Anti-scabies and mosquito repellent activity of Crinum asiaticum Linn. leaves extracts

 

Bharat Sharma, Neeru Vasudeva*, Sunil Sharma

Department of Pharmaceutical Sciences, Guru Jambheshwar University of Science and Technology,

Hisar, Haryana, India

 

ABSTRACT:

Background: Scabies is an enervating parasitic infestation of skin caused by Sarcoptes scabiei, affecting besides 130 million people at any time. Globally, this neglected tropical disease is amenable for 0.07% of the total burden of disease. In India and other West Asian countries, Anopheles stephensi is primary vector of malaria and occurrence of 200million to 450million infections causes up to 2.7million deaths annually worldwide. Crinum asiaticum Linn. (Amaryllidaceae) plant parts are used in traditional medicines for curing pain, arthritis, swelling carbuncle, piles, throat disorder, skin disease (leprosy), worms infestation, cold and cough disorders, vomiting, bowel complains, dysuria, polyuria and as insects repellents, emetic, purgative and rubifacients. Objective: The objective of this study was to evaluate the anti-scabies and mosquito repellent activity of ethanol and aqueous extracts of Crinum asiaticum leaves. Materials and Methods: The leaves of C. asiaticum were extracted in ethanol and aqueous solvents using soxhlet apparatus, evaluated for anti-scabies potential against Sarcoptes scabiei using contact bioassay method and mosquito repellent activity against Anopheles stephensi by repellency test. Results: Ethanol extract of C. asiaticum showed 100.00±0.00% mortality of S. scabiei at 10% concentration within 80 min. of contact and also at its 10% concentration, it provided 97.00±0.42% protection effect and 78.25±0.53% after 6 h treatment comparable to DEET. Conclusion: This present study revealed that the ethanol extract of C. asiaticum exhibited excellent scabicidal activity against adult S. scabiei mites and mosquito repellent activity against mosquito vector An. stephensi.

 

 

INTRODUCTION:

The Sarcoptes scabiei, ectoparasitic mite, (Itch mite, family Sarcoptidae), cause a contagious pruritic skin infestation with lesions, known as mange in animals and scabies in humans^1,2. These mites lay two or three eggs in burrow created at stratum corneum of epidermis. The larvae emerge out of eggs after 50-72 h and make new burrows. Then the larvae molt to nymph form and after two further molts, reach to adult life stage. S. scabiei takes 10-17 days to complete its life cycle³.

 

Scabies affects more than 130 million people at any point of time and lists as a neglected tropical disease according to World Health Organization⁴. In tropical regions, it is epidemiologically evidenced that, scabies generally cause pyoderma, dermatitis, eczema, urticaria and eventually serious illness due to invasion by opportunistic bacteria. Such infections can lead to cellulitis, bacteraemia or sepsis, impetigo, kidney and rheumatic heart disease that will increase health burden in resource-poor communities³.

 

Mosquito-borne disease, such as malaria, dengue, filariasis and encephalitis are major cause of morbidity and mortality worldwide and also lead to economic loss^5,6,7,8,9. In the context of control of mosquito-borne disease vector management plays a significant role in reducing the transmission of disease. Therefore mosquito repellents bearing a major role in prevention of human-mosquito contact and thus minimize the chance of infection¹⁰. The most commonly used drug for prevention of bite and as mosquito repellent is N, N-diethyl-3-methylbenzamide (DEET), an organochloride^10,11. Therefore, traditional uses of biologically active compounds derived from plant sources and insecticides and natural repellents are of great interest in the search of new active substance for vector control^12,13,14.

 

The genus Crinum possess all preeminent chemical features of family Amaryllidaceae and are also best known for biofactories of Amaryllidaceae alkaloids¹⁵. Crinum species have been used by traditional medical practitioners for treatment and prevention of various diseases throughout the world from ancient times. It is worth mentioned that out of approximately 180 alkaloids have been isolated and identified from Crinum, about 120 bases belong to crinine- and lycorine-types¹⁶. Crinum species are used in a number of countries for medicinal purposes probably due to their alkaloids constituents¹⁷. C. asiaticum Linn. is a herbaceous plant with greenish-feathery leaves. It is widely distributed to India, Hongkong, Myanmar, China, Srilanka, Malaysia, Thailand, Ryukyu Islands and Mainland Japan¹⁸. In Ayurveda, the plant is efficacious in the treatment of tumors, biliousness, urinary discharges, vomiting, and also in diseases related to blood, abdomen and vagina. According to the Unani system of medicine, this plant is used in the treatment of lung, chest and spleen disorders, bronchitis, gonorrhoea, urinary concretions, night-blindness, anuria, lumbago and toothache¹⁹.

 

Ethnomedically, it is used for the treatment of an pain, earache, swelling carbuncle, arthritis, piles, throat disorder, skin disease (leprosy), cold and cough disorders, worms infestation, vomiting, dysuria, polyuria, bowel complains, flatulence, fever colic and dyscrasia²⁰. Roots and leaves of this plant used as diaphoretic, emetic and purgative. The best remedy for treating inflammations and excrescence at the end of fingers and toes is leaves of smeared C. asiaticum with warmed castor oil. For treatment of earache and ear related problems, salt mixed with slightly warmed juice of the leaves has been used. In rheumatism roasted bulb is efficacious as rubefacient. For efficient insect repellent property bruised leaves of this plant are used^18,21,22.

 

The aim of this study is to explore the repellent and scabicidal activities of ethanol and aqueous extracts of leaves of C. asiaticum against An. stephensi and S. scabiei, respectively.

 

MATERIAL AND METHODS:

Plant material:

The leaves of C. asiaticum were collected from CCS Haryana Agricultural University, Hisar, Haryana, India. The leaf samples were identified and authenticated by Dr. Satish Kumar, Taxonomist at Department of Botany, Government College of Girls, Bhodia Khera, Fatehabad, Haryana. A voucher specimen (GJUPCOG160015 III) was preserved in the Herbarium of Department of Pharmaceutical sciences, Guru Jambheshwar University of Science and Technology, Hisar, Haryana, India. The leaves were firstly washed with tap water, shade dried and finely ground. The finely ground plant material was loaded in soxhlet apparatus and extracted with two different solvents namely ethanol and aqueous. The solvent from the extract was removed using rotary vacuum evaporator to collect the crude extract. All the chemicals and reagents used were of analytical grade.

 

In vitro anti-scabies activity:

Collection of mites:

The Sarcoptes scabiei mites were isolated from scabes and ear cerumen of infested legs and ears of rabbits under clinical examination by Dr. Snahil Gupta, Assistant Professor, Department of Veterinarian Parasitology, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, Haryana. The morphologically characterized mites were placed in petri dishes and motile adult mites were collected for testing.

 

Contact bioassay:

The ethanol and aqueous extract of C. asiaticum were diluted with 10% glycerin to get concentrations of 1%, 5% and 10%. Ten mites were placed in each petri dish and then in petri dishes 1 mL of diluted solution was added in direct contact with adult mites. Three replicates were performed for each concentration of oil. Permethrin 5% was used as a positive control and 10% glycerin was used as a negative control. The mites were inspected under stereomicroscope (Olympus) 20, 40, 60, 80 min after inoculation. Mites were considered dead when no movement was seen even after touching it with needle and no gut movement was observed over 2 min².

 

Mosquito repellent activity:

Mosquito strains:

Mosquito species Anopheles stephensi maintained at National Institute of Malaria Research laboratory were used for these studies. Adult mosquitoes were provided with 10% sucrose solution. The 6 days old females starved for 12 h before the experiment were used for repellent properties.

 

Preparation of the repellent and control replicates:

500mL of 10% sugar solution was prepared in distilled water. Sufficient quantity of bleached cotton was taken to be stacked in to a 460mL Styrofoam glass. 450mL of the above sugar solution was poured into the glass and the cotton was soaked. The cotton at the top was stretched out side in to circular foam. Remaining 40mL was used to prepare repellent formulation. To 40mL of the sugar solution required quantity of the leaves extracts concentrate was mixed to arrive at the desired concentrations, namely, 2.5%, 5%, and 10% and was poured evenly on the sugar soaked cotton in the above Styrofoam glass. For a positive control, DEET 2.5% in 10% sugar soaked cotton was prepared and 10% sugar soaked cotton was used as negative control. Standard stock solutions (10% (w/v) of ethanol and aqueous extracts of leaves of C. asiaticum made by redissolving known quantity of residue extract in corresponding solvents. By using freshly made stock solution, various test concentrations ranging between 2.5%, 5%, and 10% were prepared in double distilled water. Controls were supplemented with the equal amount required for the experiment without extracts. Tween-80 was used as an emulsifier at 0.05% concentration in the final test solution^23,24.

 

Repellency test:

These studies were carried out in a room maintained at 27ºC and 70% RH following the procedure described in Protocols for Uniform Evaluation of Insecticides for use in Vector Control (NIMR 2005). The prepared cages with the mosquitoes were placed in the room. In these cages, the Styrofoam glasses with cotton soaked with three different concentrations of extracts of leaves of C. asiaticum namely 2.5%, 5%, and 10% sugar solution, DEET 2.5% (positive control) in 10% sugar solution and 10% sugar solution (negative control) were placed in four different corners and one in the centre of the cage. After five-minute landing counts were made at 0, 1, 2, 4, 5, and 6h . The cups were removed from the cage after the five min observation at each interval of time. For subsequent exposure the position of the cups were inter changed to different corners. Landing rates of the mosquitoes on different concentrations of extracts of leaves of C. asiaticum (2.5%, 5%, and 10%), DEET (2.5%) and sugar (10%) were recorded. Data was reported as mean of the observations for each of the extract. Percent repellency was calculated by using the following formula:

 

% Protection = [(Control-Treated)/Control] x100

 

Where Control is the mean number of mosquitoes landing on negative control (10% sugar solution); and Treated is the mean number of mosquitoes landing on the repellents (DEET and leaves extracts of C. asiaticum).

 

RESULTS:

Anti-scabies activity:

The ethanol and aqueous extract of C. asiaticum with three concentrations of 1%, 5% and 10% showed remarkable % mortality of the S. scabiei mites presented in table 1. Ethanol extract of C. asiaticum gave 100.00±0.00% mortality of S. scabiei at 10% concentration within 80 min of contact whereas aqueous extract at same concentration showed 62.23±0.21% mortality. Based on % mean mortality study, it was found that Permethrin (reference) killed all the mites within 60 min but in negative control group, mortality was only 1.58±0.08% and most of mites remained alive after 80 min of treatment.

 

Table 1:Anti-scabies activity (mean mortality (%)±SD) of ethanol and aqueous extract of leaves of C. asiaticum against S. scabiei

Test agent	Concentration	Mean mortality (%)±SD
		20 min	40 min	60 min	80 min
Crinum asiaticum ethanol extract	1%	26.58±0.20	39.30±0.49	57.70±0.90	60.72±1.30
	5%	48.81±0.19	60.12±0.39	72.33±0.79	86.41±0.43
	10%	57.71±0.57	75.68±0.93	96.34±0.54	100.00±0.00
Crinum asiaticum aqueous extract	1%	8.09±0.80	15.51±0.19	19.00±0.35	21.78±0.90
	5%	16.67±0.70	22.30±0.49	30.53±0.75	42.34±0.85
	10%	24.43±0.58	35.57±0.35	48.90±0.37	62.23±0.21
Positive control		65.00±0.20	80.00±0.40	100.00±0.00	100.00±0.00
Negative control		00.00	00.00	1.58±0.08	1.58±0.08

 

Mosquito repellent activity:

The result of repellent efficacies of ethanol and aqueous extracts of C. asiaticum against malaria vector mosquito An. stephensi indicated that 10% concentration of ethanol extract of C. asiaticum showed 97.00±0.42% protection effect and 78.25±0.53% after 6 h treatment comparable to DEET i.e. (2.5%) produced the higher protection 100.00±0.00% during the entire period of study of 6 hr while aqueous extract of C. asiaticum exhibited <63.00% repellency within 6 h post treatment. The lowest protection 28.45±0.25% and 66.71±0.52% was observed in 2.5% concentration of aqueous and ethanol extract of C. asiaticum, respectively. Study revealed that both ethanol and aqueous extract of C. asiaticum showed significant repellent activity against A. stephensi as presented in table 2 and 3. Also, the repellency effect was observed to increase as the concentration of C. asiaticum extract increases from 2.5% to 10%.

Table 2: Repellency of Crinum asiaticum leaves ethanol extract against Anopheles stephensi

Dose (%)	Repellency % ±SD
	0h	1h	2h	4h	6h
DEET 2.5%	100.00±0.00	100.00±0.00	95.49±0.24	95.28±0.20	100.00±0.30
Tre*2.5%	86.23±0.23	87.50±0.72	72.71±0.58	66.71±0.41	66.71±0.52
Tre*5%	92.58±0.51	91.75±0.38	82.88±0.53	82.88±0.32	74.78±0.25
Tre*10%	97.00±0.42	95.70±0.63	89.40±0.31	82.30±0.74	78.25±0.53

 

Table 3: Repellency of Crinum asiaticum leaves aqueous extract against Anopheles stephensi

Dose (%)	Repellency %±SD
	0h	1h	2h	4h	6h
DEET 2.5%	100.00±0.00	100.00±0.00	95.49±0.24	95.28±0.20	100.00±0.30
Tre*2.5%	54.62±0.10	50.21±0.15	43.58±0.54	38.64±0.14	28.45±0.25
Tre*5%	57.36±0.37	52.39±0.71	50.15±0.33	49.25±0.50	39.16±0.41
Tre*10%	62.22±0.21	60.36±0.54	54.16±0.43	53.40±0.26	44.18±0.65

 

DISCUSSION:

From the ancient times, the plant kingdom has been of great interest as a potential source of insecticidal and repellent products because many species in the plant kingdom synthesize various types of secondary metabolites. Plants constitutes are rich source of bioactive chemicals so can be used as an alternative source for mosquito repellents²⁵. Mosquito repellents used in bite prevention treatment are the most efficacious tools for protection of human from vector-borne diseases¹⁰. Repellent compounds should be characterized as non-irritating, non toxic and long lasting²⁶. A various number of plant extracts and essential oil have been reported to have repellent and scabicidal potential which is also comparable to our study of C. asiaticum.

 

The ethanol extract of Ligularia virgaurea at high concentration of 2g/ml killed all the mites within 2 h and at a concentration of 1g/ml it took 6 h to kill all the mites²⁷. Tinospora cordifolia lotion down regulated Interleukin 1, 6 and 8 levels in skin infestations which lead to inhibition of hyperkeratosis and infiltration of inflammatory cells into scabietic lesion²⁸. Tinospora cordifolia lotion exhibited anti-scabies activity comparable to permethrin bearing same cure rate against Sarcoptes scabiei²⁹. Khan et al.³ determined the potential of methanol extract of Vitex negundo at three concentrations of 10%, 20%, 30% as scabicidal agents and showed 70%, 80% and 90% mortality of the S. scabiei mites while ivermectin (as positive control) and methyl alcohol (as negative control) displayed 85 and 5% mortality of S. scabiei.

 

Heyndrickx et al.³⁰ tested 15 plants for their anti-scabies potential and inferred that the ethanol extract of roots of Neorautanenia mitis and Pentas longiflora exhibited maximum anti-scabies potential against Psoroptes cuniculi. Dried flower heads of camomile, Matricaria chamomilla L. showed significant acaricidal activity compared to control against the mite Psoroptes cuniculi³¹. The acaricidal activity of Eupatorium adenophorum was assessed against Psoroptes cuniculi and Sarcoptes scabiei. Ethanol thermal circumfluence extract was found to be more toxic against S. scabiei at 0.5 and 1.0g/ml concentration while at 1g/ml extract killed all P. cuniculi³².

 

It was also found that eucalyptol enhance the superoxide dismutase and glutathione-s-transferase enzymatic activity, which play an important role in protection mechanism of S. scabiei mites³³. Fang et al. studied ten essential oils and reported that 1% clove and palmarosa oil killed all the motile mites within 20 and 50 min, respectively². Using contact bioassay, clove oil (1.56%) killed all the mites after exposure of 15 min while nutmeg oil showed moderate toxicity against scabies mites³⁵. Aboelhadid et al.³⁵ stated that 20% lemon oil caused 100% mortality of mites after 24 h and also investigated the elevation in hydrogen peroxide level that leads to considerable cellular damage. Elsholtzia densa also found to possess acaricidal potential against S. scabiei and at 16 mg/mL concentration killed all the mites within 16 h period³⁶.

 

Datura stramonium and Ocimum grastissimum were found to protect the exposed arms for 120 min at 5mg/l against Anopheles gambiae as inferred from the study of Afolabi et al.³⁷. Govindarajan M et al.³⁸ assessed repellent efficacy of various extracts of Delonix elata against malaria vector mosquito An. stephensi and reported that methanol extract of seed and leaves of D. elata at 5.0mg/cm² showed protection for the time 180 and 210 min., respectively. Stachytarpheta indica leaves methanol extract showed maximum protection (90.47%) at 40mg/ml against Aedes aegypti adult mosquitoes in human volunteers and Moringa oleifera methanol extract was also found to have good repellent effect against Aedes aegypti³⁹. Shankar et al.²⁵ study revealed that among the five plants Azadirachta indica, Murraya koenigii and Citrus medica gave protection for 6 h against mosquitoes followed by Ocimum tenuifloreum and Ricinus communis which provided protection for 4 h. Singh and Mittal,²³ reported the mosquito repellent and oviposition deterrent activities of hexane extract of Solanum nigrum against malaria vector Anopheles stephensi. S. nigrum at 10% concentration provided 100 % protection in beginning and 81% of 6 h post treatment. Kazembe and Makusha,⁴⁰ reported that the mixture of three extracts like Capsicum frutescens, Carica papaya, Cyanidon dactylo effectively protected (82%) for 4 h of post treatment than the mixtures of two or individual extract.

 

Various solvent extracts of Cardiospermum halicacabum exhibited promising repellency against Culex quinaquefasciatus, Ades aegypti, Anopheles stephensi at 5.0mg/cm² for 180 min⁴¹. Petroleum ether extract of Tribulus terrestris was found to have 100% protection at a dose of 1.5mg/cm² during the entire study period of 4 h against Aedes aegypti same as DEET. Ethanol and acetone extract of T. terrestris showed 32% and 73% repellency at same concentration⁵. The methanol extracts of Eclipta alba and Andrographis paniculata were also found to have repellent effect against An. stephensi⁴². Govindarajan M et al.⁴³ evaluated the repellent activity of Ervatamia coronaria, Caesalpinia pulcherrima against Culex quinaquefasciatus, ades aegypti, Anopheles stephensi and found more repellency in methanol extract of Ervatamia coronaria that provided 100% protection upto 210 min of study. Kamaraj et al.⁴⁴ evaluated larvicidal and repellent activities of methanol and ethyl acetate extracts of various plants against An. stephensi and Cx. quinquefasciatus. Methanol extract of Nelumbo nucifera, Trachyspermum ammi, ethyl acetate and methanol extract of Piper nigrum exhibited maximum repellency at 500ppm with the mean complete protection time varied from 30 to 150 min.

 

Sida acuta Burm. F. leaves extract have also been assessed for repellent activity against Culex quinquefasciatus, Anopheles stephensi, Aedes aegypti and found that at 5.0mg/cm² (higher concentration) provided 100% protection upto 120, 150, 180 min of treatment⁴⁵. The repellent efficacy of hexane extracts of tubers of Cyperus rotundus has also been reported against three mosquito species Anopheles stephensi, Anopheles culicifacies and Culex quinquefasciatus which varied from 80%-100% at different concentrations (2.5%, 5%, 10%) under different observation period²⁴. Yang et al.⁴⁶ have also reported repellent efficiency of 23 aromatic medicinal plants against female blood-starved Aedes aegypti by skin test. With comparison to DEET (82%), extracts of Cinnamomum cassia bark, Nardostachys chinensis rhizome, Paeonia suffruticosa root bark and Cinnamomum camphora steam distillate at 0.1mg/cm² provided protection effect 91%, 81%, 80%, 94%, respectively. Other plant extracts also showed <70% repellency as compared to DEET.

 

In conclusion, an attempt has been made to determine the scabicidal and mosquito repellent potential of extracts of C. asiaticum leaves against adult S. scabiei mites and An. stephensi mosquitoes vector, respectively. From this study, it is inferred that dose of 10% of ethanol extract of C. asiaticum leaves could be used for controlling An. stephensi mosquitoes and as a scabicidal agent against adult S. scabiei mites. The anti-scabies and mosquito repellent activity of C. asiaticum plant in this study might be useful for developing safer alternative to synthetic scabicidal and mosquito repellent compounds.

 

ACKNOWLEDGEMENT:

The authors sincerely acknowledge to Chairperson, Department of Pharmaceutical Sciences, Guru Jambheshwar University of Science and Technology, Hisar for providing necessary facilities to carry out this research work. The authors are also thankful to Dr. Snahil Gupta, Assistant Professor, Department of Veterinarian Parasitology, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, Haryana for helping us and providing all facilities to carry out anti-scabies activity

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Accepted on 30.09.2019           © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(2):895-900.