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Cytotoxic, Phytotoxic and Insecticidal Potential of Achillea millefolium L. and Chaerophyllum villosum wall. ex dc.

Potencial citotóxico, fitotóxico e insecticida de Achillea Millefolium L. e Chaerophyllum Villosum

Abstract

The methanolic, chloroformic and aqueous extract of Achillea millefolium and Chaerophyllum villosum were investigated for cytotoxicity, phytotoxic and insecticidal activities. Cytotoxicity was investigated by brine shrimp lethality assay indicating that the crude methanolic extract of A.millefolium and chloroformic extract of C.villosum revealed highest mortality of brine shrimps with (LD50 of 52.60 µg/ml) and (14.81 µg/ml). Phytotoxicity was evaluated using the Lemna minor bioassay which revealed that the crude methanolic extract of A.millefolium and C.villosum extract has maximum inhibition of Lemna minor with (Fl50 6.60 µg/ml) and (0.67 µg/ml).The insecticidal activity showed that among all the insects studied it was observed that methanolic extract of A. millefoliumand C. villosum was highly toxic to Sphenoptera dadkhani with (LD50=4.17 µg/ml) and (0.34 µg/ml). From the present study it can be concluded that different extracts from A. millefolium and C. villosum showed good cytotoxic, phytotoxic and insecticidal activity in a dose dependent manner.

Keywords:
cytotoxic; phytotoxic; insecticidal activity; Achillea millefolium ; Chaerophyllum villosum

Resumo

Neste estudo, os extratos metanólico, clorofórmico e aquoso de Achillea millefolium e Chaerophyllum villosum foram analisados em relação à citotoxicidade, atividade fitotóxica e inseticida. A citotoxicidade foi analisada através do ensaio de letalidade de artémia, indicando que o extrato metanólico bruto de A. millefolium e o extrato clorofórmico de C. villosum revelaram maior mortalidade de artêmias com DL50 de 52,60 µg/ml e 14,81 µg/ml. A fitotoxicidade foi avaliada utilizando o bioensaio de Lemna minor que revelou que o extrato metanólico bruto de A. millefolium e extrato de C. villosum têm inibição máxima de Lemna minor com Fl50 6,60 µg/ml e 0,67 µg/ml. A atividade inseticida mostrou que dentre todos os insetos estudados, o extrato metanólico de A. millefolium e de C. villosum foi altamente tóxico para Sphenoptera dadkhanicom DL50 = 4,17 µg/ml e 0,34 µg/ml . Por outro lado, diferentes extratos, como A. millefolium e C. villosum apresentaram boa atividade citotóxica, fitotóxica e inseticida de forma dose-dependente.

Palavras-chave:
citotóxico; fitotóxico; atividade inseticida; Achillea millefolium ; Chaerophyllum villosum

1. Introduction

Traditional medicines are extensively used from many years due to their security. In many developing countries a large number of the population uses medicinal plants for treatment of several ailments (Vendruscolo et al., 2022VENDRUSCOLO, I., VENTURELLA, S.R.T., BRESSIANI, P.A., MARCO, I.G., NOVELLO, C.R., ALMEIDA, I.V., VICENTINI, V.E.P., MELLO, J.C.P., and DÜSMAN, E., 2022. Cytotoxicity of extracts and compounds isolated from Croton echioides in animal tumor cell (HTC). Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 82, p. e264356. https://doi.org/10.1590/1519-6984.264356.
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; Nguta et al., 2011NGUTA, J.M., MBARIAA, J.M., GAKUYA, D.W., GATHUMBIC, P.K. and KIAMA, S.G., 2011. Biological screening of Kenyan medicinal plants using Artemia Salina L. (Artemiidae). Pharmacology, vol. 2, no. 7, pp. 458-478.). Plants contain certain active constituents which are important medicinally. These phytochemical constituents are present in storage organs of the botanicals (Himesh et al., 2011HIMESH, S., SARVESH, S., SHARAN, P.S. and MISHRA, K., 2011. Preliminary phytochemical screening and HPLC analysis of flavonoid from methanolic extract of Leaves of Annona squamosa. International Research Journal of Pharmacy, vol. 5, pp. 242-246.). Different varieties of compounds extracted from plant parts are used as protective agents against viral, fungal, bacterial, and insecticidal diseases in plants. They may also act as scavengers of free radicals, absorbing UV lights, act against proliferative stimuli along with acting as being antioxidant in nature (Lillo et al., 2023LILLO, A., CARVAJAL-CAICONTE, F., VITAL, W., SILVA JUNIOR, P. I. D., PAREDES-GAMERO, E. J., ALVEAR, M., &MIRANDA, A., 2023. Bioactive properties of Persea lingue Ness (Lauraceae) fruit and leaf extracts. Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 83, p. e248063. https://doi.org/10.1590/1519-6984.248063.
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).

In order to detect the antitumor compounds and the toxicity of plants towards cancer cells brine shrimp lethality bioassay is used (Olowa and Nuñeza, 2013OLOWA, L.F. and NUÑEZA, O.M., 2013. Brine shrimp lethality assay of the ethanolic extracts of three selected species of medicinal plants from iligan City, Philippines. International Research Journal of Biological Sciences, vol. 2, no. 4, pp. 74-77.). Phytochemicals are synthesized during secondary metabolic processes possessing immense potential as biological activity enhancers (Alves et al., 2024ALVES, T.A., SPADETO, M.S., VASCONCELOS, L.C., SOUZA, J.R.C.L., MENINI, L., FERREIRA, M.F.S. and PRAÇA-FONTES, M.M., 2024. Phytotoxicity and cytogenetic action mechanism of leaf extracts of Psidium cattleyanum Sabine in plant bioassays. Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 84, p. e260985. http://dx.doi.org/10.1590/1519-6984.260985. PMid:35674589.
http://dx.doi.org/10.1590/1519-6984.2609...
; Oszmiański et al., 2020OSZMIAŃSKI, J., WOJDYŁO, A., JUSZCZYK, P. and NOWICKA, P., 2020. Roots and leaf extracts of Dipsacus fullonum L. and their biological activities. Plants, vol. 9, no. 1, p. 78. http://dx.doi.org/10.3390/plants9010078. PMid:31936189.
http://dx.doi.org/10.3390/plants9010078...
).The toxicity of the plant extracts is assessed by this method. Several advantages of this assay are rapidness, simplicity and low requirements. However, for standardized experimental conditions, several conditions need to be completed, especially (pH of the medium, temperature, aeration and light and salinity) (Hamidi et al., 2014HAMIDI, M.R., JOVANOVA, B. and PANOVSKA, T.K., 2014. Toxicological evaluation of the plant products using Brine Shrimp (Artemia salina L.) model. Macedonian Pharmaeutical Bulletin, vol. 60, no. 3, pp. 9-18. http://dx.doi.org/10.33320/maced.pharm.bull.2014.60.01.002.
http://dx.doi.org/10.33320/maced.pharm.b...
). Certain anti-tumor and cytotoxic agents can be isolated from plants with the help of this bioassay and can be used against abnormal division of cells (Urmi et al., 2013URMI, K.F., MOSTAFA, S., BEGUM, G. and HAMID, K., 2013. Comparative brine shrimp lethalitybioassay ofdifferent plant parts of Bauhinia purpureaL. Journal of Pharmaceutical Sciences and Research, vol. 5, no. 4, pp. 190-192.). Lemna minor bioassay is helpful in investigating new plant growth stimulants (Hussain et al., 2010HUSSAIN, F., AHMAD, B., HAMEED, I., DASTAGIR, G., SANAULLAH, P. and SADIQ, A., 2010. Antibacterial, antifungal and insecticidal activities ofsome selected medicinal plants of polygonaceae. African Journal of Biotechnology, vol. 9, no. 5, pp. 5032-5036.). With the increase in number of pests, management of pests becomes more complex and vice versa (Hyder et al., 2024HYDER, M., LI, Y., WANG, M., MAO, J., MARI, J. M., BUKERO, A., SOOMRO, H.U., BUKERO, A.A., and ZHANG, L., 2024. Insecticidal activity, Chemical Constituents of Trachyspermum ammi, Withania coagulans and Murraya koenigii ethanloic extracts against Bemisia tabaci. Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 84, p. e260298. https://doi.org/10.1590/1519-6984.260298.
https://doi.org/10.1590/1519-6984.260298...
). Weeds have adverse effects on the production of crops. For controlling these weeds different chemical herbicides are used. But these chemical herbicides cause environmental problems and are very expensive to use (Shahnoor et al., 2014SHAHNOOR, D., KHAN, F., AHMED, M., KHAN, R.A., KHAN, M.I. and KHAN, S., 2014. Determination of antifungal and phytotoxic activities of three different fractions of Loniceraquinque locularis(Translucent Honeysuckle) plant. Journal of Pharmacognosy and Phytochemistry, vol. 2, no. 5, pp. 167-172.). Therefore natural herbicides should be investigated which are safe and eco-friendly.

Interest in the use of therapeutic plants as insecticides has increased these days due to the environmental concerns and development of resistance to the synthetic insecticides in insects. These insects are one of the major causes of damage to fruits and vegetables throughout the world (Nazir et al., 2022NAZIR, N., IMRAN, M., BODLAH, I., MAHMOOD, K., KHAN, M. R., OSMAN, K., RASOOL, A., USMAN, M., and DIN, A.U. (2022). Distribution, host range and toxicity assessment of different insecticides on Bactrocera diversa Coquillett, 1904 (Diptera: Tephritidae). Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 84, p. e263261. https://doi.org/10.1590/1519-6984.263261.
https://doi.org/10.1590/1519-6984.263261...
). The search for attaining products from plants which may act as effective against certain plant diseases with less toxicity effect and less adverse effects on environment as well is being stressed upon by researchers (Dilkin et al., 2024DILKIN, E.R.S., MATIAS, R., OLIVEIRA, A.K.M., &CORRÊA, B.O., 2024. Fungitoxic effect and phytochemical characteristics of Brazilian Cerrado weeds against Rhizoctonia solani and Macrophomina phaseolina fungi. Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 84, p. e263114. https://doi.org/10.1590/1519-6984.263114.
https://doi.org/10.1590/1519-6984.263114...
). Plants contain the naturally occurring insecticides. Active constituents of plants are found to be effective against certain insects causing diseases in biological organisms (Santana et al., 2021SANTANA, C.B., SOUZA, J.G.L., TOLEDO, A.G., ALVES, L.F.A., ALVES, D.S., CORRÊA, J.M. and PINTO, F.G.S., 2021. Antimicrobial and insecticidal effects of essential oil and plant extracts of Myrcia oblongata DC in pathogenic bacteria and Alphitobius diaperinus. Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 82, p. e233425.PMid:34105655.). The mono-terpenoids are the bioactive agents present in medicinal plant extracts which cause mortality of insects. Due to their high volatile nature they have insecticidal activity which is useful for controlling stored-product insects (Ahmad et al., 2013AHMAD, F., SAGHEER, M., HAMMAD, A., RAHMAN, S.M. and MASOOR, U.H., 2013. Insecticidal activity of some plant extracts against Trogoderma granarium (E.). The Agriculturists, vol. 11, no. 1, pp. 103-111.).

Tribolium castaneum (red four beetle) is the main pest of flour and certain other products and has severely damaged stored grains like wheat (Suresh et al., 2001SURESH, S., WHITE, N.D.G., JAYAS, D.S. and HULASARE, R.B., 2001. Mortality resulting from interactions between the red flour beetle and the rusty grain beetle. Proceedings of the Entomological Society of Manitoba, vol. 57, pp. 11-18.), affecting the quantity and quality of these food grains (Smith Junior et al., 1971SMITH JUNIOR, L.W., PRATT JUNIOR, J.J., NII, I. and UMINA, A.P., 1971. Baking and taste properties of bread made from hard wheat flour infested with species of Tribolium, Tenebrio, Trogoderma and Oryzaphilus. Journal of Stored Products Research, vol. 6, no. 7, pp. 307-316. http://dx.doi.org/10.1016/0022-474X(71)90043-9.
http://dx.doi.org/10.1016/0022-474X(71)9...
). Trogoderma granarium (Khapra beetle) has caused major loss of stored grains in certain regions of Pakistan and is also one of the harmful insect pests (Bell and Wilson, 1995BELL, C.H. and WILSON, S.M., 1995. Phosphine tolerance and resistance in TrogodermagranariumEverts (Coleoptera:Dermestidae). Journal of Stored Products Research, vol. 3, pp. 199-205.). Due to stored grains insect pests about 2- 6% food grains of Pakistan are lost each year during storage (Avesi, 1983AVESI, G.M., 1983. Post harvest losses in rice. Programm Farm, vol. 3, pp. 11-12.). Sphenoptera dadkhani (Peach flat-headed borer) caused severe damage to the plum and peach orchards in Pakistan (Zahid, 2014ZAHID, M., 2014. Integrated management of peach flat-headed borer, Sphenoptera dadkhani (Oben.) damaging trees of stone fruit orchards. Peshawar: First Ann. Tech. Rep. Pak. Sci. Foundation Project, vol. 171, pp. 01-58. Final Tech. Report, PSF Project No. PSF/NSLP/KP-NIFA.). In Pakistan, yield of cotton crop decreases to about 28.13% due to pests (Javed et al., 2021JAVED, Z., ASIM, M.S., ISHAQ, A.R., YOUNIS, T., MANZOOR, M., HUSSAIN, D., and ASRAR, M., 2021. Monitoring of Insecticides resistance against cotton Jassid (Amrasca biguttutla) under laboratory conditions. Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 83, p. e247676. https://doi.org/10.1590/1519-6984.247676.
https://doi.org/10.1590/1519-6984.247676...
). Potential negative effect of pesticides on pollinators has been a cause of concern too (El Helaly et al., 2021EL-HELALY, A.A., EL-MASARAWY, M.S. and EL-BENDARY, H.M., 2021. Using citronella to protect bees (honeybee Apis mellifera L.) from certain insecticides and their nano formulations. Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 81, no. 4, pp. 899-908. http://dx.doi.org/10.1590/1519-6984.230140. PMid:33053125.
http://dx.doi.org/10.1590/1519-6984.2301...
)

Achillea millefolium L. (Asteraceae) is known as Baranjasif, and its flowers and leaves are used medicinally (Ahmed, 2015AHMED, M., 2015. Some medicinal plant resources and traditional uses in Pakistan. Journal of Plant Breeding and Crop Science, vol. 7, no. 5, pp. 158-162.). Plant has been used as antioxidant, antimicrobial, analgesic, anti-inflammatory, anti-hypersensitive, anti-diabetic, anticancer and anti-diarrheal (Presena, 2016PRESENA, J., 2016.An investigation on the pharmacognosy, phytochemistry and pharmacology of Adenia wightiana (Wall. ex Wight & Arn.) Engl. (Passifloraceae). Kalapet: Pondicherry University, Kanchi Mamunivar Centre for Postgraduate Studies, Department of Botany. Doctoral dissertation.). It is useful in hepatitis, jaundice and is a hepato-protective herb. It occurs in Azad Kashmir, Swat, Hazara and Kaghan (Fazal et al., 2013FAZAL, H., AHMAD, N. and HAIDER ABBASI, B., 2013. Identification, characterization, and palynology of high-valued medicinal plants. TheScientificWorldJournal, vol. 2013, p. 283484. http://dx.doi.org/10.1155/2013/283484. PMid:23844389.
http://dx.doi.org/10.1155/2013/283484...
).

Chaerophyllum villosum L. (Apiaceae) is known as Jangali Gajar (Mehta and Bhatt, 2007MEHTA, P.S. and BHATT, K.C., 2007. Traditional soap and detergent yielding plants of Uttaranchal. Indian Journal of Traditional Knowledge, vol. 6, no. 6, pp. 279-284.). The leaves and seeds are used for the treatment of stomach pain, cough and cold (Aziz et al., 2015AZIZ, U.I., ZAHRA, N.B., SHINWARI, Z.K. and QAISER, M., 2015. Ethno-medicinal review of folklore medicinal plants belonging to family Apiaceae of Pakistan. Pakistan Journal of Botany, vol. 47, no. 3, pp. 1007-1014.). It occurs at an altitude of (5000-6000 ft) (Khan et al., 2014KHAN, M.A., KHAN, M.A., HUSSAIN, M. and MUJTABA, G., 2014. Plant diversity and conservation status of Himalayan Region Poonch Valley Azad Kashmir (Pakistan). Pakistan Journal of Pharmaceutical Sciences, vol. 27, no. 5, pp. 1215-1239. PMid:25176378.). It grows in moist and cold environment on the road sides or open areas at height of (2100-3500 m) and is extensively distributed in East Asia Himalayas including India to Bhutan, Nepal and China (Joshi and Mathela, 2013JOSHI, R.K. and MATHELA, C.S., 2013. Volatile oil composition and antioxidant activity of leaf of Chaerophyllum villosum Wall. ex DC from Uttrakh. Recent Research in Science and Technology, vol. 5, no. 1, pp. 25-28.). According to Flora of Pakistan it is found in the hills from (2500-4000m). The present study investigates the cytotoxic, phytotoxic and insecticidal activities of Achellia millefolium and Chaerophyllum villosum.

2. Materials and Methods

2.1. Plant materials

Both the plants Achillea millefolium and Chaerophyllum villosum were collected from Merajani top, Abbottabad District, Khyber Pakhtunkhwa, Pakistan at an altitude of (2,992 m) during August-September 2013.They were recognized by a taxonomist named Prof. Dr. Abdur Rashid at Botany Department, University of Peshawar, Pakistan. Voucher specimen numbers i.e. M. AdilBot.2244 (PUP) and M. Adil Bot. 2245 (PUP) were given and specimens were deposited in Herbarium, Botany Department, University of Peshawar. At room temperature plants were dried and ground with a mechanical grinder. The powdered plant materials (500g) were soaked in (1,000 ml), (97% methanol and chloroform) for two weeks. Both extracts were passed through (Whatman filter paper No.1823). The resulting methanolic and chloroformic extracts were subjected to rotary evaporator at 40ºC to get concentrated crude extracts. The aqueous extract was prepared by soaking 20 g powdered plant material in 80ml distilled water for 48 hrs. It was filtered to get the filtrate (Dastagir and Hussain, 2010DASTAGIR, G. and HUSSAIN, F., 2010. Phytotoxic and insecticidal activity of plants of family Zygophyllaceae and Euphorbiaceae. Sarhad Journal of Agriculture, vol. 29, no. 1, pp. 83-91.).

2.2. Cytotoxic activity

The cytotoxic activity was done using the method of (Meyer et al., 1982MEYER, B., FERRIGNI, N., PUTNAM, J., JACOBSEN, L., NICHOLS, D. and MCLAUGHLIN, J., 1982. Brine shrimp, a convenient general bioassay for active plant constituents. Planta Medica, vol. 45, no. 05, pp. 31-34. http://dx.doi.org/10.1055/s-2007-971236.
http://dx.doi.org/10.1055/s-2007-971236...
). The stock solution was prepared when the methanolic, chloroformic and aqueous extracts of plant (10 mg) were dissolved in 1ml of dimethyl sulphoxide (DMSO)and then three concentrations i.e., 10µl, 100µl, 1,000µl were taken and were shifted to sterilized vials from this stock solution. At 85 ºC the vials were sterilized for 2 days. There were three replicates for each concentration. At low temperatures (4°C) the brine shrimp eggs were stored to maintain sustainability. The brine shrimp eggs were hatched in a tray (22x32 cm). It was half-filled with filtered brine solution (sea salt solution) and 50 mg eggs of brine shrimp was sprinkled and was subjected to incubation at 37°C.After 2 days the brine shrimp eggs hatched and 10 larvae/vial was placed. The volume was made to 10 ml with seawater and it was incubated at 25-27°C for 24 hours under illumination. In the other vials solvent was added, which served as negative controls. The (Etoposide) was used as standard drug and as a positive control.

2.3. Phytotoxic activity

Phytotoxic potential of the plant extracts was investigated against the Lemna minor by following McLaughlin (1991)MCLAUGHLIN, J.L., 1991. Crown-gall tumours in potato discs and brine shrimp lethality: two simple bioassays for higher plant screening and fractionation. In K. HOSTETT-MANN, ed. Methods in plant biochemistry. London: Academic Press, pp. 1-31.. In (1,000 ml) distilled water several constituents were dissolved and the pH (5.5-6.0) was adjusted through addition of KOH pellets. The stock solution was prepared by dissolving extracts (30mg) in methanol and chloroform (1.0 ml). The three concentration 10 μg/ml, 100 μg/ml and 1,000 μg/ml, were taken from stock solution/standard solution and were shifted to Petri dishes (for each concentration 3 replicates were used). The petri dishes were left for sometime so that the solvents evaporate from Petri dishes. Then petri dishes were filled up with 20 ml E-medium and in each petri dish lemna minor plants with three fronds were added. The E-medium was added to other Petri dish for control. The initial reading of the Lemna minor bioassay was taken and then it was kept for a week. After a week the numbers of fronds in all petri dishes were counted and the data were arranged and were analyzed statistically (Steel et al.,1997STEEL, R.G.D., TORRIE, J.H. and DICKEY, D.A., 1997. Principles and procedures of statistics. A biometrical approach. 3rd ed. New York: McGraw Hill Book Co. Inc., pp. 172-177.). The following formula was used to calculate the percent inhibition of the Lemna minor (Equation 1).

% i n h i b i t i o n = 100 Number of fronds in tests Number of fronds in negative control × 100 (1)

Insecticidal activity: The insecticidal activity was carried out as described by Bashir et al. (2010)BASHIR, A., SADIA, S., SADIQ, A., SHUMAILA, B. and IBRAR, K., 2010. Biological screening of aerial parts of Sarcococcasaligna. Journal of Medicinal Plants Research, vol. 4, no. 22, pp. 2404-2410.. The stock solution was prepared by mixing 20mg of the plant sample in 3ml of methanol and chloroform. In order to make the aqueous extract 20g of powdered plant was soaked in 80 ml of distilled water for 48 hrs. After 48 hrs it was filtered by means of standard filter paper. In controlled conditions of humidity and temperature (25-27ºC) the test insects such as Tribolium castanium, Trochoderma granarium and Sphenoptera dadkhani were raised in the plastic bottles. For insecticidal activity insects of same size and age were used. The insecticidal activity was done following (Bashir et al., 2010BASHIR, A., SADIA, S., SADIQ, A., SHUMAILA, B. and IBRAR, K., 2010. Biological screening of aerial parts of Sarcococcasaligna. Journal of Medicinal Plants Research, vol. 4, no. 22, pp. 2404-2410.). On first day the petri dishes (90mm) were sterilized at 104 ºC for 4 hours and filter papers were shaped as per the dimensions of Petri plates. After that filter papers were placed in petri plates and then stock solutions of test samples were poured into it with micropipettes. The Petri dishes were left overnight for the evaporation of methanol and chloroform. On the second day ten small and equal sized healthy insects of each species were selected and were shifted to the labeled Petri dishes. In an incubator at 27°C in growth chamber the Petri dishes were placed for 24hrs with relative humidity of 50%. After 24 hrs incubation results were noted by calculating the number of survived insects in each Petri dish. Mortality percentage was calculated following the formula as follows (Equation 2).

% m o r t a l i t y = 100 Number of insects in tests Number of insects in negative control × 100 (2)

2.4. Phytochemical screening

The phytochemical tests of methanolic, chloroformic and aqueous extracts of Achillea millefolium and Chaerophyllum villosum were done to find out tannins, glycosides, saponins, triterpenoids, phytosterols, phenols,, alkaloids, steroids, flavonoids and oils following the standard methods of Sofowora (1993)SOFOWORA, A., 1993. Medicinal plants and traditional medicines in Africa. New York: John Wiley & Sons., Trease and Evans (1989)TREASE, G.E. and EVANS, W.C., 1989. Pharmacognosy. 13th ed. London: J.J. and A. Churchill Ltd., Iyenger (1995)IYENGER, M.A., 1995. Study of crude drugs. 8th ed. Manipal: Manipal Power Press., Kokate (2010)KOKATE, C.K., 2010. Plant constituents practical pharmacognosy. 14th ed. Delhi: Vallabh Prakashan., Edeoga et al. (2005)EDEOGA, H.O., OKWU, D.E. and MBAEBIE, B.O., 2005. Phytochemical constituents of some Nigerian medicinal plants. African Journal of Biotechnology, vol. 4, no. 7, pp. 685-688. http://dx.doi.org/10.5897/AJB2005.000-3127.
http://dx.doi.org/10.5897/AJB2005.000-31...
.

3. Results and Discussion

3.1. Cytotoxic activity

The results revealed that different extracts from both plants showed dose dependent toxicity to brine shrimps. Methanolic extract of Achellia millefolium was most toxic to brine shrimps (73.4%) with LD50 value of 52.60 followed by chloroform extract (66.7%) at higher doses (Table 1). The result agrees with Naeem Qaisar et al. (2013)NAEEM QAISAR, M., CHAUDARY, B.A., UZAIR, M. and HUSSAIN, S.N., 2013. Evaluation of antioxidant and cytotoxic capacity of Croton bonplandianum. Baill. American Journal of Plant Sciences, vol. 4, no. 3, pp. 1709-1712. http://dx.doi.org/10.4236/ajps.2013.49208.
http://dx.doi.org/10.4236/ajps.2013.4920...
who also reported lower LD50 value for methanolic extract of Croton bonplandianum. Similarly, chloroform extract of Chaerophyllum villosum measured maximum toxicity to brine shrimps (96.67%) with LD50 value of 14.81 followed by aqueous extract (83.4%) at higher doses (Table 1). These results are in accordance with Misonge et al. (2015)MISONGE, J.O., KINYANJUI, J.G., KINGORI, W.M. and MWALUKUMBI, J.M., 2015. Phytochemical screening and cytotoxicity evaluation of LaunaeacornutaH. (Asteraceae) using brine shrimp. Merit Research Journal of Medicineand Medical Sciences, vol. 3, no. 6, pp. 116-120. who also revealed highest toxicity of brine shrimps to the chloroformic extract of Launaea cornuta. This indicates that the toxicological activity shown by A. millefolium and C. villosum was due to the presence of cytotoxic agents. Earlier studies showed that saponins, alkaloids, tannins and flavonoids are some of the cytotoxic agents present in different plants (Huang et al., 2012HUANG, W.Y., ZHANG, H.C., LIU, W.X. and LI, C.Y., 2012. Survey of antioxidant capacity and phenolic composition of blueberry, blackberry, and strawberry in Nanjing. Journal of Zhejiang University. Science. B, vol. 13, no. 2, pp. 94-102. http://dx.doi.org/10.1631/jzus.B1100137. PMid:22302422.
http://dx.doi.org/10.1631/jzus.B1100137...
; Mungenge et al., 2014MUNGENGE, C., ZIMUDZI, C., ZIMBA, M. and NHIWATIWA, T., 2014. Phytochemical screening, cytotoxicity and insecticidal activity of the fish poison plant Synaptolepis alternifolia Olive. (Thymelaeaceae). Journal of Pharmacognosy and Phytochemistry, vol. 2, no. 5, pp. 15-19.).

Table 1
Cytotoxic activity of Achellia millefolium and Chaerophyllum villosum Wall. ex DC.

3.2. Phytotoxic activity

The results revealed that methanolic extract of Achellia millefolium had showed profound growth inhibition (90.0%) of growth of Lemna minor with FI50 value of 6.60 μg/ml followed by chloroform (73.4%) and aqueous extract (63.4%) at higher doses (Table 2). The data also showed that methanolic extracts of Chaerophyllum villosum reduced the growth of Lemna minor by 76.7% with FI50 value of 0.67 μg/ml followed by chloroform (73.4%) and aqueous extract (70.0%).The methanolic extracts of A. millefolium and C. villosum caused greater growth inhibition of Lemna minor as compared to the chloroform and aqueous extract. The results are strengthened by the findings of Ghaffari et al. (2013)GHAFFARI, M., BANO, A. and HAYAT, K., 2013. Antimicrobial and phytotoxic effects of the plant Heliotropiumdasycarpum. International Journal of Pharma and Bio Sciences, vol. 4, no. 4, pp. 339-345. who also revealed maximum toxicity of Lemna minor plants due to methanolic extract of Heliotropium dasycarpum. Hameed et al. (2013)HAMEED, I., HUSSAIN, F., SHER, Z., BACHA, N. and KHAN, A.A., 2013. Cytotoxicity and phytotoxicity of some selected medicinal plants of family Solanaceae. Pakistan Journal of Botany, vol. 45, pp. 1749-1754. reported high toxicity of Lemna minor to methanolic extract of Datura innoxia. Different extracts of both the plants exhibited that % inhibition of the fronds of Lemna minor was dose dependent. Romero-Romero et al. (2002)ROMERO-ROMERO, T., ANAYA, A.L. and CRUZ-ORTEGA, R., 2002. Screening for effects of phytochemical variability on cytoplasmic protein synthesis pattern of crop plants. Journal of Chemical Ecology, vol. 28, no. 3, pp. 617-629. http://dx.doi.org/10.1023/A:1014504531418. PMid:11944837.
http://dx.doi.org/10.1023/A:101450453141...
reported that the phyto-toxins hamper the enzymatic activity, permeability of membrane, respiratory chains, division of cell and electron transport chain in photosynthesis. The herbicidal potential of both the plants might be due to the presence of phytotoxins.

Table 2
Phytotoxic activity of Achellia millefolium and Chaerophyllum villosum Wall. ex DC.

3.3. Insecticidal activity

Results regarding insecticidal activity of different extracts of Achellia millefolium and Chaerophyllum villosum are shown in Table 3. The results indicated that chloroform extract of Achellia millefolium was most toxic (66.7%) to Triboliumcastaneum with LD50 value of 31.41 followed by methanolic extract (56.7%) at higher doses. Minimum toxicity (46.7%) was exhibited by aqueous extract at higher doses (Table 3). The methanolic extract of Achellia millefolium was most toxic (60.0%) to Trochoderma granarium with LD50 value of 242.22 followed by chloroform extract (50.0%) at higher doses. The low (40.0%) toxicity was measured by aqueous extract at higher doses. The study further revealed that methanolic extract of Achillea millefolium showed greater toxicity (90.0%) to Sphenoptera dadkhani (LD50 value of 4.17) followed by chloroform extract (80.0%) at high doses. Aqueous extracts of the same plant noted low (66.7%) toxicity to Sphenoptera dadkhani at high doses (Table 3). The methanolic extract of Chaerophyllum villosum was most active (93.4%) against Tribolium castaneum with LD50 value of 8.90 followed by chloroform extract (90.0%) at high doses. The low (46.7%) mortality was exhibited by aqueous extract at high doses (Table 4). Methanolic extract of Chaerophyllum villosum was most toxic (83.4%) to Trochoderma granarium (LD50 value of 0.76) followed by chloroform extract (60.0%). The low (53.4%) toxicity was shown by aqueous extract at high doses (Table 4). Methanolic extract of Chaerophyllum villosum showed greater toxicity (83.4%) to Sphenoptera dadkhani with LD50 value of 0.34 followed by chloroform extract (80.0%) at high doses (Table 4).The methanolic extracts of A. millefolium and C. villosum showed maximum toxicity to Sphenopteradadkhani. In the present study the LD50 values for Tribolium castaneum and Trochoderma granarium were high and hence these insects showed resistance to the A. millefolium extracts. These results are supported by Hussain et al. (2010)HUSSAIN, F., AHMAD, B., HAMEED, I., DASTAGIR, G., SANAULLAH, P. and SADIQ, A., 2010. Antibacterial, antifungal and insecticidal activities ofsome selected medicinal plants of polygonaceae. African Journal of Biotechnology, vol. 9, no. 5, pp. 5032-5036. who reported that T. castaneumand T. granarium were resistant to methanolic extract of Rumex hastatus. The growth inhibition of Lemna minor might be due to the occurrence of triterpenoids, glycosides, amino acids and saponins in these plants (Table 5). Similar results are also reported by Fazal et al. (2013)FAZAL, H., AHMAD, N. and HAIDER ABBASI, B., 2013. Identification, characterization, and palynology of high-valued medicinal plants. TheScientificWorldJournal, vol. 2013, p. 283484. http://dx.doi.org/10.1155/2013/283484. PMid:23844389.
http://dx.doi.org/10.1155/2013/283484...
and Saleem et al. (2014)SALEEM, S., MANSOOR, U.H., SAGHEER, M. and SAHI, S.T., 2014. Insecticidal activity of essential oils of four medicinal plants against different stored grains insect pests. Pakistan Journal of Zoology, vol. 46, no. 2, pp. 1407-1414.. Mostafa et al. (2012)MOSTAFA, M., HOSSAIN, H., HOSSAIN, M.A., BISWAS, P.K. and HAQUE, M.Z., 2012. Insecticidal activity of plant extracts against Tribolium castaneum Herbs. Journal of Advance Scientific Research, vol. 3, no. 7, pp. 80-84., Gonzalez et al. (2013)GONZÁLEZ, A., FERREIRA, F., VAZQUEZ, A., MOYNA, P. and PAZ, E.A., 2013. Biological screening of Uruguayan Medicinal Plants. Journal of Ethnopharmacology, vol. 39, no. 3, pp. 217-220. http://dx.doi.org/10.1016/0378-8741(93)90040-C. PMid:8258980.
http://dx.doi.org/10.1016/0378-8741(93)9...
and Jide-Ojo et al. (2013)JIDE-OJO, C.C., GUNGULA, D.T., and OJO, O.O., 2013. Extracts of Jatropha curcas L. exhibit significant insecticidal and grain protectant effects against maize weevil, Sitophilus zeamais (Coleoptera: curculionidae). Journal of Stored Products and Postharvest Research, vol. 4, no. 3, pp. 44-50. http://dx.doi.org/10.5897/JSPPR2013.0147.
http://dx.doi.org/10.5897/JSPPR2013.0147...
reported that steroids, phenolics and flavonoids have shown toxicity against pathogens, pests and tested insects. Similar compounds were also present in the tested plants (Table 5). These results showed that different extracts from A. millefolium and C. villosum are potential sources of insecticides against the three tested insects. This also indicated that toxic phytochemicals are methanolic soluble that might be responsible for insecticidal activity.

Table 3
Insecticidal activity of Achillea millefolium L.
Table 4
Insecticidal activity of Chaerophyllum villosum Wall. ex DC.
Table 5
Qualitative phytochemical screening of Achillea millefolium L. and Chaerophyllum villosumWall. ex DC.

4. Conclusion

The results concluded that different extracts from A. millefolium and C. villosum showed good cytotoxic, phytotoxic and insecticidal activity in a dose dependent manner. The cytotoxicity exhibited by the present plants clearly indicates the presence of potent bioactive compounds and they might be helpful in future for the treatment of cancer. The phytotoxic effect as shown by both the plants revealed that these plants are rich sources of phytotoxic compounds and might serve as a good source of natural herbicide for the control and management of weeds in agriculture in order to improve crops yield. The insecticidal activity of both plants indicated that plant extracts which are eco and user friendly, play vital role in protection of storage commodities. Therefore, these extracts may be potential candidates for their use in the formulation of commercial repellents and insecticides that serve as effective control option, in the management of stored product insects responsible for huge loss of food commodities during storage.

Acknowledgements

The research was supported by The Deanship of Scientific Research (DSR) at King Abdul Aziz University, Jeddah, Grant No. [IFPRC-187-130-2020]. The authors are highly grateful for this support.

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Publication Dates

  • Publication in this collection
    20 Jan 2023
  • Date of issue
    2024

History

  • Received
    27 Mar 2022
  • Accepted
    09 Nov 2022
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