Phytochemical Analysis and Nematicidal Activity of Ethanolic Leaf Extracts of Datura metel, Datura innoxia and Brugmansia suaveolens Against Meloidogyne incognita

The present study was aimed to screen the phytochemicals and quantification of alkaloids, phenolic compounds and flavonoids and to evaluate the nematicidal activity of ethanolic leaf extracts of Datura metel, Datura innoxia and Brugmansia suaveolens against Meloidogyne incognita. Phytochemical screening of leaf extracts of D. metel, D. innoxia and B. suaveolens was carried out by qualitative analysis and the results revealed that the presence of bioactive compounds like alkaloids, steroids, flavonoids, terpenoids, phenolic compounds, tannins, anthroquinone glycosides, Original Research Article Nandakumar et al.; AJOB, 2(4): 1-11, 2017; Article no.AJOB.34241 2 saponins and triterpenes. The quantitative phytochemical analysis showed that the maximum amount of alkaloids 12.12 mg/g in leaf of D. innoxia, phenolic compounds 5.675 mg/g in leaf of D. metel and flavonoids 4.945 mg/g in leaf of B. suaveolens. The nematicidal activity of extracts of D. metel, D. innoxia and B. suaveolens were studied against the root knot nematode M. incognita. For nematicidal activity, the leaf extracts were used at different concentrations like 250 ppm, 500 ppm, 750 ppm and 1000 ppm in different exposure time like 24 hrs, 48 hrs and 72 hrs. The in vitro nematicidal activity results suggested that the extracts of D. metel, D. innoxia and B. suaveolens were possessed nematicidal activity. From this study, we concluded that the extracts of D. metel, D. innoxia and B. suaveolens contain phytochemicals and the plants were exhibited nematicidal activity against root knot nematode M. incognita. The present study may be useful for the identification and isolation of novel active nematicidal compounds from the leaf of D. metel, D. innoxia and B.


INTRODUCTION
Plant-derived substances have recently become a great interest owing to their versatile applications. Medicinal plants are the local heritage with global importance and the world is endowed with a rich wealth of medicinal plants [1]. Secondary metabolites are the substances produced by plants as defense chemicals. They include alkaloids, flavonoids, essential oils, phenols, saponins etc. Recently, many pharmaceutical companies have spent a lot of time and money in developing natural products extracted from plants, but the information available is quite meagre [2]. Plant pathogens consist of viruses, bacteria, parasitic green algae, protozoans, fungi and nematodes [3]. Plants may represent a source of natural pesticides [4]. Phytochemicals that extracted from different parts of plant such as alkaloids, glycosides, limonoids, quassinoids and phenolics become new and promising tools as fungicides, bactericides and nematicides [5]. Phytonematodes are root feeders, which completing their life cycles in or on the root zone or even shoot zone in some cases. The most wide spread plant nematodes that parasitize crops are the root-knot nematodes which consist of more than 150 species. Among the root-knot nematodes, M. javanica, M. incognita, M. arenaria and M. hapla are of major agronomic importance, being responsible for 95% of infestations [6] and responsible for at least 90% of all damage caused by nematodes [7]. Plant parasitic nematodes, especially root-knot nematodes (Meloidogyne species), are among the most important soil borne pathogens of economically important agricultural crops, affecting both the quantity and quality of marketable yields [8,9].
Plant parasitic nematodes cause diseases to nearly all crop of economic importance with estimated losses of US$ 125 billion per year worldwide [10]. Meloidogyne species root knot nematode is one of the most harmful nematode pests in both tropical and subtropical crop production regions and cause extensive economic damage worldwide [11]. The presence of root-knot nematodes causes the deformation of root system when galls are formed; nutrient and water uptake by plant roots are then affected [12]. M. incognita has been reported to constitute about 47% of the total root-knot nematodes population [13]. There is increasing interest in the development and adoption of environmental friendly tactics for managing nematodes, particularly as fumigants and other chemical nematicides become more limited [14]. Organic amendments offer an alternative or supplementing control tactic to chemical or cultural control of plant-parasitic nematodes, but confirmation of their potential effect requires more detailed studies [15].
Biological control of root knot nematode is a promising method of management, especially by the bacterial endospore-forming parasite Pasteuria penetrans [16]. The applications of natural products are of priority to control the rootknot nematodes infecting vegetable crops. Several workers have reported different plant parts for their nematicidal properties against plant parasitic nematodes which were used as soil amendments or as extracts [17,18]. The nematicidal properties of natural products have been identified by testing the effect of plant parts and extracts (leaves, stem, fruits and seeds), oil extracts, plant exudates and plant volatiles on nematodes that infect plants [19,20]. Various nematicidal compounds of plant origin, including triglycerides, sesquiterpenes, alkaloids, steroids, tannins, diterpenes, flavonoids, phenolics and many essential oils, have been identified in this way [21]. Numerous plant species, representing 57 families, have been found to contain biologically active phytochemicals which are able to control plant diseases [22]. Alcoholic and aqueous extracts from as many 39 plant species have been shown to possess nematicidal activity against M. incognita. Additionally, many of these plant extracts have been reported to possess broad-spectrum activity against diverse insect pests and pathogens [23]. The effective nematicidal activity was reported in extract of Gliricidia sepium [24] and Azadirachta indica (Neem), Withania somnifera (Ashwagandha), Tagetes erecta (Marigold) and Eucalyptus citriodora (Eucalyptus) against nematodes (M. incognita) associated with papaya (Carica papaya) [25].
D. metel, D. innoxia and B. suaveolens belongs to the family Solanaceae are distributed worldwide. All species of Datura and Brugmansia contain certain biologically active tropane alkaloids. The main alkaloids present in the plants are scopolamine and hyoscyamine. The effects of these alkaloids include stimulation of the central nervous system and simultaneous depression of the peripheral nerves typical for a parasympathomimetic. The medicinal qualities of alkaloids include spasmolytic, antiasthmatic, anticholinergic, narcotic and anesthetic properties [26]. There is no study on Datura and Brugmansia species in controlling nematodes of crop plants. The use of synthetic nematicidal chemicals for the management of nematodes is an expensive and highly toxic. For the above mentioned reasons, there is a need to search of cheaper and less toxic alternative control measure on nematodes in crop plants. So, the present study was aimed to analyse the phytochemicals and to evaluate the nematicidal activity of extracts of D. metel, D. innoxia and B. suaveolens against root knot nematode M. incognita.

Preparation of Ethanolic Extract for Phytochemical Screening
Fifty gram of leaf powder of D. metel, D. innoxia and B. suaveolens were soaked in 500 mL of ethanol separately and then kept in orbital shaker for 48 hrs at room temperature. After 48 hrs, the mixture was filtered through a clean muslin cloth and the filtrate again filtered by using Whatmann No.1 filter paper. Then the extracts were concentrated and dried in a rotary evaporator at 37°C [27] till a sticky mass was obtained. After evaporation, the dried extracts were stored at 4°C until further use.

Preliminary Phytochemical Screening
Phytochemical tests for the screening of bioactive constituents in extracts of D. metel, D. innoxia and B. suaveolens were carried out by using the standard procedures [28][29][30].

Test for alkaloids
A few mL of the extract was taken and a drop of Mayer's reagent was added by the side of the test tube. A creamy or white precipitate formed indicates the presence of alkaloids.

Test for steroids
Two mL of acetic anhydride was added to 0.5 gram of extract with two mL of H 2 SO 4 . The colour changed from violet to blue or green, which indicates the presence of steroids.

Test for flavonoids
A few drops of 1% aluminium chloride solution was added to a portion of plant extract. A yellow colouration was observed, which indicates the presence of flavonoids.

Test for terpenoids
Five mL of plant extract was mixed with two mL of chloroform and three mL of concentrated H 2 SO 4 was carefully added to form a layer. A reddish brown colouration in the interface was formed, which indicates the presence of terpenoids.

Test for phenolic compounds
Fifty milligram of extract was dissolved in five mL of distilled water and then added three mL of 10% lead acetate solution and formed bulky white precipitate, which indicates the presence of phenolic compounds.

Test for tannins
About ten mL of plant extract was taken in a test tube and a few drops of 0.1% ferric chloride was added and observed the brownish green or a blue-black colouration, which indicates the presence of tannins.

Test for cardiac glycosides
Five hundred milligram of extract was diluted to five mL with distilled water and to this two mL of glacial acetic acid and one drop of ferric chloride were added. Then one mL of conc.H 2 SO 4 was added. A brown ring at the interface indicates the presence of deoxysugar characteristic of cardenolides. A violet ring may appear below the ring while in the acetic acid layer, a greenish ring may form, which indicates the presence of cardiac glycosides.

Test for anthroquinone glycosides
Two hundred milligram of extract was dissolved in diluted H 2 SO 4 and boiled. Then it was filtered and cooled. To the filtrate, three mL of benzene or chloroform or ether was added and mixed well. The chloroform layer was separated and two mL of ammonia solution was added and ammonia layer was allowed to form of rose pink (or) cherry red color development, which indicates the presence of anthroquinone glycosides

Test for saponins
Ten mL of plant extract was taken in a test tube and then mixed with five mL of distilled water and shaken vigorously for a stable persistent froth.
The frothing was mixed with 3 drops of olive oil and again shaken vigorously, then observed the formation of emulsion, which indicates the presence of saponins.

Test for triterpenes
Ten milligram of the extract was dissolved in one mL of chloroform and then one mL of acetic anhydride was added and followed the addition of two mL of concentrated H 2 SO 4 . Formation of reddish violet colour indicates the presence of triterpenes.

Quantitative Phytochemical Content Analysis
The quantitative determination of phytochemicals such as alkaloids, total phenolic compounds and flavonoids in the extracts of D. metel, D. innoxia and B. suaveolens were analysed by standard methods.

Determination of alkaloids
Five gram of the sample was weighed and transferred to 250 mL beaker and 10% acetic acid in ethanol was added and covered and then allowed to stand for 4 hours. This was filtered and the extract was concentrated on a water bath to one quarter of the original volume. Concentrated ammonium hydroxide was added drop wise to the extract until precipitation was complete. The whole solution was allowed to settling. Then the precipitate was collected and washed with dilute ammonium hydroxide and filtered. The residue is the alkaloid, which was dried and weighed. All determinations were carried out in triplicates [30,31].

Determination of phenolic compounds
Hundred milligram of extract was weighed accurately and dissolved in 100 mL of triple distilled water (TDW). One mL of this solution was transferred to a test tube, then 0.5 mL of the Folin-Ciocalteu reagent and 1.5 mL of 20% Na 2 CO 3 solution were added and ultimately the volume was made up to eight mL with TDW and shaking the content vigorously and finally allowed to stand for 2 hours after which the absorbance was measured at 765 nm. These data were used to estimate the total phenolic content using a standard calibration curve obtained from various diluted concentrations of gallic acid. All determinations were carried out in triplicates [32].

Determination of flavonoids
The method of determination of flavonoids is based on the formation of the flavonoidsaluminium complex which has an absorptive maximum at 415 nm. 100 µL of the plant extract in methanol (10 mg/mL) was mixed with 100 µL of 20% aluminum trichloride in methanol and a drop of acetic acid and then diluted to five mL with methanol. The absorption at 415 nm was read after 40 minutes. Blank samples were prepared from 100 µl of plant extract and a drop of acetic acid, and then diluted to five mL with methanol. The absorption of standard rutin solution (0.5 mg/mL) in methanol was used as above condition. All determinations were carried out in triplicates [33].

Preparation of Ethanolic Extract for Nematicidal Activity
One kilogram leaf powder of D. metel, D. innoxia and B. suaveolens were taken separately and soaked in 3 liters of ethanol for 15 days at room temperature in the orbital shaker. The ethanolic extracts were filtered and then concentrated under low pressure at 40°C using a rotary evaporator and the final yield of extracts were stored at 4°C prior to use.

Statistical Analysis
The results of this study were subjected to statistical analysis and the results were expressed as mean percentage ± standard deviation of three replicates. Mortality percent value of nematicidal activity was calculated and significance was determined by using Duncan's Multiple Range Test (DMRT) at 5% level (P = .05).  Table 1. The secondary metabolites alkaloids, steroids, flavonoids, terpenoids, phenolic compounds, tannins, anthroquinone glycosides, saponins and triterpenes were present and cardiac glycosides were absent in extracts of D. metel, D. innoxia and B. suaveolens. Phytochemical screening helps to identify the chemical constituents of the plant extracts and also used to search for bioactive agents for starting products used in the synthesis of some useful drugs [36]. Similarly, the phytochemical analysis and antibacterial activity of leaf of Vitex negundo and D. metel against bacterial pathogens were reported [37,38].

RESULTS AND DISCUSSION
The extracts of D. metel, D. innoxia and B. suaveolens were subjected to quantitative analysis of alkaloids, phenolic compounds and flavonoids and the results were presented in Table 2. The extracts of D. metel, D. innoxia and B. suaveolens showed different amount of phytocompounds. Among the quantification of secondary metabolites, the alkaloids content was maximum in all three tested extracts when compared with other compounds like phenolic compounds and flavonoids. The extract of D. innoxia contain alkaloids 12.12 ± 0.0194 mg/g followed by D. metel 7.314 ± 0.0164 mg/g and B. suaveolens 5.903 ± 0.0133 mg/g. Phenolic compounds was observed maximum in D. metel 5.675 ± 0.0111 mg/g when compared with D. innoxia 3.625 ± 0.0147 mg/g and B. suaveolens 3.435 ± 0.0110 mg/g. The flavonoids content in extract of B. suaveolens 4.945 ± 0.0256 mg/g, D. innoxia 4.118 ± 0.0167 mg/g and D. metel 3.701 ± 0.0249 mg/g were observed. Similarly, the screening and quantification of phytocompounds in Dendrobium ovatum [39] and the alkaloids, phenolic compounds and flavonoids content quantified in many plant species such as Pteris argyreae, Pteris confusa, Pteris vittata, Pteris biaurita and Pteris multiaurita [40] and the total phenolic content of Indigofera aspalathoides [41] were reported. In the present study, the content of alkaloids, phenolic compounds and flavonoids in leaf of D. metel, D. innoxia and B. suaveolens was observed in lesser amount than other plants like D. ovatum, P. argyreae, P. confusa, P. vittata, P. biauria, P. multiauria and I. aspalathoides. Nematicidal potential of botanicals obtained from Andrographis affinis and Adhatoda vasica is possibly due to presence of phytocompounds. The secondary metabolites, alkaloids and flavonoids of plants were reported to have ovicidal and nematicidal properties against root knot nematodes [42][43][44].
Alkaloids play some metabolic role and in control of development in living system. They are also involved in protective function in animals and are used as medicine especially the steroidal alkaloids [45]. Phenolic compounds are characterized by the presence of several phenol groups and act as chemical barriers against invading pathogens and protecting the plant species [46]. Flavonoids are a group of polyphenolic compounds and they widely distributed in plants. Almost 6000 flavonoids have been identified in plants. Flavonoids are free radical scavengers to the major role of inhibition of hydrolytic, oxidative enzymes, antiinflammatory action, anticarcinogenic, antifungal, antiviral and antibacterial activities and inhibit the initiation, promotion and progression of tumours [47]. Triterpenoids are a large class of natural isoprenoids present in higher plants, which exhibit a wide range of biological activities. Steroids have been observed to promote nitrogen retention in osteoporosis and in animals with wasting illness [48]. Anthraquinones are considered to be one of the most active agents in metastatic breast cancer. The mechanism of action of tannins is based on their ability to bind proteins thereby inhibiting cell protein synthesis [39]. D. stramonium is a species possess various active biomolecules with medicinal and pharmacological properties and are responsible for antibacterial, antiviral and anti-diabetic activities [49] and the analgesic and CNS depression of the species is attributed to the presence of high alkaloids contents. The atropine biomolecules isolated from this species is used for treating parkinson's disease, peptic ulcers, diarrhea and bronchial asthma [50].
Traditionally, leaves of D. stramonium are externally used for injuries, wounds, bleeding and pain. Juice of flower petals of D. stramonium is used in ear pain and seeds are used as purgative in cough, fever and asthma [51]. The wound healing property of B. suaveolens contain bioactive compounds alkaloids, triterpenoids and flavonoids are well known and are attributed to their antimicrobial  property and also responsible for wound contraction and increased rate of epithelialization [52]. The plant D. metel, D. innoxia and B. suaveolens were examined and observed that they possess secondary metabolites including alkaloids and flavonoids with high concentrations, which may serve as potent sources of bioactive molecules are using as remedies for physiological disorders and pathological diseases.
In this study, the nematicidal activity of extracts of D. metel, D. innoxia and B. suaveolens were recorded at different concentrations such as 250 ppm, 500 ppm, 750 ppm and 1000 ppm when exposed to different time intervals like 24 hrs, 48 hrs and 72 hrs. It is seen from the data presented in Tables 3 to 5 revealed that all three tested extracts were showed nematicidal activity against root-knot nematode M. incognita. The nematicidal activity of extract of D. metel was analyzed and the results were presented in Table  3. The maximum 53% of larval mortality was recorded at 1000 ppm concentration of extract of D. metel after 72 hrs of exposure time.
The extract of D. innoxia showed nematicidal activity and the results were presented in Table  4. The maximum 44% of larval mortality of M. incognita was recorded at 1000 ppm concentration in 72 hrs of exposure time. The nematicidal activity of extracts of B. suaveolens was determined and the results were showed in Table 5. There was observed the maximum 64% of nematicidal activity was recorded at 1000 ppm concentration of extract of B. suaveolens in 72 hrs of exposure time. All the three selected plant extracts were showed gradual increase of nematicidal activity to the plant extracts with the increase of concentration and exposure time. The statistical analysis was performed for the determination of the significance between concentrations of extract (250 ppm 500 ppm 750 ppm and 1000 ppm) and nematicidal activity at different time intervals (24 hrs, 48 hrs and 72 hrs).
The significance was calculated by ANOVA using DMRT at 5% level of P value (P = .05). Table 3, 4 and 5 showed significant difference in nematicidal activity among different concentrations and various time intervals. So, the nematicidal activity of the present study was concentrations and time dependent. Because the higher concentration of the extract 1000 ppm showed maximum nematicidal activity than lower concentration 250 ppm. Similarly, 72 hrs of exposure time showed maximum nematicidal activity than 24 hrs of exposure time. The significance was also mentioned in the results as superscript letters. The table value possess superscript a, which indicates the maximum nematicidal activity than other values carry alphabetic letters b -k in the superscript.    [21]. Datura species contains rich source of alkaloids like atropine, meteloidine, nicotine, scopolamine, hyoscyamine, terpenoids and flavonoids which have high rate of nematicidal activity [56,57].
Similarly, the Marigold (Tagetes spp.) contains polyacetylenes and polythienyls that proved their nematicidal activity [58]. In this study, the secondary metabolites like alkaloids, steroids, flavonoids, terpenoids, phenolic compounds, tannins, anthroquinone glycosides, saponins and triterpenes were observed in the leaf of D. metel, D. innoxia and B. suaveolens and they may be responsible for the nematicidal activity.

CONCLUSION
From this study, we concluded that the medicinal plants D. metel, D. innoxia and B. suaveolens possess phytochemicals and nematicidal activity. So, these plants would serve as source for novel nematicidal agents. Further studies are needed to identify and isolate the active nematicidal compounds from these plants, which may be useful as bionematicide for controlling nematode diseases.
Tiruchirappalli, Tamilnadu, India for her help in collection and maintenance of nematodes.