Parasitol Res DOI 10.1007/s00436-015-4563-3
ORIGINAL PAPER
Phytochemical profile and larvicidal properties of seed essential oil from Nigella sativa L. (Ranunculaceae), against Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus (Diptera: Culicidae) Gnanaprakasam Adaikala Raj 1 & Manivachagam Chandrasekaran 1 & Shanmugam Krishnamoorthy 1 & Mahalingam Jayaraman 1 & Venugopalan Venkatesalu 2
Received: 22 May 2015 / Accepted: 27 May 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstracts The present study deals with investigation of larvicidal activity and their chemical constituents of the essential oil from the seeds of Nigella sativa L. (Ranunculaceae). Totally, 18 chemical compounds were identified by GC and GC-MS analysis. Thymol (19.13 %) and α-phellandrene (14.9 %) were identified as major chemical components followed by camphor (12.14 %), borneol (11.31 %), and carvacrol (8.65 %). The larval mortality was observed after 12 and 24 h of exposure period. The results revealed that the essential oil were evaluated against the fourth instar larvae of Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus. After 12 h of exposure period, the larvicidal activities were LC50 =196.9 and LC90 = 523.5 ppm (A. aegypti), LC50 =88.1 and LC90 =272.4 ppm (A.stephensi), and LC 50 = 241.6 and LC 90 = 545.4 ppm (C. quinquefasciatus), and the larvicidal activities after 24 h of exposure period were LC50 =99.9 and LC90 =300.8 ppm (A. aegypti), LC50 =53.9 and LC90 =172.6 ppm (A. stephensi), and LC50 =141.7 and LC90 =364.0 ppm (C. quinquefasciatus). The results of the present study showed that the essential oil from seeds of N. sativa is inexpensive food formulation and new source of natural larvicidal agent. Keywords Nigella sativa . Phytochemical composition . Aedes aegypti . Anopheles stephensi . Culex quinquefasciatus
* Manivachagam Chandrasekaran [email protected] 1
Department of Botany, Annamalai University, Annamalainagar 608 002, Tamil Nadu, India
2
Botany Wing - DDE, Annamalai University, Annamalainagar 608 002, Tamil Nadu, India
Introduction The species of Anopheles, Culex, and Aedes are important vectors that are capable of transmitting potential pathogens to human beings, and they are responsible for several infectious diseases like malaria, filariasis, Japanese encephalitis, yellow fever, dengue, and chikungunya (Nauen 2007). They have, therefore, become a challenging problem to public health worldwide, and it has a serious social and economical impact especially in tropical and subtropical countries (Bossche and Coetzer 2008). Tropical areas are more prone to parasitic diseases, and the risk has increased due to climate change and intensifying globalization (Karunamoorthi et al. 2010). Also, owing to poor drainage system, especially during rainy seasons, the presence of many fish ponds, irrigation ditches, and the rice fields provide abundant mosquito breeding places. Mosquito vector-borne diseases contribute to the major disease burden in India (Prabhu et al. 2011). In India, A. stephensi is responsible for malaria transmission in urban areas (Senthilkumar et al. 2009). Dengue fever has in recent years seen a great resurgence in tropical climates and spreading to new areas transmitted predominantly by one of the vectors, A. aegypti (WHO 2012). Mosquito-borne diseases are endemic over 100 countries, causing mortality of nearly two million people every year and at least one million children die of such diseases each year, leaving as many as 2100 million people at risk around the world (Klempner et al. 2007). The control of mosquito is an important public health concern around the world. Mosquitoes not only are a nuisance but also transmit several diseases like filariasis, dengue fever, Japanese encephalitis, etc. Despite an array of control measures taken to suppress the mosquito population, the latter continue to take heavy toll of human life every year, particularly in developing countries having poor socio-economic
Parasitol Res
conditions (Sagar and Schgal 1997). Because of resistance, insecticides against mosquitoes are becoming in effective (Vincent 2000). Mosquito bites may also cause allergic responses including local skin reactions and systemic reactions such as urticarial. Personal protection is one approach to prevent mosquito bites (Senthilkumar and Venkatesalu 2012; Sakulku et al. 2009). Most common mosquito repellents available contain N, N-diethyl-3-methylbenzamide or also called DEET that has shown strong protection from mosquitoes. However, it may exert toxic reaction under some circumstances and age groups and damage plastic, synthetic materials; thus, the alternative new products need to be explored (Revay et al. 2012; Chio and Yang 2008). In this context, essential oils have received much attention as potentially useful bioactive compounds against insects (Cheng et al. 2003) showing a broad spectrum of activity against insects, low mammalian toxicity, and degrading rapidly in the environment. Previous studies of essential oils obtained from the plants Murraya exotica (Krishnamoorthy et al. 2015), Feronia limonia (Senthilkumar et al. 2013), Mentha spicata, Govindarajan et al. (2012), and many other plants (Cheng et al. 2003; Traboulsi et al. 2005, 2002) have demonstrated promising larvicidal activity against mosquito vectors. Nigella sativa L. (Ranunculaceae), commonly known as “black cumin”, is a herbaceous plant that grows in Mediterranean countries and is also cultivated in Turkey. Seeds of N. sativa have been employed for thousands of years as a spice and food preservative. The oil and seed constituents have shown potential medicinal properties in traditional medicine (Salem 2005). During the past few decades, many phytochemical and pharmacological studies have been conducted on N. sativa seeds because of its marked biological activities, e.g., antioxidant, anti-inflammatory, antiulcer, anticarcinogenic, immunological and antiparasitic effects (Ali and Blunden 2003). The seeds have been reported to contain mainly fixed oils, proteins, alkaloids, saponins, and essential oil that previously characterized by a higher percentage of monoterpenes, the main constituents being thymoquinone and p-cymene (Ali and Blunden 2003). Uses for cough, bronchitis, headache, rheumatism, fever, influenza and eczema too and for the treatment of dyslipidemia, hyperglycaemia and related abnormalities have been recorded (Zaoui et al. 2002). The seeds contain a yellowish volatile oil, a fixed oil, proteins, amino acids, reducing sugars, mucilage, alkaloids, organic acids, tannins, resins, toxic glucoside, metarbin, bitter principles, glycosidal saponins, crude fiber, minerals and vitamins (Ramadan 2007). Black cumin crude fixed seed oil is a valuable source of essential fatty acids, glycolipids, phospholipids and bioactive phytosterols (Ramadan 2007; Ramadan et al. 2012). This oil has been reported to possess antitumor activity, antioxidant activity, anti-inflammatory activity, antibacterial activity and a stimulatory effect on the immune system. Actually, a great deal of attention has been focused on
black cumin seed oils and thus their consumption has increased, especially in Middle East countries. The recently, the cold-pressing procedure has been used to obtain black cumin seed oil (Ramadan et al. 2012). The present study was made to analyze the chemical composition and to study the mosquito larvicidal activity of seeds essential oil from N. sativa against early fourth instar larvae of A. aegypti, A. stephensi, and C. quinquefasciatus.
Materials and methods Plant material The seeds of N. sativa were purchased from Jothi Herbal Store Chidambaram, Cuddalore District, Tamil Nadu, India during month of April, 2014. Extraction of essential oil The healthy seeds of N. sativa were subjected to hydrodistillation using Clevenger ×77 type of apparatus for 10 h. The obtained essential oil was dried over anhydrous sodium sulfate, and the purified essential oil was stored in amber color vial (sealed with parafilm) at 4 °C for larvicidal assay. GC and GC-MS analysis of essential oil Gas chromatography (GC) analysis was carried out using Thermo GC-Trace Ultra Ver: 5.0, Thermo MS DSQ II. The chromatograph was fitted with DB 5-MS capillary non-polar column. The injector temperature was set at 300 °C and the oven temperature initially at 80 °C then programmed to 200 °C at the rate of 5 °C/min and held at 200 °C for 10 min. Then, the temperature was increased to 300 °C at the rate of 20 °C/min and finally held at 300 °C for 5 min. Helium was used as a carrier gas with the flow rate of 1.0 ml/ min. The sample was injected in the split mode in the ratio of 1:100. The percentage of composition of the essential oil was calculated by the GC peak areas. GC-mass spectrometry (GC-MS) analysis of essential oil was performed by using Varian 3800 Gas chromatography equipped with Varian 1200 -L single quadrupole mass spectrometer. GC conditions were same as reported for GC analysis, and the same column was used. The mass spectrometer was operated in the Electron Impact (EI) mode at 70 eV. Ion source and transfer line temperature was kept at 300 °C. The mass spectrum was obtained by centroid scan of the mass range from 40 to 800 amu. Identification of components of the essential oil was matching their recorded spectra with the data bank mass spectra of NIST and WILEY library provided by the instrument software, and the components were confirmed by comparing with previous literature.
Parasitol Res
Mosquito larvicidal bioassay The eggs of A. aegypti and A. stephensi were received from the Field Station, Center for Research in Medical Entomology (ICMR-Government of India), Virudhachalam, and the egg rafts of C. quinquefasciatus were collected from drainage of local residential area of Annamalai Nagar (11° 23′ 17 N, 79° 42′ 57 E) and reared in the laboratory (29±3 °C, 75 to 85 % RH). The larvae were fed with Brewer’s yeast/dog biscuit (1:3). The larvae at the early fourth instar stage were used for larvicidal assay. The larvicidal effect of seed essential oil from N. sativa against A. aegypti, A. stephensi, and C. quinquefasciatus was studied with the standard procedures recommended by the WHO (1981). The essential oil was dissolved with 1 ml of methanol and prepared into different concentrations viz., 12.5, 25, 50, 100, 200, and 400 ppm with distilled water. Twenty larvae (in a 100-ml beaker) of the early fourth instar stage were used for larvicidal assay, and three replicates were maintained for each concentration. During this experiment, no food was offered to the larvae. The larval mortality was calculated after 12 and 24 h of the exposure periods. All moribund mosquito larvae were considered as dead. The larval mortality was also checked for water and DMSO individually. Statistical analysis The results are expressed as the mean±SD. All statistical analyses were performed using SPSS version 11.5 statistical software (SPSS Inc., Chicago, IL, USA). The average larval mortality data probit analysis calculating LC50, LC90 and other statistics, 95 % confidence limits, and chi-square values were calculated.
Results and discussion Yield and chemical composition of essential oil The hydro-distillation of seeds of N. sativa yielded 0.03 % (v/ w) of essential oil with white pale greenish yellow color. Table 1 shows the chemical constituents of the essential oil analyzed by GC (Fig. 1) and GC-MS. In total, 18 compounds were identified by GC-MS and representing 100 % of essential oil, whereas thymol (19.13 %) and α-phellandrene (14.9 %) were identified as major chemical components followed by camphor (12.14 %), borneol (11.31 %), and carvacrol (8.65 %) that were identified as the major chemical compounds. Similarly, our results coincide with the chemotype with 33.0 % p-cymene and 26.8 % thymol, and the preponderance of monoterpenes was reported for N. sativa essential oil from Morocco (Moretti et al. 2004). Our results with previous data on the variability seed volatile oils depend
on the origin of the samples and environmental and climatic conditions. A variety of chemotypes have been described in the literature. An Iranian N. sativa essential oil was found to be dominated by phenylpropanoids components and displayed a trans-anethole chemotype (Nickavar et al. 2003). Singh et al. (2005) reported the presence of two major chemical compounds viz., p-cymene and thymoquinone from the seed essential oil of N. sativa. The chemotype with 60.2 % p-cymene and 12.9 % γ-terpinene was reported by Wajs et al. (2008) for N. sativa from Poland. Burits and Bucar (2000) reported the chemical composition of the essential oil of N. sativa from Austria. This observed differences in the chemical composition may be attributed to occurrence of chemotypes, geographical locations, season at the time of collection, stage of development, culture climate, and other culture conditions, which may affect biological activities (Runyoro et al. 2010). Mosquito control at the larval stage is an effective procedure because they are localized in space and time (Howard et al. 2007) resulting in less dangerous outcomes to non-target organisms, while the fight against adult is temporary and unsatisfactory. During the last three decades, mosquito controls were directed to the use of insecticide of plant origin. Environmental safety of insecticides is of first and foremost criterion for mosquito control programs (Rajkumar and Jebanesan 2005). In general, plant essential oils have been recognized as an important natural resource of insecticides (Gbolade et al. 2000). The results of the present study are also comparable to the earlier reports on the larvicidal activity of plant essential oils. N. sativa seeds contain a large amount of fixed oils (Ko¨ kdil and Yilmaz 2005), and the main constituent of the seed extract is thymoquinone (Aboul-Ela 2002). Several pharmacological effects have been attributed to active principles of N. sativa which includes thymoquinone, thymohydroquinone, dithymoquinone, thymol, carvacrol, nigellicine, nigellimine-x-oxide, nigellidine, and alphahedrin (Aljabre et al. 2005). Immunomodulatory and therapeutic properties of the N. sativa have been reviewed (Salem 2005). N. sativa seed extract inhibits fungal growth in dermatophytes (Aljabre et al. 2005), antioxidant activity (Burits and Bucar 2000), antitumor activity (Worthen et al. 1998), and a stimulatory effect on the immune system (Salem and Hossain 2000), Finally, N. sativa and its active ingredients have been shown to play potent anti-tumor activities in different mammalian cell lines and animal tumor models (Ali and Blunden 2003; Salem 2005), explaining the traditional use of N. sativa as a natural therapeutic product in treating various types of cancer. The larvicidal activity of seed essential oil from N. sativa was investigated. The essential oil had a remarkable larvicidal activity, and the results are presented in Table 2. The larvicidal activity and the larval mortality were observed against the early fourth instar larvae; after 12 h of exposure periods, the larvicidal activities were LC50 =196.9 and LC90 =523.5 ppm
Parasitol Res Table 1
Chemical constituents of seed essential oil from Nigella sativa
Peak no.
Retention time (min)
Chemical compositiona,b
%
1 2 3 4
1.58 5.87 6.43 7.35
α-Thujene α-Pinene Sabinene α-Phellandrene
0.16 0.33 0.16 14.9
5 6 7 8 9 10 11 12 13 14 15 16 17 18 Total
8.12 8.88 9.38 10.37 10.7 11.2 11.37 11.93 12.58 12.73 13.28 13.4 13.6 15.2
β-Pinene p-Cymene α-Terpinene α-Terpinolene Unidentified Linalool Ocimene Camphor Borneol Thymoquinone Thymol Carvacrol β-Caryophyllene α-Eudesmol
0.12 3.32 0.33 7.48 0.49 1.33 0.83 12.14 11.31 5.99 19.13 8.65 7.98 5.82 100.00
a
Compounds listed in order of elution from DB 35-MS Capillary standard non-polar column
b Components identified based on computer matching of the mass peaks with WILEY and NIST Library
Fig. 1 Gas Chromatogram of seed essential oil from Nigella sativa
(A. aegypti), LC50 =88.1 and LC90 =272.4 ppm (A. stephensi), and LC50 =241.6 and LC90 =545.4 ppm (C. quinquefasciatus), and the larvicidal activities after 24 h of exposure period were LC50 =99.9 and LC90 =300.8 ppm (A. aegypti), LC50 =53.9 and LC90 =172.6 ppm (A. stephensi), and LC50 =141.7 and LC90 =364.0 ppm (C. quinquefasciatus). Senthilkumar et al. (2013) reported the larvicidal activities of essential oil of Feronia limonia leaf against A. stephensi with LC50 38.93 and LC90 108.64 ppm (after 12 h), LC50 15.03 and LC90 36.69 ppm (after 24 h); A. aegypti with LC50 37.60 and LC 90 104.69 ppm (after 12 h), LC 50 11.59 and LC 90 42.95 ppm (after 24 h); and C. quinquefasciatus with LC50 52.08 and LC90 124.33 ppm (after 12 h), LC50 22.49 and LC90 60.90 ppm (after 24 h). The leaf essential oil from M. exotica had a strong larvicidal activity with LC50 =74.7 and LC90 = 152.7 (after 12 h); LC50 =35.8 and LC90 =85.4 ppm (after 24 h) against A. aegypti, LC50 =56.3 and LC90 =107.8 (after 12 h); LC50 =31.3 and LC90 =75.1 ppm (after 24 h) against A. stephensi and LC50 =74.4 and LC90 =136.9 ppm (after 12 h); LC50 =43.2 and LC90 =103.2 ppm (after 24 h) against C. quinquefasciatus (Krishnamoorthy et al. 2015). Many researchers have examined the larvicidal activity of various plant seed oils and extracts against mosquitoes (Sagar and Sehgal 1996). Elbanna (2006) reported that the extract from Eucalyptus seeds contained toxic compounds against Culex pipiens larvae. Knio et al. (2008) found that anis and parsley seed oils had strong larvicidal activity against Ochlerotatus
Parasitol Res Table 2 Larvicidal properties of seed essential oil from Nigella sativa against the larvae of A. aegypti, A. stephensi, and C. quinquefasciatus after 12 and 24 h of exposure period Name of the mosquito species
Time
Concentration (ppm)
A. aegypti
After 12 h
12.5 25 50 100 200 400 12.5 25 50
14±0.28 20±0.57 32±0.73 47±0.34 55±0.50 74±0.81 17±0.28 32±0.50 48±0.37
100 200 400 12.5 25 50 100 200 400 12.5 25 50 100 200 400 12.5 25 50
53±0.57 74±0.81 96±1.15 18±0.40 32±0.28 47±0.50 59±0.57 82±0.31 96±0.87 23±0.76 39±0.50 58±0.50 73±1.15 86±1.12 100±0.0 8±0.57 14±0.25 24±0.28
100 200 400 12.5 25 50 100 200 400
35±0.57 48±0.45 70±0.76 13±0.28 22±0.25 38±0.50 45±0.45 68±0.28 90±0.76
After 24 h
A. stephensi
After 12 h
After 24 h
C.quinquefasciatus
After 12 h
After 24 h
% mortality ± SE
LC50 (LCL-UCL)a
LC90 (LCL-UCL)a
χ2 (df=4)b
196.9 (121.3–336.7)
523.5 (368.2–1066.1)
15.2
99.9 (52.4–150.6)
300.8 (226.5–482.2)
12.4
88.1 (35.9–141.1)
272.4 (199.6–473.26)
15.4
53.9 (25.2–79.0)
172.6 (133.5–262.1)
9.2
241.6 (168.8–399.3)
545.4 (394.3–1009.1)
14.8
141.7 (92.8–204.0)
364.0 (278.1–562.0)
12.5
LCL lower confidence level, UCL upper confidence level a
95 % Confidence interval
b
Degrees of freedom; χ2 chi-square value
caspius (Pallas), with LC50 values of
ORIGINAL PAPER
Phytochemical profile and larvicidal properties of seed essential oil from Nigella sativa L. (Ranunculaceae), against Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus (Diptera: Culicidae) Gnanaprakasam Adaikala Raj 1 & Manivachagam Chandrasekaran 1 & Shanmugam Krishnamoorthy 1 & Mahalingam Jayaraman 1 & Venugopalan Venkatesalu 2
Received: 22 May 2015 / Accepted: 27 May 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstracts The present study deals with investigation of larvicidal activity and their chemical constituents of the essential oil from the seeds of Nigella sativa L. (Ranunculaceae). Totally, 18 chemical compounds were identified by GC and GC-MS analysis. Thymol (19.13 %) and α-phellandrene (14.9 %) were identified as major chemical components followed by camphor (12.14 %), borneol (11.31 %), and carvacrol (8.65 %). The larval mortality was observed after 12 and 24 h of exposure period. The results revealed that the essential oil were evaluated against the fourth instar larvae of Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus. After 12 h of exposure period, the larvicidal activities were LC50 =196.9 and LC90 = 523.5 ppm (A. aegypti), LC50 =88.1 and LC90 =272.4 ppm (A.stephensi), and LC 50 = 241.6 and LC 90 = 545.4 ppm (C. quinquefasciatus), and the larvicidal activities after 24 h of exposure period were LC50 =99.9 and LC90 =300.8 ppm (A. aegypti), LC50 =53.9 and LC90 =172.6 ppm (A. stephensi), and LC50 =141.7 and LC90 =364.0 ppm (C. quinquefasciatus). The results of the present study showed that the essential oil from seeds of N. sativa is inexpensive food formulation and new source of natural larvicidal agent. Keywords Nigella sativa . Phytochemical composition . Aedes aegypti . Anopheles stephensi . Culex quinquefasciatus
* Manivachagam Chandrasekaran [email protected] 1
Department of Botany, Annamalai University, Annamalainagar 608 002, Tamil Nadu, India
2
Botany Wing - DDE, Annamalai University, Annamalainagar 608 002, Tamil Nadu, India
Introduction The species of Anopheles, Culex, and Aedes are important vectors that are capable of transmitting potential pathogens to human beings, and they are responsible for several infectious diseases like malaria, filariasis, Japanese encephalitis, yellow fever, dengue, and chikungunya (Nauen 2007). They have, therefore, become a challenging problem to public health worldwide, and it has a serious social and economical impact especially in tropical and subtropical countries (Bossche and Coetzer 2008). Tropical areas are more prone to parasitic diseases, and the risk has increased due to climate change and intensifying globalization (Karunamoorthi et al. 2010). Also, owing to poor drainage system, especially during rainy seasons, the presence of many fish ponds, irrigation ditches, and the rice fields provide abundant mosquito breeding places. Mosquito vector-borne diseases contribute to the major disease burden in India (Prabhu et al. 2011). In India, A. stephensi is responsible for malaria transmission in urban areas (Senthilkumar et al. 2009). Dengue fever has in recent years seen a great resurgence in tropical climates and spreading to new areas transmitted predominantly by one of the vectors, A. aegypti (WHO 2012). Mosquito-borne diseases are endemic over 100 countries, causing mortality of nearly two million people every year and at least one million children die of such diseases each year, leaving as many as 2100 million people at risk around the world (Klempner et al. 2007). The control of mosquito is an important public health concern around the world. Mosquitoes not only are a nuisance but also transmit several diseases like filariasis, dengue fever, Japanese encephalitis, etc. Despite an array of control measures taken to suppress the mosquito population, the latter continue to take heavy toll of human life every year, particularly in developing countries having poor socio-economic
Parasitol Res
conditions (Sagar and Schgal 1997). Because of resistance, insecticides against mosquitoes are becoming in effective (Vincent 2000). Mosquito bites may also cause allergic responses including local skin reactions and systemic reactions such as urticarial. Personal protection is one approach to prevent mosquito bites (Senthilkumar and Venkatesalu 2012; Sakulku et al. 2009). Most common mosquito repellents available contain N, N-diethyl-3-methylbenzamide or also called DEET that has shown strong protection from mosquitoes. However, it may exert toxic reaction under some circumstances and age groups and damage plastic, synthetic materials; thus, the alternative new products need to be explored (Revay et al. 2012; Chio and Yang 2008). In this context, essential oils have received much attention as potentially useful bioactive compounds against insects (Cheng et al. 2003) showing a broad spectrum of activity against insects, low mammalian toxicity, and degrading rapidly in the environment. Previous studies of essential oils obtained from the plants Murraya exotica (Krishnamoorthy et al. 2015), Feronia limonia (Senthilkumar et al. 2013), Mentha spicata, Govindarajan et al. (2012), and many other plants (Cheng et al. 2003; Traboulsi et al. 2005, 2002) have demonstrated promising larvicidal activity against mosquito vectors. Nigella sativa L. (Ranunculaceae), commonly known as “black cumin”, is a herbaceous plant that grows in Mediterranean countries and is also cultivated in Turkey. Seeds of N. sativa have been employed for thousands of years as a spice and food preservative. The oil and seed constituents have shown potential medicinal properties in traditional medicine (Salem 2005). During the past few decades, many phytochemical and pharmacological studies have been conducted on N. sativa seeds because of its marked biological activities, e.g., antioxidant, anti-inflammatory, antiulcer, anticarcinogenic, immunological and antiparasitic effects (Ali and Blunden 2003). The seeds have been reported to contain mainly fixed oils, proteins, alkaloids, saponins, and essential oil that previously characterized by a higher percentage of monoterpenes, the main constituents being thymoquinone and p-cymene (Ali and Blunden 2003). Uses for cough, bronchitis, headache, rheumatism, fever, influenza and eczema too and for the treatment of dyslipidemia, hyperglycaemia and related abnormalities have been recorded (Zaoui et al. 2002). The seeds contain a yellowish volatile oil, a fixed oil, proteins, amino acids, reducing sugars, mucilage, alkaloids, organic acids, tannins, resins, toxic glucoside, metarbin, bitter principles, glycosidal saponins, crude fiber, minerals and vitamins (Ramadan 2007). Black cumin crude fixed seed oil is a valuable source of essential fatty acids, glycolipids, phospholipids and bioactive phytosterols (Ramadan 2007; Ramadan et al. 2012). This oil has been reported to possess antitumor activity, antioxidant activity, anti-inflammatory activity, antibacterial activity and a stimulatory effect on the immune system. Actually, a great deal of attention has been focused on
black cumin seed oils and thus their consumption has increased, especially in Middle East countries. The recently, the cold-pressing procedure has been used to obtain black cumin seed oil (Ramadan et al. 2012). The present study was made to analyze the chemical composition and to study the mosquito larvicidal activity of seeds essential oil from N. sativa against early fourth instar larvae of A. aegypti, A. stephensi, and C. quinquefasciatus.
Materials and methods Plant material The seeds of N. sativa were purchased from Jothi Herbal Store Chidambaram, Cuddalore District, Tamil Nadu, India during month of April, 2014. Extraction of essential oil The healthy seeds of N. sativa were subjected to hydrodistillation using Clevenger ×77 type of apparatus for 10 h. The obtained essential oil was dried over anhydrous sodium sulfate, and the purified essential oil was stored in amber color vial (sealed with parafilm) at 4 °C for larvicidal assay. GC and GC-MS analysis of essential oil Gas chromatography (GC) analysis was carried out using Thermo GC-Trace Ultra Ver: 5.0, Thermo MS DSQ II. The chromatograph was fitted with DB 5-MS capillary non-polar column. The injector temperature was set at 300 °C and the oven temperature initially at 80 °C then programmed to 200 °C at the rate of 5 °C/min and held at 200 °C for 10 min. Then, the temperature was increased to 300 °C at the rate of 20 °C/min and finally held at 300 °C for 5 min. Helium was used as a carrier gas with the flow rate of 1.0 ml/ min. The sample was injected in the split mode in the ratio of 1:100. The percentage of composition of the essential oil was calculated by the GC peak areas. GC-mass spectrometry (GC-MS) analysis of essential oil was performed by using Varian 3800 Gas chromatography equipped with Varian 1200 -L single quadrupole mass spectrometer. GC conditions were same as reported for GC analysis, and the same column was used. The mass spectrometer was operated in the Electron Impact (EI) mode at 70 eV. Ion source and transfer line temperature was kept at 300 °C. The mass spectrum was obtained by centroid scan of the mass range from 40 to 800 amu. Identification of components of the essential oil was matching their recorded spectra with the data bank mass spectra of NIST and WILEY library provided by the instrument software, and the components were confirmed by comparing with previous literature.
Parasitol Res
Mosquito larvicidal bioassay The eggs of A. aegypti and A. stephensi were received from the Field Station, Center for Research in Medical Entomology (ICMR-Government of India), Virudhachalam, and the egg rafts of C. quinquefasciatus were collected from drainage of local residential area of Annamalai Nagar (11° 23′ 17 N, 79° 42′ 57 E) and reared in the laboratory (29±3 °C, 75 to 85 % RH). The larvae were fed with Brewer’s yeast/dog biscuit (1:3). The larvae at the early fourth instar stage were used for larvicidal assay. The larvicidal effect of seed essential oil from N. sativa against A. aegypti, A. stephensi, and C. quinquefasciatus was studied with the standard procedures recommended by the WHO (1981). The essential oil was dissolved with 1 ml of methanol and prepared into different concentrations viz., 12.5, 25, 50, 100, 200, and 400 ppm with distilled water. Twenty larvae (in a 100-ml beaker) of the early fourth instar stage were used for larvicidal assay, and three replicates were maintained for each concentration. During this experiment, no food was offered to the larvae. The larval mortality was calculated after 12 and 24 h of the exposure periods. All moribund mosquito larvae were considered as dead. The larval mortality was also checked for water and DMSO individually. Statistical analysis The results are expressed as the mean±SD. All statistical analyses were performed using SPSS version 11.5 statistical software (SPSS Inc., Chicago, IL, USA). The average larval mortality data probit analysis calculating LC50, LC90 and other statistics, 95 % confidence limits, and chi-square values were calculated.
Results and discussion Yield and chemical composition of essential oil The hydro-distillation of seeds of N. sativa yielded 0.03 % (v/ w) of essential oil with white pale greenish yellow color. Table 1 shows the chemical constituents of the essential oil analyzed by GC (Fig. 1) and GC-MS. In total, 18 compounds were identified by GC-MS and representing 100 % of essential oil, whereas thymol (19.13 %) and α-phellandrene (14.9 %) were identified as major chemical components followed by camphor (12.14 %), borneol (11.31 %), and carvacrol (8.65 %) that were identified as the major chemical compounds. Similarly, our results coincide with the chemotype with 33.0 % p-cymene and 26.8 % thymol, and the preponderance of monoterpenes was reported for N. sativa essential oil from Morocco (Moretti et al. 2004). Our results with previous data on the variability seed volatile oils depend
on the origin of the samples and environmental and climatic conditions. A variety of chemotypes have been described in the literature. An Iranian N. sativa essential oil was found to be dominated by phenylpropanoids components and displayed a trans-anethole chemotype (Nickavar et al. 2003). Singh et al. (2005) reported the presence of two major chemical compounds viz., p-cymene and thymoquinone from the seed essential oil of N. sativa. The chemotype with 60.2 % p-cymene and 12.9 % γ-terpinene was reported by Wajs et al. (2008) for N. sativa from Poland. Burits and Bucar (2000) reported the chemical composition of the essential oil of N. sativa from Austria. This observed differences in the chemical composition may be attributed to occurrence of chemotypes, geographical locations, season at the time of collection, stage of development, culture climate, and other culture conditions, which may affect biological activities (Runyoro et al. 2010). Mosquito control at the larval stage is an effective procedure because they are localized in space and time (Howard et al. 2007) resulting in less dangerous outcomes to non-target organisms, while the fight against adult is temporary and unsatisfactory. During the last three decades, mosquito controls were directed to the use of insecticide of plant origin. Environmental safety of insecticides is of first and foremost criterion for mosquito control programs (Rajkumar and Jebanesan 2005). In general, plant essential oils have been recognized as an important natural resource of insecticides (Gbolade et al. 2000). The results of the present study are also comparable to the earlier reports on the larvicidal activity of plant essential oils. N. sativa seeds contain a large amount of fixed oils (Ko¨ kdil and Yilmaz 2005), and the main constituent of the seed extract is thymoquinone (Aboul-Ela 2002). Several pharmacological effects have been attributed to active principles of N. sativa which includes thymoquinone, thymohydroquinone, dithymoquinone, thymol, carvacrol, nigellicine, nigellimine-x-oxide, nigellidine, and alphahedrin (Aljabre et al. 2005). Immunomodulatory and therapeutic properties of the N. sativa have been reviewed (Salem 2005). N. sativa seed extract inhibits fungal growth in dermatophytes (Aljabre et al. 2005), antioxidant activity (Burits and Bucar 2000), antitumor activity (Worthen et al. 1998), and a stimulatory effect on the immune system (Salem and Hossain 2000), Finally, N. sativa and its active ingredients have been shown to play potent anti-tumor activities in different mammalian cell lines and animal tumor models (Ali and Blunden 2003; Salem 2005), explaining the traditional use of N. sativa as a natural therapeutic product in treating various types of cancer. The larvicidal activity of seed essential oil from N. sativa was investigated. The essential oil had a remarkable larvicidal activity, and the results are presented in Table 2. The larvicidal activity and the larval mortality were observed against the early fourth instar larvae; after 12 h of exposure periods, the larvicidal activities were LC50 =196.9 and LC90 =523.5 ppm
Parasitol Res Table 1
Chemical constituents of seed essential oil from Nigella sativa
Peak no.
Retention time (min)
Chemical compositiona,b
%
1 2 3 4
1.58 5.87 6.43 7.35
α-Thujene α-Pinene Sabinene α-Phellandrene
0.16 0.33 0.16 14.9
5 6 7 8 9 10 11 12 13 14 15 16 17 18 Total
8.12 8.88 9.38 10.37 10.7 11.2 11.37 11.93 12.58 12.73 13.28 13.4 13.6 15.2
β-Pinene p-Cymene α-Terpinene α-Terpinolene Unidentified Linalool Ocimene Camphor Borneol Thymoquinone Thymol Carvacrol β-Caryophyllene α-Eudesmol
0.12 3.32 0.33 7.48 0.49 1.33 0.83 12.14 11.31 5.99 19.13 8.65 7.98 5.82 100.00
a
Compounds listed in order of elution from DB 35-MS Capillary standard non-polar column
b Components identified based on computer matching of the mass peaks with WILEY and NIST Library
Fig. 1 Gas Chromatogram of seed essential oil from Nigella sativa
(A. aegypti), LC50 =88.1 and LC90 =272.4 ppm (A. stephensi), and LC50 =241.6 and LC90 =545.4 ppm (C. quinquefasciatus), and the larvicidal activities after 24 h of exposure period were LC50 =99.9 and LC90 =300.8 ppm (A. aegypti), LC50 =53.9 and LC90 =172.6 ppm (A. stephensi), and LC50 =141.7 and LC90 =364.0 ppm (C. quinquefasciatus). Senthilkumar et al. (2013) reported the larvicidal activities of essential oil of Feronia limonia leaf against A. stephensi with LC50 38.93 and LC90 108.64 ppm (after 12 h), LC50 15.03 and LC90 36.69 ppm (after 24 h); A. aegypti with LC50 37.60 and LC 90 104.69 ppm (after 12 h), LC 50 11.59 and LC 90 42.95 ppm (after 24 h); and C. quinquefasciatus with LC50 52.08 and LC90 124.33 ppm (after 12 h), LC50 22.49 and LC90 60.90 ppm (after 24 h). The leaf essential oil from M. exotica had a strong larvicidal activity with LC50 =74.7 and LC90 = 152.7 (after 12 h); LC50 =35.8 and LC90 =85.4 ppm (after 24 h) against A. aegypti, LC50 =56.3 and LC90 =107.8 (after 12 h); LC50 =31.3 and LC90 =75.1 ppm (after 24 h) against A. stephensi and LC50 =74.4 and LC90 =136.9 ppm (after 12 h); LC50 =43.2 and LC90 =103.2 ppm (after 24 h) against C. quinquefasciatus (Krishnamoorthy et al. 2015). Many researchers have examined the larvicidal activity of various plant seed oils and extracts against mosquitoes (Sagar and Sehgal 1996). Elbanna (2006) reported that the extract from Eucalyptus seeds contained toxic compounds against Culex pipiens larvae. Knio et al. (2008) found that anis and parsley seed oils had strong larvicidal activity against Ochlerotatus
Parasitol Res Table 2 Larvicidal properties of seed essential oil from Nigella sativa against the larvae of A. aegypti, A. stephensi, and C. quinquefasciatus after 12 and 24 h of exposure period Name of the mosquito species
Time
Concentration (ppm)
A. aegypti
After 12 h
12.5 25 50 100 200 400 12.5 25 50
14±0.28 20±0.57 32±0.73 47±0.34 55±0.50 74±0.81 17±0.28 32±0.50 48±0.37
100 200 400 12.5 25 50 100 200 400 12.5 25 50 100 200 400 12.5 25 50
53±0.57 74±0.81 96±1.15 18±0.40 32±0.28 47±0.50 59±0.57 82±0.31 96±0.87 23±0.76 39±0.50 58±0.50 73±1.15 86±1.12 100±0.0 8±0.57 14±0.25 24±0.28
100 200 400 12.5 25 50 100 200 400
35±0.57 48±0.45 70±0.76 13±0.28 22±0.25 38±0.50 45±0.45 68±0.28 90±0.76
After 24 h
A. stephensi
After 12 h
After 24 h
C.quinquefasciatus
After 12 h
After 24 h
% mortality ± SE
LC50 (LCL-UCL)a
LC90 (LCL-UCL)a
χ2 (df=4)b
196.9 (121.3–336.7)
523.5 (368.2–1066.1)
15.2
99.9 (52.4–150.6)
300.8 (226.5–482.2)
12.4
88.1 (35.9–141.1)
272.4 (199.6–473.26)
15.4
53.9 (25.2–79.0)
172.6 (133.5–262.1)
9.2
241.6 (168.8–399.3)
545.4 (394.3–1009.1)
14.8
141.7 (92.8–204.0)
364.0 (278.1–562.0)
12.5
LCL lower confidence level, UCL upper confidence level a
95 % Confidence interval
b
Degrees of freedom; χ2 chi-square value
caspius (Pallas), with LC50 values of
