Research Report

Larvicidal and Growth-Regulatory Activities of Methanolic and N-hexane Extracts of Leaf of Ficus vallis-choudae Delile (Rosales: Moraceae) against Culex quinquefasciatus (Diptera: Culicidae)  

Joy Isoken Olikiabo , Israel Kayode Olayemi , Azubuike Christian Ukubuiwe , Kamoru Abdulazeez Adeniyi , Adesewa Aina , Oghenekevwe Juliet Oluwafemi , Mariah Oyiza Samuel
Applied Entomology Unit, Department of Animal Biology, Federal University of Technology, Minna, 920102, Nigeria
Author    Correspondence author
Medicinal Plant Research, 2018, Vol. 8, No. 3   doi: 10.5376/mpr.2018.08.0003
Received: 10 Jan., 2018    Accepted: 22 Feb., 2018    Published: 02 Mar., 2018
© 2018 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Olikiabo J.I., Olayemi I.K., Ukubuiwe A.C., Adeniyi K.A., Aina A., Oluwafemi O.J., and Samuel M.O., 2018, Larvicidal and growth-regulatory activities of methanolic and n-hexane extracts of leaf of Ficus vallis-choudae delile (rosales: moraceae) against Culex quinquefasciatus (diptera: culicidae), Medicinal Plant Research, 8(3): 14-20 (doi: 10.5376/mpr.2018.08.0003)

Abstract

The study aimed at determining the larvicidal and Insect-Growth-Regulating (IGR) potentials of n-hexane and methanolic extracts of Leaf of Ficus vallis-choudae against Culex quinquefasciatus mosquitoes in a bid to develop a cost effective and eco-friendly control lead. Collected leaves of the plants were air-dried, pulverized and sohxlet-extracted. Phytochemical screening of the crude extracts was done following standard protocols. Graded concentrations of 0.00625 to 0.05 mg/L (n-hexane extract) and 0.2 to 0.5 mg/L (methanolic extract) of the crude extract were assayed against fourth larval instar of the mosquito species. The mosquitoes were monitored from zero to 48 hours post-exposure to determine mortality. Sub lethal concentrations of 0.00625 (n-hexane) and 0.2 mg/L (methanol) were assayed for IGR activities. The phytochemical screening of crude extracts of the leaf revealed presence of seven secondary metabolites, whose presence was dependent on extraction solvent. They include flavonoids, tannins, saponins, cardiac glycosides, steroids, anthraquinones and terpenoids. After 48 hours post-exposure, analyses revealed a time and dose-dependent toxicity of the leaf extracts. The LC50 and LC90, respectively, of the n-hexane and methanolic extracts were, 0.022 and 0.042 mg/L, and 0.355 and 0.463 mg/L, respectively. IGR studies revealed significant increase in duration of development by the sub lethal concentrations. This study revealed larvicidal and IGR potential of n-hexane and methanolic extracts of the leaf of Ficus vallis-choudae, and therefore, a promising lead agent for development of larvicides for mosquito vector control.

Keywords
Phytochemicals; Bioassay; Toxicity; Lethal concentrations

Background

Vector-borne disease still remains and represents a great threat to public health. Among these, are those transmitted by mosquitoes, precisely, the female mosquitoes. These diseases transmitted by mosquitoes cause millions of death every year (Kamaraj et al., 2011). Several mosquitos’ species belonging to the genera Anopheles, Culex and Aedes carry disease pathogens of malaria, filariasis, encephalitis, dengue and yellow fever (Hubalek and Halouzka, 1999). Mosquito-transmitted diseases have remained the major cause of morbidity and mortality in Sub-Sahara Africa (Taubes, 2000; Jang et al., 2002). For instance, there are up to 500 million clinical cases and about one million death due to malaria globally, and sub-Sahara, respectively; with Africa recording over 90% of these cases (Snow et al., 2005).

 

Culex quinquefasciatus is a peridomestic mosquito rarely found far from human residences and/or his activities, and feed on birds, mammals and human hosts. Having complete metamorphosis comprising of egg, larva, pupa and adult, the mosquito species has four larval instars, namely, Larval stages 1-4 (i.e., L1-L4) (Ukubuiwe at al., 2012). The mosquito species are the primary vector of lymphatic filariasis, which have remained one of the most prevalent disease widely distributed in the tropics, affecting about 120 million people worldwide, with 44 million people manifesting chronic symptoms (Bernhard et al., 2003). In recent times, increased insecticide resistance by the vector and destruction of non-target organisms in the environment have been a major drawback in the eradication of these diseases (Zaim and Guillet, 2002; Omena et al., 2007).

 

Plants are very rich source of alternate reservoir of bioactive chemicals that are capable of controlling disease vectors, especially, mosquitoes, hence curbing the spread of vectored diseases (Olayemi et al., 2017). Plant pesticides have be found to be less toxic, possessing growth-delaying capabilities, easily biodegradable (Ignacimuthu, 2000) and target-specific (Sukumar et al., 1991). The phytochemicals derived from plant parts can act as larvicides and repellents against various target organisms (Pedro et al., 2014). A great number of plants in Africa, including Nigeria, have been, traditionally, observed (or noted) for their medical and pesticide properties (Okwute, 1992).

 

Ficus vallis-choudae, a plant widely spread in the tropical Africa including Nigeria, is one of such plants. It belongs to the Moraceae family, and has immense ethno-medicine value. The plant possess antifungal activities and extensively used in the treatment of jaundice, gastrointestinal problem and epilepsy (Adekunle, 2006). Despite these acclaimed properties of the plant, its larvicidal and growth-regulating potentials have not been investigated, this study, thus aims at elucidating these potentials systematically and updating current literature on the plant.

 

1 Materials and Methods

1.1 Collection and maintenance of larvae

Mosquito egg rafts were collected from stagnant water bodies in Minna, Niger State. The egg rafts were brought to the Laboratory of the Department of Biological Sciences, Federal University of Technology (FUT) Minna, Niger State. Hatched-out mosquito larvae were fed fish feed (Cuppens®) until they developed into the fourth instar (Ukubuiwe et al., 2016).

 

1.2 Collection, authentication and processing of leaves of Ficus vallis-choudae

The leaves were collected from various locations in Minna, Niger state, Nigeria (longitude 6033ꞌE and latitude 9027ꞌN). The leaves were authenticated by a Botanist in the Department of Biological Sciences, FUT, Minna, Niger State, and shade-dried in the laboratory at room temperature of 28oC for a period of three weeks.

 

1.3 Preparation of crude extracts of F. vallis-choudae

The dried leaves were weighed (50 g) and placed in the extracting flask of the Sohxlet apparatus. Two extracting solvents, n-hexane and methanol, were put in turn in the conical flask of the apparatus, for the production of n-hexane and methanol extracts of the plant, respectively. The liquid extracted was transferred into an evaporating dish, where water bath was used to dry-up the solvents. The extract was scooped and preserved in the refrigerator at 4°C until needed for bioassay (WHO, 2005; Olayemi et al., 2017).

 

1.4 Phytochemical screening of F. vallis-choudae

Qualitative phytochemical screenings of the crude extracts were carried out using standard procedures described by Trease and Evans (1989), Evans (1996) and Harborne (1998).

 

1.5 Preparation of stock solution and working concentrations

Working solutions for the bioassay were done according to the methods of Olayemi et al. (2017). Briefly, stock solutions of both extracts were prepared by dissolving the crude extracts in their respective solvent of extraction in the ratio 1:10 (for example, one gram of n-hexane crude extract was dissolved in 10 ml of n-hexane). This resulted in working solutions of 0.2, 0.3 and 0.4 mg/L for the methanol extract, and 0.00125, 0.0025, and 0.005 mg/L for the n-hexane extract.

 

1.6 Data analysis

The results obtained were expressed as mean ± Standard error of mean. Differences between mean were analyzed using analysis of variance (ANOVA), while significant differences between treatments were separated using Duncan multiple range test (DMRT), using Statistical Packages for Social Sciences (SPSS) Version 23. All decisions were taken at p = 0.05 using. The larval mortality data were subjected to probit analysis for calculating LC50 and LC90.

 

2 Results

2.1 Phytochemical screening of crude extracts of leaf of F. vallis-choudae

Seven (7) bioactive secondary metabolites were encountered in the Crude extracts of the leaf of the plant. They include flavonoids, tannins, cardiac glycosides, steroids, saponins, terpenoids and anthraquinones. Five (5) of these were encountered in the n-hexane extract, while six (6) in the methanolic extract. All metabolite components were present in the former extract, except tannins and cardiac glycoside, while only anthraquinones was absent in the methanolic extract (Table 1).

 

 

Table 1 Phytochemicals components of Leaf of Ficus vallis-choudae

Note: + = represents present; - = represents absent

 

2.2 Toxicity of crude extracts of leaf of F. vallis-choudae against Culex quinquefasciatus

Both extracts types were found to be toxic against the fourth larval instar of the mosquito species. The toxicity of both extracts was concentration and time-dependent, i.e., greater mortality was achieved at higher concentrations and with increase in time. For the n-hexane extract, 0.025 mg/L elicited 100% mortality (30.00 ± 0.00) at 48 hours, while similar feat was obtained by 0.05 mg/L within 12 hours (Table 2).

 

 

Table 2 Mortality of Culex quinquefasciatus larvae exposed to n-hexane extracts of leaf of Ficus vallis-choudae

Note: N = 30 larvae Positive Control = 0.00625 mg/ L n-hexane, Negative Control = Distilled water

Values are presented in mean ± standard error of mean 4 of replicates

*Values followed by the same alphabets in a column are not significantly different at p < 0.05 level of significance; **Values followed by the same alphabets in a row are not significantly different at p < 0.05 level of significance; *** - = 100% mortality attained before 24-hour exposure

 

Similarly, in the methanolic extract, 0.4 mg/L concentration cause greatest mortality at the 48th hour post-exposure, while similar toxicity was achieved by 0.5 mg/L before 24 hours of exposure (Table 3). Lethal concentrations of the extracts of the n-hexane and methanol leaf extracts were, respectively, 0.022 and 0.042 mg/L and 0.355 and 0.463 mg/L for the LC50 and LC90 (Table 4).

 

 

Table 3 Mortality of Culex quinquefasciatus larvae exposed to methanolic extracts of leaf of Ficus vallis-choudae

Note: N = 30 larvae; Positive Control = 0.2 mg/ L methanol, Negative Control = Distilled water

Values are presented in mean ± standard error of mean of four replicates

*Values followed by the same alphabets in a column are not significantly different at p<0.05 level of significance; **Values followed by the same alphabets in a row are not significantly different at p < 0.05 level of significance; *** - = 100% mortality attained before 24 hour exposure.

 

 

Table 4 Lethal concentrations (mg/ L) of Leaf Extracts Ficus vallis-choudae against 4th instar larvae of Culex quinquefasciatus

Note: R2 = coefficient of determination that shows the relationship between the extract concentration and mortality

 

2.3 Growth regulatory activities of crude extracts of Leaf of F. vallis-choudae against Cx. quinquefasciatus

There was no significant (p > 0.05) effect of both crude extract type on survivorship of the mosquito species. Average Larval and Survivorship were, respectively, 94.59 ± 2.18 and 99.17 ± 0.54 % and 96.03 ± 1.12 and 99.67 ± 0.18 % for n-hexane and methanol extract, respectively (Figure 1).

 

 

Figure 1 Effects of Sub-lethal concentrations of methanolic and n-hexane leaf extracts of Ficus vallis-choudae on Survival rate (%) of Immature Life Stages of Culex quinquefasciatus mosquitoes L1-4 = Larval Stages, ALS= Average Larval Survivorship, PSS= Pupal Stage Survivorship, AIS = Average Immature Survivorship

 

Duration of development, on the other hand, was significantly (p < 0.05) affected by the plant extracts. Total larval duration (TLD) was delayed from 7.24 ± 0.21 days in the negative control to 10.63 ± 0.16 and 10.63 ± 0.16 days, respectively, in n-hexane and methanol leaf extracts. Similarly, pupal stage duration (PSD) of development was slowed increased from 1.24 ± 0.08 days in the negative control to 2.65 ± 0.04 and 2.79 ± 0.09 days, respectively, in n-hexane and methanol leaf extracts (Table 5).

 

 

Table 5 Effects of Sub-lethal concentrations of methanolic and n-hexane leaf extracts of Ficus vallis-choudae on duration of development (Days) of Culex quinquefasciatus mosquitoes

Note: Values are presented in mean ± standard error of mean of four replicates. TLD =Total Larval Duration, PSD = Pupal Stage Duration, TID = Total Immature Duration

* Values followed by the same alphabets in the similar column are not significantly different at p < 0.05 level of significance

 

These led to a significant increase in Total Immature Duration (TID) of development from 8.48 ± 0.29 days to 13.28 ± 0.20 and 13.09 ± 0.47 days, respectively, and in n-hexane and methanol leaf extracts (Table 5).

 

3 Discussions

This study revealed the presence of seven secondary metabolites; these were flavonoids, tannins, cardiac glycosides, steroids, saponins, terpenoids and anthraquinones. These bioactive compounds have been reported to possess therapeutic (Lawan et al., 2008), toxicity (Shaalan et al., 2005) and larvicidal effects (Pedro et al., 2014) on mosquitoes. There were disparity in the numbers of these phytochemical constituents encountered in the two extract types (five in n-hexane extract and six in methanolic extract). These differences could be due to solvent of extraction (Raghavendra et al., 2011). El Tayeb et al. (2009) had observed variations in the phytochemical constituents of the plant part. Earlier, Olayemi et al. (2017) had reported similar observation in the numbers of phytochemical constituents in F. sur (a close relative) using similar solvents of extraction, although, variation in phytochemical constituents of the extract types were encountered. This difference could have been due to the plants species (Kamaraj et al., 2011).

 

In the present study, the two extract types of leaf of Ficus vallis-choudae (n-hexane and methanolic) were found to be toxic to fourth larval instar of the mosquito species; this effect was, largely, concentration- and time-dependent. As concentration of extracts and duration of exposure of the mosquito to the extracts increase, mortality also increases. Imam and Tajuddeen (2013) and Olayemi et al. (2013; 2017) observed similar trends. This could be due to increased toxicity associated with increase in concentration of the extract (Shaalan et al., 2005; Kishore et al., 2011).

 

Further, the n-hexane extract of the leaf of Ficus vallis-choudae was more toxic than the methanolic extract: as the highest value tested for methanol was 10 times the highest for n-hexane. This could have been due to polarity of the solvents used (Kishore, 2011). Lethal concentrations of the extracts of the n-hexane and methanol leaf extracts were, respectively, 0.022 and 0.042 mg/L and 0.355 and 0.463 mg/L, respectively, for the LC50­ and LC90. These values were lower than those reported for Carica papaya (Olayemi et al., 2013), Jatropha curcas (Olayemi et al., 2014) and propolis (Adeniyi et al., 2017). Although, with higher LC50 values, the extracts of this plant (Ficus vallis-choudae) had lower LC90s than F. sur and Adansonia digitata (Olayemi et al., 2017).

 

In the present study, though, survivorship of immature life stages of Cx. quinquefasciatus was not affected by sub-lethal concentrations of the plant extracts, duration of development was significantly increased. This finding is epidemiologically important as delay or elongation of duration of development would ensure suppressed metamorphosis, with its attendant reduction in population below threshold required to sustain disease transmission (Olayemi et al., 2013). The plant extracts could have delayed development by acting as a growth hormone suppressant of (MacRae, 2010), could have disrupted feeding activities by mosquito species. The latter have resultant effects on the accumulation of threshold-biomass required for metamorphosis of life stages (Timmerman and Briegel, 1993).

 

4 Conclusions

This study has revealed that crude extracts of the leaf of F. vallis-chaude contains secondary metabolites with biomedical importance. The efficacies of these extracts are dependent on solvent of extraction, duration of exposure to mosquitoes and test concentration of the extracts. More so, the extracts were toxic to Cx. quinquefasciatus mosquitoes, acting as a larvicide and a growth regulator. Therefore, this plant could be a viable lead agent in the development of natural bio-pesticide for the control of mosquito disease vector.

 

Further studies, however, are advocated to evaluate the efficacies of fractionated portions of these extracts and other extract types of the leaf against the mosquito species. Further, mosquitocidal potency of other parts of the plant should be investigated and toxicity of these fractions against non-target aquatic and terrestrial organisms appraised.

 

Author’s contributions

OJL, OIK and UAC conceived and designed the experiment. OJL, AA, OJO and SOM performed the experiments. OIK, UAC and AKA analysed the data. OJL and UAC wrote the first draft of the manuscript. OIK and AKA contributed to writing of the manuscript. All Authors agreed with manuscript results and conclusion. All authors read and approved the final manuscript.

 

Acknowledgements

We express our deepest appreciation goes to the Staff members of the Department of Biological Sciences, Federal University of Technology for material support in carrying out the study.

 

References

Adekunle A.A., Familoni O.B. and Okoli S.O., 2006, Antifungal activity of bark extract of Ficus vallis-Choudae and Detarium microcarpum, Acta SATECH, 2(2), 64-67

https://doi.org/10.4314/njtr.v12i2.8

 

Adeniyi K.A., Olayemi I.K., Shittu K.O., Ukubuiwe A.C., Salihu I.M. and Garba Y., 2017, Mosquito-larvicidal Efficacy of the Extract of Musca domestica Maggot against Culex pipiens (Diptera: Culicidae), an important Vector of Filariasis, Nigerian Journal of Technological Research, 12(2), 54-58

https://doi.org/10.1016/S0031-9406(05)60500-7

 

Bernhard L., Bernhard P. and Magnussen P., 2003, Management of patients with lymphoedema caused by Filariasis in north-eastern Tanzania: alternative approaches, Physiotherapy, 89 (12), 743-749

 

El Tayeb F.M., Taha A.K., Mardi H.G. and Sid Ahmed O.A., 2009, Water extracts of Hargal, Solenostem maargel (Del.), and Usher, Calotropis procera (A.), leaves as natural insecticides against mosquito larvae, Journal of Science Technology, 10(3), 67-76

 

Evans W.C., 1996, Trease and Evans Pharmacognosy, 14th Ed., London: Saunders, pp. 78

 

Harborne J.B., 1998, Phytochemical methods, A guide to modern technique of plant analysis, Third edition Chapman and Hall Publication, London, UK

 

Hubalek Z. and Halouzka J., 1999, West Nile Fever-A reemerging mosquito-borne viral disease in Europe, Emerging Infectious Diseases, 5, 650-653

https://doi.org/10.3201/eid0505.990506

PMid:10511521 PMCid:PMC2627716

 

Ignacimuthu S., 2000, the root of botanicals in combating mosquitoes, Loyola College, Chennai, India,19

 

Imam T.S. and Tajuddeen U.M., 2013, Qualitative phytochemical screening and larvicidal potencies of ethanolic extracts of five selected macrophyte species against Anopheles mosquitoes, Journal of Research in Environmental Science and Toxicology, 2(6), 121-125

 

Jang Y.S., Kim M.K., Ahn, Y.S. and Lee H.S., 2002, Larvicidal activity of Brazilian plant against Aedes aegypti and Culex pipiens pallens, Journal of American Mosquito Control Association, 18: 210-213

PMid:12322944

 

Kamaraj C., Bagavan A., Elango G., Zahir A.A., Rajakumar G. and Marimuthu S., 2011. Larvicidal activity of medicinal plant extracts against Anopheles subpictus and Culex tritaeniorhynchus. Indian Journal of Medical Research, 134, 101-106

PMid:21808141 PMCid:PMC3171902

 

Kishore N., Mishra B.B., Tiwari V.K. and Tripathi V., 2011, A review on natural products with mosquitosidal potentials, In: Tiwari VK, editor, Opportunity, challenge and scope of natural products in medicinal chemistry, Kerala: Research Signpost, 335-365

 

Lawan A., Katsayal U.A. and Yaro A.H., 2008, Anti-inflammatory and anti-nociceptive effects of the methanolic extract of the stem bark of Ficus vallis-Choudae (Moraceae). African Journal of Pharmacy and Pharmacology, 2 (10), 200-203

 

MacRae T.H., 2010, Gene expression, metabolic regulation and stress tolerance during diapause. Cellular and Molecular Life Sciences, 67(14), 2405-2424

https://doi.org/10.1007/s00018-010-0311-0

PMid:20213274

 

Okwute S.K., 1992, Plant-derived Pesticidal and Antimicrobial Agents for Use in Agriculture: A Review of Phytochemical and Biological Studies on some Nigerian Plants, Journal of Agricultural Science Technology, 2(1), 62-70

 

Olayemi I.K., Yakubu H. and Ukubuiwe A.C., 2013, Larvicidal and insect growth regulatory (IGR) activities of leaf-extract of Carica papaya against the filariasis vector mosquito, Culex pipiens pipiens (Diptera: Culicidae), Acta Malaysiana, 2(3), 100-106

 

Olayemi I.K., Busari J., Adeniyi K.A. and Ukubuiwe A.C., 2014, Comparative Larvicidal Efficacy of Leaf and Stem Extracts of Jatropha Curcas Plant, against the Mosquito Vector of Filariasis, Culex pipiens pipiens (Diptera: Culicidae), Malaya Journal of Biosciences, 1(2):104-108 ISSN 2348-6236 print /2348-3075 online

 

Olayemi I.K., Samuel O.M., Ukubuiwe A.C., Ande A.T., Adeniyi K.A. and Shittu K.O., 2017, Larvicidal Activities of Leaf Extracts of Adansonia digitata L. (Malvales: Malvaceae) and Ficus sur Forssk (Rosales: Moraceae) against Culex quinquefasciatus Mosquito (Diptera: Culicidae), Journal of Mosquito Research, 7(15), 115-124

https://doi.org/10.5376/jmr.2017.07.0015

 

Omena C., de Navarro F., Paula de E. and Luna S., 2007, Larvicidal activities against Aedes aegypti of some Brazilian medicinal plants, Biology Resource Technology, 98, 2549-2456

 

Pedro M.G.J., Auvrey N.A., Bryle A.L. and Maria F.L.S., 2014, Larvicidal activity of selected plant extracts against the Dengue vector Aedes aegypti mosquito, International Research Journal of Biological Sciences, 3(4), 23- 32

 

Raghavendra B.S., Prathibha K.P. and Vijayan V.A., 2011, Larvicidal Efficacy of Eugenia jambolana Linn, Extracts in three mosquito species at Mysore, Journal of Entomology, 8 (5), 491-496

https://doi.org/10.3923/je.2011.491.496

 

Shaalan E.A.S., Canyonb D., Younesc M.W.F., Abdel-Wahaba H. and Mansoura A.H., 2005, A review of botanical phytochemicals with mosquitocidal potential, Environmental Interest, 3, 1149-1166

https://doi.org/10.1016/j.envint.2005.03.003

PMid:15964629

 

Snow R., Guerra A., Abdisalan M. and Myint Y., 2005, The global distribution of clinical episodes of Plasmodium falciparum malaria, Nature, 343, 214- 217

https://doi.org/10.1038/nature03342

PMid:15759000 PMCid:PMC3128492

 

Sukumar K., Perich M.J. and Boobar L.R., 1991, Botanical derivatives in mosquito control: a review, Journal of America Mosquito Control Association, 7(2), 210-237

 

Taubes G., 2000, Vaccine, Searching for a parasite’s weak spot. Science, 290 (5491):434-437

https://doi.org/10.1126/science.290.5491.434

PMid:11183756

 

Timmermann S.E. and Briegel H., 1993, Water depth and larval density affect development and accumulation of reserves in laboratory populations of mosquitoes, Bulletin of the Society of Vector Ecology, 18,174-187

 

Trease G.E. and Evans W.C., 1989, Pharmacognosy, 11th ed., Bailliere Tindall, London, 45-50

 

Ukubuiwe A.C., Olayemi I.K., Omalu I.C.J., Odeyemi M.O., Jibrin A.I. and Oyibo-Usman K.A., 2012, Comparative assessment of immature survivorship and developmental duration of Culex pipiens pipiens (Diptera: Culicidae) populations in north central Nigeria. Biomed Central Epidemiology, 3(10): WMC003753

 

Ukubuiwe A.C., Olayemi I.K. and Jibrin A.I., 2016, Genetic Variations in Bionomics of Culex quinquefasciatus (Diptera: Culicidae) Mosquito Population in Minna, North-central Nigeria, International Journal of Insect Science, 8: 9-15 (doi:10.4137/IJIS.S32516)

https://doi.org/10.4137/IJIS.S32516

 

World Health Organization Guidelines, 2005, For Laboratory and Field Testing of Mosquito Larvicides

 

Zaim M. and Guillet P., 2002, Alternative insecticides: an urgent need, Trends in Parasitology, 18,161-163

https://doi.org/10.1016/S1471-4922(01)02220-6

Medicinal Plant Research
• Volume 8
View Options
. PDF(392KB)
. FPDF
. HTML
. Online fPDF
Associated material
. Readers' comments
Other articles by authors
. Joy Olikiabo
. Israel Olayemi
. Azubuike Ukubuiwe
. Kamoru Adeniyi
. Adesewa Aina
. Oghenekevwe Oluwafemi
. Mariah Samuel
Related articles
. Phytochemicals
. Bioassay
. Toxicity
. Lethal concentrations
Tools
. Email to a friend
. Post a comment

ass fuck sexo anal Gruppen Pornos Blondine Pornos inzest porn hd