan Pvt. Ltd., Mumbai. 1976; 867-868.

7.      Sujogya KP. Laxmipriya P. Pieter L. Maoxuan L. Johan N. Walter L. Antimicrobial, Anthelmintic, and Antiviral Activity of Plants Traditionally Used for Treating Infectious Disease in the Similipal Biosphere Reserve, Odisha, India. Front. Pharmacol. 2017; 8:658. doi: 10.3389/fphar.2017.00658

8.      Rahmatullah M. Azam MN. Khatun Z. Seraj S. Islam F. Rahman MA et al. Medicinal plants used for treatment of diabetes by the Marakh sect of the Garo tribe living in Mymensingh district, Bangladesh. Afr. J. Tradit. Complement Altern. Med. 2012; 9(3):  380–385. doi: 10.4314/ajtcam.v9i3.12

9.      Zheng XL. Xing FW. Ethnobotanical study on medicinal plants around Mt. Yinggeling, Hainan Island, China. J. Ethnopharmacol. 2009; 124(2):197–210. doi: 10.1016/j.jep.2009.04.042

10.   Mulaudzi RB. Ndhlala AR. Kulkarni MG. Staden J. Pharmacological properties and protein binding capacity of phenolic extracts of some venda medicinal plants used against cough and fever. J. Ethnopharmacol. 2012; 143(1):185–193. doi: 10.1016/j.jep.2012.06.022

11.   Kadir MF. Sayeed MSB. Mia MMK. Ethnopharmacological survey of medicinal plants used by traditional healers in Bangladesh for gastrointestinal disorders. J. Ethnopharmacol. 2013; 147(1):148–156.                        doi: 10.1016/j.jep.2013.02.023

12.   Sameer W. Rajesh G. Swati J. Sushma S. Kalpana J. Lannea coromandelica attenuates glucagon and oxyntomodulin mediated cAAMP formation in HEK cells stably expressing human glucagon receptor. Journal of herbal Medicine 2015; 5(3):153-157. doi:10.5530/ijper.55.2.88

13.   Imam MZ. Moniruzzaman M. Antinociceptive effect of ethanol extract of leaves of Lannea coromandelica. Journal of Ethnopharmacology 2014; 154(1):109-115. doi: 10.1016/j.jep.2014.03.032

14.   Sawarkar HA. Khadabadi SS. Banarase NB. Mulaey BP. Aswar PB. Anthelmintic activity of extracts of Trigonella Foenum Graecum Linn. Research J. Pharm and Tech. 2009; 2(1): 128-130.

15.   Khandelwal KR. Practical Pharmacognosy Techniques and Experiments. Nirali Prakashan; Pune. 2003.

16.   Kokate CK. Practical Pharmacognosy. Vallabh prakashan; Delhi. 2005:107–111.

17.   Ajaiyeoba EO. Onocha PA. Olarenwaju OT. Invitro anthelmintic properties of Buchholzia coriaceae and Gynandropsis gynandra extract. Pharm Biol 2001; 39(3):217- 220. doi.org/10.1076/phbi.39.3.217.5936

18.   Das SS. Dey M. Ghosh AK. Determination of Anthelmintic Activity of the Leaf and Bark Extract of Tamarindus Indica Linn. Indian J Pharm Sci. 2011; 73(1): 104-107. doi: 10.4103/0250-474X.89768

19.   Sabin S. Dijo D. Jahanara H. Anjusha MK. Merin B. An in vitro evaluation on anthelmintic activity of different extracts of Hemigraphis colorata leaves. Research J. Pharm and Tech. 2019; 12(9): 4394-4396. doi:10.5958/0974-360X.2019.00755.8

20.   Kousalya M. Geetha P. Jesuraja A. Vinoth KM. In-vitro study of anthelmintic activity of Eclipta prostrata (L) y various extracts. Research J. Pharm and Tech. 2017; 10(1):58-60. doi: 10.5958/0974-360X.2017.00014.2

21.   Rajesh R. Chitra K. Padmaa MP. In vitro anthelmintic activity of aerial parts of Aerva lanata Linn Juss. International Journal of Pharmaceutical Sciences and Drug Research 2010; 2(4): 269-271.

22.   Murugamani V. Raju L. Baska AR. Manjir SK and Girija GS. The new method developed for evaluation  of anthelmintic activity by housefly worms and compared with conventional earthworm method. ISRN pharmacology 2012; Article ID 709860. doi:10.5402/2012/709860

23.   Siju EN. Rajalakshmi GR. Hariraj N. Sreejith KR. Soumya S. Muneer EK. Premalatha K. In vitro anthelmitic activity of Mussaenda frondosa. Research J. Pharm and Tech. 2010; 3(1): 151-153.

24.   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

25.   Martin RJ. Mode of action of anthelmintic drugs. Vet J 1997; 154:11-34. doi: 10.1016/s1090-0233(05)80005-x

26.   Athnasiadou S. Kyriazakis I. Jackson F. Coop RL. Direct anthelmintic effects of condensed tannins towards different gastrointestinal nematodes of sheep: In vitro and in vivo studies. Vet Parasitol. 2001; 99:205–219. doi: 10.1016/s0304-4017(01)00467-8

27.   Sreesha NN. Neeraja ED. Greeshma R. Meenu B. Alexeyena V. Comparative evaluation of the anthelmintic activity of Coriandrum sativum Linn. and Apium graveolens Linn. Research J. Pharm and Tech. 2017; 10(11): 3857- 3859. doi: 10.5958/0974-360X.2017.00699.0

 

 

 

 

 

 

Received on 26.11.2020           Modified on 30.08.2021

Accepted on 11.12.2021         © RJPT All right reserved

Research J. Pharm. and Tech. 2022; 15(6):2539-2542.

DOI: 10.52711/0974-360X.2022.00424